Genregulation in Rhodobacter capsulatus durch Stickstoff

Transcription

Genregulation in Rhodobacter capsulatus durch
Stickstoff, Molybdän, Kupfer und Schwefel
Dissertation zur Erlangung des Grades
eines Doktors der Naturwissenschaften
der Fakultät für Biologie und Biotechnologie
der Ruhr-Universität Bochum
angefertigt im
Lehrstuhl für Biologie der Mikroorganismen
vorgelegt von
Jessica Wiethaus
aus
Dortmund
Referent: Prof. Dr. Franz Narberhaus
Korreferent: PD Dr. Mathias Lübben
Bochum
2007
Danksagungen
Herrn Prof. Franz Narberhaus danke ich für die wissenschaftliche Betreuung meiner Doktorarbeit sowie für die wertvollen Anregungen und Ratschläge.
Bei Herrn PD Dr. Mathias Lübben möchte ich mich für die Übernahme des Korreferates bedanken.
Mein besonderer Dank gilt Herrn Dr. Bernd Masepohl für sein wissenschaftliches Engagement und die sehr gute Betreuung innerhalb der letzten drei Jahre. Durch zahlreiche Diskussionen ergaben sich immer wieder hilfreiche Ideen und Anregungen.
Bei allen jetzigen und ehemaligen Mitgliedern des Lehrstuhls möchte ich mich für die gute
Arbeitsatmosphäre bedanken. Insbesondere danke ich Britta Schubert, welche als Mitglied der
Rhodobacter-Gruppe aber auch danach zum gelingen dieser Arbeit beigetragen hat.
Abschließend danke ich vor allem meiner Familie für ihre Unterstützung und Geduld während
der vergangenen Jahre.
Inhaltsverzeichnis
I
Inhaltsverzeichnis
I
Inhaltsverzeichnis
Seite
I
II
Abkürzungen
III
A
Einleitung
B
C
D
E
F
1
1. Adaptation an eine dynamische Umwelt – der Schlüssel zum Erfolg
1
2. Stickstoff-Kontrolle durch Zwei-Komponenten-Regulationssysteme
2.1 Zwei-Komponenten-Regulationssysteme
2.2 Das enterobakterielle Ntr System
2
2
3
3. Metall-abhängige Repressoren und Aktivatoren
3.1 Metalle in biologischen Systemen
3.2. Molybdat
3.2.1 Molybdat-Transport und –Speicherung
3.2.2 Der Molybdat-abhängige Regulator ModE
3.3 Kupfer
3.3.1 Mechanismen der Kupfer-Homöostase
3.3.2 Kupfer-abhängige Regulatoren
5
5
6
6
8
9
9
11
4. Regulation der Taurin-Schwefel-Assimilation
4.1 Taurin als Schwefel-Quelle
4.2 Regulation der Taurin-Assimilation
13
13
14
5. Das phototrophe Purpurbakterium Rhodobacter capsulatus
5.1 Stickstoff-Kontrolle durch „cross-talk“ von Zwei-KomponentenRegulationssystemen
5.2 Molybdat-abhängige Regulation durch MopA und MopB
5.3 Regulation der Kupfer-Toleranz durch CutR
5.4 Regulation der Taurin-Assimilation durch TauR
15
16
18
19
19
Cross-talk towards the response regulator NtrC controlling
nitrogen metabolism in Rhodobacter capsulatus
21
Overlapping and specialized function of the molybdenumdependent regulators MopA and MopB in Rhodobacter capsulatus
29
Protein-protein interactions between MopA, MopB and Mop
from Rhodobacter capsulatus
41
The multicopper oxidase CutO confers copper tolerance to
Rhodobacter capsulatus
63
The GntR-like regulator TauR activates expression of taurine
utilization genes in Rhodobacter capsulatus
72
I
Inhaltsverzeichnis
G
Diskussion
90
1. Stickstoff-Kontrolle durch „cross-talk“ von NtrY und NtrC
1.1 NtrY ist eine bifunktionelle Sensorkinase für NtrC
1.2 NtrY registriert den periplasmatischen Stickstoff-Status
1.3 Der Responseregulator NtrX
90
90
91
93
2. Die Molybdat-abhängigen Regulatoren MopA und MopB
2.1 MopA und MopB regulieren den Molybdat-Metabolismus
2.2 MopA und MopB sind DNA-bindende Regulatoren
2.3 Molybdat erhöht die DNA-Affinität von MopA- und
MopB-Dimeren
2.4 MopA und MopB: mehr als nur Regulatoren
94
94
96
98
100
3. Der neuartige Kupfer-abhängige Regulator CutR
103
4. TauR, der chimäre Aktivator der Taurin-Assimilation
105
H
Zusammenfassung
108
I
Summary
110
J
Literaturliste
112
K
Publikationen
125
1. Artikel
125
2. Kongress-Beiträge
125
L
Anhang
127
1. Erklärung
127
2. Lebenslauf
128
II
Abkürzungen
II
Abkürzungen
Abb.
Abbildung
ABC
ATP-binding cassette
ADP
Adenosin-5`-Diphosphat
AMP
Adenosin-5`-Monophosphat
Asp
Aspartat-Rest
ATP
Adenosin-5`-Triphosphat
ATPase
Adenosintriphosphatase
C-Terminus
Carboxyl-Terminus
Cu
Kupfer
DNA
Desoxyribonukleinsäure
DR
direct repeat
EBP
enhancer binding protein
FeFeco
Eisen-Eisen-Cofaktor
FeMoco
Eisen-Molybdän-Cofaktor
GABA
γ-Aminobuttersäure
GOGAT
Glutamatsynthase
GS
Glutaminsynthetase
H
Wasserstoff
HTH
helix-turn-helix
MCO
Milticopper-Oxidase
Moco
Molybdopterin-Cofactor
mop
molybdenum protein
N
Stickstoff
N-Terminus
Amino-Terminus
Ntr
nitrogen regulatory system
Pi
Phosphat-Rest
PLP
Pyridoxal-5`-Phosphat
RNA
Ribonukleinsäure
ROS
reactive oxygen species
TCS
two-component regulatory system
Trp
Tryptophan-Rest
u. a.
unter anderem
UMP
Uridin-5`-Monophosphat
wHTH
winged helix-turn-helix
z. B.
zum Beispiel
III
Einleitung
A
Einleitung
1.
Adaptation an eine dynamische Umwelt – der Schlüssel zum
Erfolg
Das Habitat eines Bakteriums ist keineswegs ein immer gleich bleibendes, statisches System.
Verschiedene Parameter wie Temperatur, Lichtintensität, Ionenstärke oder Nährstoffangebot
können zum Teil großen Schwankungen unterliegen. Eine schnelle Adaptation kann zum
einen kurzfristig das Überleben der Zelle sichern, zum anderen aber auch langfristig einen
Selektionsvorteil gegenüber anderen Bakterien darstellen. Zudem wird eine hohe Effektivität
des Stoffwechsels erzielt, indem energieaufwendige Reaktionen und Syntheseleistungen nur
bei Bedarf durchgeführt werden. Die hierfür benötigte Überwachung einzelner Umweltparameter und der Transfer der eingegangenen Information wird durch ein Netzwerk signalsensorischer und -transduzierender Systeme geleistet. Angriffspunkt zur Auslösung der Zellantwort
stellen dabei DNA-, RNA- oder Protein-Moleküle dar. Durch Modifikation ihrer Struktur
oder Aktivität kommt es letztlich zur Adaptation des Stoffwechsels, der Struktur oder der
Verhaltensweise an veränderte Umweltbedingungen.
Eine adäquate Zellantwort wird auf Ebene der DNA oftmals durch signalmodulierte, DNAbindende Transkriptionsregulatoren ausgelöst. Die Signaltransduktion auf den Regulator kann
direkt oder indirekt erfolgen. So kann der Regulator selbst durch Bindung eines Signalmoleküls den Reiz aufnehmen oder ein Sensorprotein modifiziert den Regulator in Abhängigkeit
von einem Signal. Beide Formen der Signaltransduktion beeinflussen die DNA-Bindeaffinität des Regulators und führen so zur Aktivierung oder Repression der Transkription von Zielgenen. Entsprechend ihrer Stoffwechselleistungen verfügen Bakterien über ein Netzwerk von
Regulatoren, um die Adaptation an verschiedenste Umweltparameter zu gewährleisten.
Im Rahmen dieser Arbeit wurde die Adaptation des Modell-Organismus Rhodobacter capsulatus an die Verfügbarkeit folgender Nährstoffe näher untersucht:
•
Stickstoff
•
Molybdän
•
Kupfer
•
Schwefel
1
Einleitung
2.
Stickstoff-Kontrolle durch Zwei-Komponenten-Regulationssysteme
2.1
Zwei-Komponenten-Regulationssysteme
Wenn es ein Maß für die Anpassungsfähigkeit eines Bakteriums, für seinen „bakteriellen IQ“
gibt, so ist dies die Anzahl seiner Zwei-Komponenten-Regulationssysteme (TCS, two-component regulatory system) (Galperin, 2005; Hutchings et al., 2004). Dementsprechend stellen
TCS das bei weitem gängigste bakterielle System zur Signaltransduktion dar und sind an so
vielfältigen Prozessen wie Chemotaxis, Sporulation, Kompetenz, Virulenz, Phosphat- und
Sticktoff-Regulation beteiligt (Galperin, 2004; Stock et al., 1989).
Im einfachsten Fall setzen sich solche Systeme aus einer Sensor-Histidinkinase und einem
Responseregulator zusammen (Stock et al., 2000). Beide Proteine zeigen dabei einen modulartigen Aufbau (Abb. 1). Die Aufnahme eines spezifischen extra- oder intrazellulären Reizes
durch die Sensorkinase erfolgt durch die N-terminale Sensordomäne. Dies bewirkt die ATPabhängige Autophosphorylierung an einem Histidin-Rest der konservierten Kinasedomäne
(West & Stock, 2001). Die Signalweiterleitung erfolgt durch Übertragung der PhosphorylGruppe auf einen Aspartat-Rest in der konservierten N-terminalen Regulatordomäne des
Responseregulators (Stock & West, 2003). Die resultierende Konformationsänderung betrifft
auch die C-terminale Effektordomäne und induziert die Auslösung der adaptiven Antwort
(Gao et al., 2007; Lee et al., 2001). So kommt es beispielsweise bei der Chemotaxis zur Interaktion des phosphorylierten Responseregulators CheY mit dem Flagellen-Apparat, was zu
Stimulus
Sensorkinase
SD
ATP
KD
M e m b ra
n
C yto p la
sm a
ADP
His
P
P
Responseregulator
Asp
RD
ED
Adaptation
Abb. 1: Genereller Aufbau
eines TCS. Eine Membran-assoziierte Sensorkinase nimmt
den Reiz auf und autophosphoryliert an einem HistidinRest (His). Durch Übertragung
der Phosphoryl-Gruppe (P) auf
einen Aspartat-Rest (Asp) des
Responseregulators wird dieser
aktiviert. ED: Effektordomäne;
KD: Kinasedomäne; RD: Regulatordomäne; SD: Sensordomäne.
2
Einleitung
einem veränderten Schwimmverhalten führt (Szurmant & Ordal, 2004). Die Dephosphorylierung des Responseregulators erfolgt entweder spontan oder wird durch die Sensorkinase, eine
Phosphatase oder eine reversible Phosphorylierungskaskade vermittelt.
Die Mehrheit der Responseregulatoren fungiert als Transkriptionsregulator, so dass die durch
das TCS hervorgerufene Zellantwort die Aktivierung und/oder Repression von Zielgenen
durch Bindung an spezifische DNA-Abschnitte umfasst. Die meisten Transkriptionsregulatoren lassen sich basierend auf Homologien in der DNA-bindenden Effektordomäne der
OmpR-, NarL-, LytR-, PrrA-, YesN- oder NtrC-Familie zuordnen (Galperin, 2006).
2.2
Das enterobakterielle Ntr-System
Stickstoff ist als Bestandteil von Proteinen und Nukleinsäuren für alle Organismen essentiell.
Mikroorganismen können ihren Stickstoff-Bedarf durch Nutzung von molekularen Stickstoff
(N2), Nitrat (NO3-) oder Aminosäuren decken. Die bevorzugte bakterielle Stickstoff-Quelle ist
jedoch Ammonium (NH4+), welches vornehmlich über den ubiquitär verbreiteten Glutaminsynthetase(GS)/Glutamatsynthase(GOGAT)-Reaktionsweg in organische Verbindungen eingebunden und somit für biosynthetische Reaktionen zur Verfügung gestellt wird (Fuchs,
1999; Suzuki & Knaff, 2005):
Glutamat + NH4+ + ATP
GS
Glutamin + α-Ketoglutarat + NADPH
Glutamin + ADP + Pi
GOGAT
2 Glutamat + NADP+
Eine niedrige intrazelluläre Glutamin-Konzentration ist daher ein Zeichen von StickstoffMangel und führt über das enterobakterielle Ntr-System (nitrogen regulatory system) zur
Adaptation des Stoffwechsels (Abb. 2) (Reitzer, 2003). Das Ntr-System leistet die primäre
Signalaufnahme und –transduktion durch GlnD und PII und die weitere Signalumsetzung
durch ein TCS, bestehend aus NtrB und NtrC (Arcondèguy et al., 2001; Merrick & Edwards,
1995).
GlnD ist ein bifunktionelles Enzym, welches das Signaltransduktionsprotein PII (kodiert durch
glnB) in Abhängigkeit von der intrazellulären Glutaminkonzentration modifiziert. Unter
Stickstoff-Mangel kommt es zur Uridylylierung des trimeren PII-Proteins durch GlnD. Bei
hohen intrazellulären Konzentrationen an gebundenem Stickstoff hingegen wird die Uridylylierungs-Aktivität durch Glutamin allostorisch gehemmt und GlnD katalysiert die Abspaltung
3
Einleitung
des UMP-Restes von PII. Die weitere Signalübertragung erfolgt in Abhängigkeit vom PIIUridylylierungszustand.
So reguliert PII über die Interaktion mit der bifunktionellen Adenylyltransferase (ATase) die
GS-Aktivität (Jiang et al., 2007). Bei hoher Stickstoff-Verfügbarkeit erfolgt die Adenylylierung und infolgedessen die Inaktivierung der GS. Deadenylylierung und damit Aktivierung
der GS hingegen wird durch PII-UMP stimuliert.
Des Weiteren reguliert PII durch das TCS NtrB/NtrC die Expression von Genen des Stickstoff-Metabolismus. Unter Stickstoff-Mangel kann PII-UMP nicht mit der bifunktionellen
Histidin-Kinase/Phosphatase NtrB interagieren. Es kommt somit zunächst zur Autophosphorylierung der dimeren Sensorkinase NtrB, gefolgt von der Übertragung der PhosphorylGruppe auf den Responseregulator NtrC. Dieser zeigt einen dreigeteilten Aufbau aus N-terminaler Regulatordomäne, zentraler AAA+-Typ ATPase-Domäne und C-terminaler Fis-Typ
DNA-Bindedomäne (Lee et al., 2003; Volkman et al., 1995). NtrC zählt zu den EnhancerBindeproteinen (EBPs), und fungiert als σ54-abhängiger Transkriptionsaktivator (Studholme
& Dixon, 2003; Xu & Hoover, 2001). Statt der durch die „housekeeping“ σ70-RNA-Polymera-
+N
GS
NtrC
ATase
NtrB
NtrC
GS
P
AMP
PII
GlnD
PII
UMP
GS
NtrC
NtrB
-N
NtrC
P
P
ATase
GS
AMP
σ54
+
gen
Abb. 2: Funktionsweise des enterobakteriellen Ntr-Systems. Das Ntr-System setzt sich aus GlnD,
PII und dem TCS NtrB/NtrC zusammen. Die Aktivität von NtrC und der GS wird in Abhängigkeit vom
Stickstoff-Status (- N: Mangel; + N: Überschuss) reguliert. NtrC-P fungiert als σ54-abhängiger
Transkriptionsaktivator (+) von Genen des Stickstoff-Metabolismus. Eine genaue Erläuterung des
Modells erfolgt im Text. ATase: Adenylyltransferase; GS: Glutaminsynthetase.
4
Einleitung
se erkannten -35/-10-Region weisen σ54-abhängige Promotoren eine konservierte -24/-12Region auf (Buck & Cannon, 1992; Paget & Helmann, 2003). Durch Phosphorylierung von
NtrC wird die DNA-Bindung an eine UAS (upstream activator sequence) sowie die Oligomerisierung des sonst dimeren Proteins induziert (Morett & Segovia, 1993; Rappas et al., 2007).
Die bei EBPs hoch konservierte ATPase-Domäne katalysiert im Anschluss die Isomerisierung
vom geschlossenen zum offenen Promotor/RNA-Polymerase-Komplex (Porter et al., 1993;
Wedel & Kustu, 1995). Bei Stickstoff-Überschuss interagiert PII mit NtrB und inhibiert selektiv die Kinase-Aktivität, was die Autophosphorylierung der Sensorkinase verhindert. Zudem
führt die Phosphatase-Aktivität von NtrB unter diesen Bedingungen zur Dephosphorylierung
von NtrC-P und somit zur Inhibierung der Transkriptionsaktivierung.
Enterobakterien verfügen meist über zwei PII-Proteine, GlnK und GlnB. In Escherichia coli
sind beide Proteine in Abhängigkeit vom Uridylylierungsstatus in der Lage, die ATase- sowie
die NtrB-Aktivität zu regulieren (Atkinson & Ninfa, 1998; van Heeswijk et al., 1996). Zusätzlich unterliegt der AmtB-Ammoniumtransporter der Kontrolle durch GlnK (Coutts et al.,
2002). Die Bindung des unmodifizierten GlnK hemmt die Aktivität des Transporters, so dass
Ammonium nicht länger aktiv aufgenommen wird. Da die Expression des glnK-amtB Operons jedoch selbst der Stickstoff-Aktivierung durch NtrC unterliegt, wird vermutet, dass GlnK
im Wesentlichen der Adaptation an einen kurzfristigen Anstieg der Stickstoff-Verfügbarkeit
dient.
3.
Metall-abhängige Repressoren und Aktivatoren
3.1
Metalle in biologischen Systemen
Die meisten Schwermetalle sind auf Grund unvollständig aufgefüllter d-Orbitale hoch reaktiv
und können redoxaktive Komplexe ausbilden (Nies, 1999). Dies befähigt sie als enzymatische
Cofaktoren in biologischen Systemen vielfältige Funktionen zu übernehmen. Dem physiologischen Nutzen einiger Metalle steht jedoch ihre zum Teil hohe Toxizität gegenüber. So vermögen freie Schwermetall-Ionen mit Thiol-, Amino- und Carboxylgruppen von Proteinen zu
interagieren und hierdurch deren Funktion zu beeinträchtigen. Zudem kann es durch Reaktionen mit molekularem Sauerstoff zur Erzeugung von oxidativem Stress kommen (Nies, 1999).
Folglich werden bevorzugt minder bis leicht toxische Schwermetalle von biologischen Systemen genutzt. Zu den für viele Organismen essentiellen Biometallen zählen u. a. Zink, Eisen,
5
Einleitung
Nickel, Kupfer und Molybdän. So lässt sich Eisen z. B. im aktiven Zentrum von Enzymen
finden, welche an grundlegenden Stoffwechselvorgängen wie Atmung (Cytochrome) und
Photosynthese (Ferredoxine) beteiligt sind.
Entsprechend des bivalenten Charakters von Biometallen müssen sowohl Mangel- als auch
Überschusssituationen vermieden werden, um ein effizientes Wachstum zu sichern. Verschiedene Homöostase-Mechanismen dienen daher der Aufrechterhaltung einer intrazellulären
Metall-Konzentration im physiologischen Bereich (Silver & Phung, 2005). In Mangelsituationen kommen vor allem Aufnahmesysteme wie ABC-Transporter (ATP-bin-ding cassette)
zum Einsatz (Rosen, 2002). Eine intrazelluläre Akkumulation freier Metall-Ionen kann durch
Bindung an Speicherproteine vermieden werden. Bei Metallen mit einer hohen Bioverfügbarkeit bewältigen zudem meist Efflux-Pumpen die Entgiftung des Cyto- oder Periplasmas (Poole, 2007). Die genannten Systeme unterliegen meist einer strengen Regulation auf genetischer
Ebene (O'Halloran, 1993; Zhu & Thiele, 1996). Die intrazelluläre Metall-Akkumulierung
dient dabei als Signal, welches durch Regulator-DNA-Interaktion in eine abgeänderte Transkription umgesetzt wird. Die Signalübertragung kann durch TCS oder direkte Bindung des
Metalls an den Regulator realisiert sein.
3.2. Molybdat
3.2.1 Molybdat-Transport und -Speicherung
Das Übergangsmetall Molybdän ist essentiell für die meisten biologischen Systeme (Hille,
2002). So findet sich Molybdän im aktiven Zentrum von über 40 redoxaktiven Enzymen,
welche Schlüsselreaktionen der Kohlenstoff-, Stickstoff- und Schwefel-Zyklen katalysieren
(Hille, 1996; Mendel & Hänsch, 2002). Dabei kann zwischen zwei Molybdän-haltigen Cofaktoren unterschieden werden (Mendel & Bittner, 2006). Lediglich Nitrogenasen weisen den
Eisen-Molybdän-Cofaktor (FeMoco) auf, bei dem das Molybdän durch einen Eisen-SchwefelCluster, Homocitrat und einen Histidin-Rest der Nitrogenase koordiniert wird. Molybdoenzyme wie die Xanthin-Dehydrogenase tragen hingegen den Molybdopterin-Cofaktor (Moco),
bei dem das Molybdän-Atom durch den organischen Cofaktor Pterin koordiniert wird (Kisker
et al., 1998).
Um die Aktivität von Molybdoenzyme zu sichern, nehmen viele Bakterien das bioverfügbare
Oxyanion Molybdat (MoO42-) über einen hoch affinen ABC-Transporter auf (Pau, 2003; Self
6
Einleitung
et al., 2001). Entsprechende Transportsysteme wurden bislang in mehr als 20 Prokaryoten,
wie z. B. E. coli, Bradyrhizobium japonicum und Xanthomonas axonopodis, identifiziert (Balan et al., 2006; Delgado et al., 2006; Rech et al., 1995). Die Aufnahme von Molybdat wird
dabei durch das Substratbindeprotein ModA, das integrale Membranprotein ModB und die
ATPase ModC vermittelt (Hollenstein et al., 2007). In der Regel sind die entsprechenden Gene in einem Operon in der Reihenfolge modA, modB und modC organisiert. Auf Grund seiner
vergleichbaren Struktur und Chemie wird auch Wolframat über den ModABC-Transporter in
die Zelle eingebracht (Rech et al., 1996). Zudem kann Wolfram Molybdän in Cofaktoren ersetzen, was in den meisten Fällen zur Inaktivierung der entsprechenden Molybdoenzyme führt
(Lei et al., 2000; Trautwein et al., 1994). Daher ist die Bioverfügbarkeit von Wolframat für
Mikroorganismen, welche Mod-Transporter kodieren, wahrscheinlich äußerst gering, so dass
hauptsächlich Molybdat aufgenommen wird (Pau, 2003). Neben der Molybdat-Aufnahme
über den hoch affinen ModABC-Transporter scheint bei E. coli und andere Organismen zusätzlich der Transport über nieder affine Systeme realisiert zu sein (Imperial et al., 1985;
Sperl & DeMoss, 1975). Ein möglicher Kandidat für die unspezifische Molybdat-Aufnahme
ist der Sulfat-Transporter, welcher Molybdat mit einer zehnfach geringeren Effizienz als
ModABC transportieren würde (Pau, 2003).
Ein weitere Komponente des Molybdat-Metabolismus sind die in einer Reihe von Bakterien
und Archaea vorkommenden mop-Domänen-Proteine (molybdenum protein). Diese hoch
konservierten Domänen von 69 Aminosäuren binden sowohl Molybdat als auch Wolframat
und lassen sich in drei cytoplasmatischen Proteinen mit distinkter Funktion finden: Molbindin, ModC und ModE (Abb. 3) (Duhme et al., 1999). Molbindine sind mit einem Molekular-
Transport
Transport
ModA
ModE
Regulation
Regulation
ModB
Membran
ModC
Cytoplasma
Mb
Abb. 3: Vorkommen von mopDomänen in Proteinen des Molybdat-Metabolismus. Molybdatbindende mop-Domänen (grau) sind
in der ATPase ModC und dem Regulator ModE C-terminal lokalisiert.
Molbindine (Mb) bestehen entweder
aus einer mono-mop- oder einer dimop-Domäne und sind putative Molybdat-Speicherproteine.
Speicherung??
Speicherung
7
Einleitung
gewicht von 7 kDa oder 14 kDa lediglich aus einer (mono-mop) oder zwei (di-mop) Molybdat-bindenden Domänen aufgebaut. Die Molbindin-Familie umfasst u. a. Mop aus Eubacterium acidaminophilum und Haemophilus influenzae (Makdessi et al., 2004, Masters et al.,
2005). Molbindine sind multimere Proteine, wobei di-mop-Molbindine Trimere und monomop-Molbindine Hexamere bilden (Delarbre et al., 2001; Wagner et al., 2000; Williams et
al., 1999). In beiden Fällen kommt es somit zu einer hexameren Anordnung von mopDomänen. Obwohl für einen Teil dieser Proteine bereits die Kristallstruktur vorliegt, ist ihre
physiologische Funktion weitestgehend unklar. Da Molbindine jedoch 8 Molybdat pro Multimer binden, wird von einer Rolle als Molybdat-Speicherproteine ausgegangen (Pau & Lawson, 2002; Schüttelkopf et al., 2002). Des Weiteren lassen sich C-terminale mop-Domänen in
dem Transporterprotein ModC (mono-mop-Domäne) und in Molybdat-abhängigen Regulatorproteinen der ModE-Familie (di-mop-Domäne; siehe 3.2.2) finden.
3.2.2 Der Molybdat-abhängige Regulator ModE
In E. coli fungiert der Molybdat-abhängige Transkriptionsregulator ModE als negativer oder
positiver Effektor für eine Reihe von Operons des Molybdat-Metabolismus (Grunden &
Shanmugam, 1997; Tao et al., 2005). So dient ModE als Molybdat-abhängiger Repressor des
modABC-Operons und passt die Aufnahme von Molybdat seiner intrazellulären Verfügbarkeit
an (Grunden et al., 1996). Im Gegensatz dazu steigert ModE im Zusammenspiel mit anderen
Transkriptionsfaktoren die Expression von Proteinen der frühen Moco-Biosynthese und von
Molybdoenzymen wie der periplasmatischen Nitratreduktase (Anderson et al., 2000; McNicholas & Gunsalus, 2002). Dies ermöglicht eine effektive Verwertung des Molybdats und
vermeidet zudem eine intrazelluläre Akkumulation freier Metall-Ionen.
Im ModE-Dimer lassen sich hinsichtlich Struktur und Funktion zwei Domänen definieren
(Abb. 4) (Hall et al., 1999). Die N-terminale DNA-Bindedomäne weist das „winged helixturn-helix“(wHTH)-Motiv der LysR-Familie bakterieller Transkriptionsregulatoren auf. Demgegenüber kann die C-terminale di-mop-Domäne der Molybdat-Bindung zugeordnet werden
und scheint entscheidend für die Dimerisierung zu sein (McNicholas et al., 1998b). ModE
bindet Molybdat mit einer Stöchiometrie von zwei Molekülen pro Dimer, was zu einer erhöhten Bindeaffinität an konservierte DNA-Abschnitte führt (Studholme & Pau, 2003). Durch
direkte Bindung an diese so genannten Mo-Boxen wird die Expression nachgeschalteter Gene
8
Einleitung
modifiziert und so eine Adaptation des Molybdat-Stoffwechsels an die vorherrschende Konzentration des Metalls erzielt.
Abb. 4: Ribbon-Diagramm des ModEDimers aus E. coli. Gezeigt ist das ModEDimer mit den beiden Untereinheiten A und
B. Die vier C-terminalen mop-Domänen
(Mop1A, Mop2A, Mop1B, Mop2B) sind
farblich voneinander abgehoben. Die beiden
N-terminalen DNA-Bindedomänen sind in
rot und orange dargestellt und die wHTHMotive sind angezeigt. Zur Unterscheidung
von A und B sind die Trp186-Reste (A186,
B186) in der „ball-and-stick“-Darstellung
gezeigt (Gourley et al., 2001).
3.3
Kupfer
3.3.1 Mechanismen der Kupfer-Homöostase
Kupfer ist sowohl in eukaryotischen als auch in prokaryotischen Organismen als katalytischer
Cofaktor von Enzymen weit verbreitet. So lässt sich Kupfer im aktiven Zentrum von über 30
Proteinen finden, die an Prozessen wie Photosynthese, Zellatmung und Eisen-Transport beteiligt sind (Arredondo & Nunez, 2005). Die Eigenschaft, Elektronen sowohl leicht aufnehmen
als auch abgeben zu können, macht Kupfer zu einem idealen Partner von Redoxreaktionen
aber auch zu einem potenten Cytotoxin. Bei einer intrazellulären Akkumulation freier Cu(I)Ionen kann es zur Übertragung von Elektronen auf Sauerstoff und somit zur Entstehung reaktiver Sauerstoffspezies (ROS, reactive oxygen species) kommen (Manzel et al., 2004; Tree et
al., 2005; Valko et al., 2005). ROS können die oxidative Schädigung von biologischen Makromolekülen wie Proteinen, Lipiden und DNA-Molekülen bewirken (Ercal et al., 2001). Darüber hinaus kann Kupfer mit verschiedenen funktionellen Gruppen von Proteinen, Polysacchariden und Lipiden interagieren und deren Struktur und Funktion ändern. Der physiologische Bedarf an Kupfer bei gleichzeitiger Toxizität stellt hohe Anforderungen an biologische
9
Einleitung
Systeme. Sowohl Mangel- als auch Überschusssituationen müssen durch eine gut regulierte
Kupfer-Homöostase vermieden werden. Entsprechende Mechanismen sind hierbei chromosomal und, im Falle von hoch Kupfer-resistenten Bakterien, plasmidär kodiert (Cooksey,
1993).
Spezifische Kupfer-Aufnahmesysteme sind nur in wenigen Prokaryoten bekannt. In Enterococcus hirae erfolgt die Kupfer-Aufnahme in Mangelsituationen über die CPx-Typ ATPase
CopA (Magnani &. Solioz, 2005). CPx-Typ ATPasen gehören zur Superfamilie der P-Typ
ATPasen und katalysieren den Transport von Übergangs- oder Schwermetallen über die Cytoplasmamembran (Solioz & Vulpe, 1996). Daneben scheint jedoch vielfach die unspezifische
Aufnahme über MIT-Systeme (metal inorganic transport) realisiert zu sein (Hassett et al.,
2000; Nies, 1999).
Die Kupfer-Entgiftung erfolgt meist über Effluxsysteme (Nies, 2003). So leisten vielfach
CPx-Typ ATPasen wie CopA aus E. coli den Transport von Kupfer über die Cytoplasmamembran (Rensing et al., 2000). Ausschließlich bei Gram-negativen Bakterien sorgen Substrat/Protonen-Antiporter der CBA-Transporter-Familie für den direkten Efflux von KupferIonen in das umgebende Medium (Nies, 2003; Poole, 2001). Dies gewährleistet den zusätzlichen Schutz des Periplasmas vor Kupfer-induzierter Schädigung. Neben Effluxsystemen tragen vereinzelt Kupfer-Chaperone zur Detoxifizierung bei. Durch Bindung des Kupfers wird
dieses zellschädigenden Reaktionen entzogen und kann darüber hinaus anderen Systemen der
Kupfer-Homöostase zugeführt werden. So leitet CusF in E. coli dem CBA-Transporter periplasmatische Kupfer-Ionen zu (Rensing & Grass, 2003).
In Organismen wie Pseudomonas syringae, Salmonella enterica und E. coli wurden Multicopper-Oxidasen (MCO) als weitere Komponente der bakteriellen Kupfer-Homöostase identifiziert (Mellano & Cooksey, 1988; Kim et al., 2002; Grass & Rensing, 2001). MCO koppeln
die Oxidation ihres Substrats in ein-Elektron-Schritten an die Reduktion von molekularem
Sauerstoff zu Wasser. Dabei bilden drei Typen von Kupfer-Bindestellen mit unterschiedlichen spektroskopischen wie funktionellen Eigenschaften eine Einheit (Solomon et al., 1996).
Typ 1 („blue copper“) ist der primäre Elektronen-Akzeptor des Substrats. Ein so genannter
trinuclearer Cluster aus einem Typ 2 („normal copper“) und zwei Typ 3 („binuclear coppers“)
Kupfern bildet die Sauerstoff-Binde- und -Reduktionsstelle. Die genaue Funktion von MCOs
in der Kupfer-Homöostase ist unklar. Es wird vermutet, dass sie als Vermittler zwischen Kupfer- und Eisen-Homöostase fungieren. So setzt die periplasmatische MCO CueO aus E. coli
zum einen Cu(I) in das weniger toxische Cu(II) um (Outten et al., 2001). Zudem konnte das
Catechol-Siderophor Enterobaktin als natürliches Substrat von CueO identifiziert werden
10
Einleitung
(Kim et al., 2001). Die Oxidation des Siderophors verhindert die Enterobaktin-abhängige Reduktion von Cu(II) zu Cu(I) (Kamau & Jordan, 2002; Li et al., 1994; Schweigert et al., 2001).
Das resultierende Oxidationsprodukt aus Enterobaktin komplexiert zudem Kupfer und könnte
zusätzlich zur Entgiftung beitragen.
Die genannten Homöostase-Mechanismen müssen letztlich einen sehr engen intrazellulären
Kupfer-Konzentrationsbereich aufrechterhalten und sich dynamisch wechselnden Umweltbedingungen anpassen. Dies wird durch Kupfer-abhängige Regulatorproteine gewährleistet.
3.3.2 Kupfer-abhängige Regulatoren
Neben relativ universellen Kupfer-Homöostase-Mechanismen haben sich verschiedene Kupfer-abhängige Regulationssysteme in Bakterien entwickelt (Abb. 5). Diese lassen sich zunächst grob in TCS und Regulatorproteine, welche direkt als Sensor fungieren, einteilen.
Das TCS CusR/CusS reguliert in E. coli die Transkription des cus-Operons (Cu-sensing),
welches für den Efflux-Komplex CusCFBA kodiert (Munson et al., 2000; Franke et al.,
2003). Die in der Cytoplasmamembran lokalisierte Histidinkinase CusS dient hierbei als Sensor für periplasmatische Cu(I)-Ionen. Bei erhöhten Cu(I)-Konzentrationen vermittelt CusS die
Phosphorylierung des Responseregulators CusR, welcher dann durch Bindung an die konservierte CusR-Box die Transkription des cus-Operons aktiviert (Yamamoto & Ishihama, 2005).
Weitere Vertreter solcher Kupfer-abhängigen TCS sind u. a. CrdR/CrdS aus Helicobacter
pylori und CopR/CopS aus P. syringae (Mills et al., 1994; Waidner et al., 2005).
Eine weitere Gruppe Kupfer-abhängiger Regulatorproteine ist der MerR-Familie bakterieller
Transkriptionsfaktoren zuzuordnen (Brown et al., 2003). So vermittelt E. coli CueR in Anwesenheit von Kupfer, Silber und Gold die Aktivierung des durch copA und cueO kodierten
Cue-Systems (Cu-efflux-system) (Stoyanov et al., 2001; Stoyanov & Brown, 2003). Bindung
von zwei Cu(I)-Ionen pro CueR-Dimer erfolgt über eine C-terminale Metall-Bindedomäne
(Changela et al., 2003; Chen et al., 2003). Das N-terminale „helix-turn-helix“(HTH)-Motiv
hingegen dient der Bindung an eine konservierte DNA-Sequenz, der so genannten CueR-Box
(Yamamoto & Ishihama, 2005). CueR-Homologe wurden u. a. in Pseudomonas putida, Salmonella enterica und Bacillus subtilis identifiziert (Adaikkalam & Swarup, 2002; Gaballa et
al., 2003; Kim et al., 2002).
Ein weiterer Kupfer-Regulator ist CopL, welcher ausschließlich in X. axonopodis und nah
11
Einleitung
CusS
CusS
+ Cu
TCS
TCS
P
P
CusR
+
cus
CopL
CueR
Aktivatoren
Aktivatoren
+ Cu
+
+
+ Cu
cue
CopY
Repressoren
Repressoren
-
copA
CsoR
- Cu
cop
-
- Cu
cso
Abb. 5: Kupfer-abhängige Genregulation. Die Wirkungsweise einzelner Regulatoren der bakteriellen Kupfer-Homöostase ist schematisch dargestellt. Es kann zwischen TCS, Aktivatoren und Repressoren unterschieden werden. In Abhängigkeit von der Kupfer-Verfügbarkeit (+ Cu: hohe Verfügbarkeit; - Cu: geringe Verfügbarkeit) beeinflussen die Regulatoren die Expression von Zielgenen (+: Aktivierung; -: Repression). Eine genaue Erklärung der Funktionsweise der dargestellten regulatorischen
Systeme erfolgt im Text.
verwandten Spezies vorkommt (Voloudakis et al., 2005). CopL vermittelt die Kupfer-abhängige Expression des copA-Gens, welches für eine MCO kodiert. Es lassen sich keine konservierten Domänen in CopL identifizieren.
Die bisher genannten Regulatoren aktivieren in Anwesenheit von Kupfer die Transkription
von Zielgenen. Im Gegensatz dazu wirken einige Regulatoren der Kupfer-Homöostase in
Abwesenheit von Kupfer als Repressoren. Ein Beispiel hierfür ist CopY, der Regulator des
copYZAB-Operons aus Enterococcus hirae (Portmann et al., 2004, 2006). Neben dem Repressor selbst werden im cop-Operon zwei Cpx-Typ ATPasen (CopA, CopB) und ein KupferChaperon (CopZ) kodiert. CopY reprimiert das Operon durch direkte Bindung eines Nterminalen wHTH-Motivs an eine konservierte DNA-Sequenz, die so genannte Cop-Box. Der
Repressor liegt dabei als Dimer mit zwei gebundenen Zn(II)-Ionen vor. Bei steigenden Kupfer-Konzentrationen werden Cu(I)-Ionen durch CopZ an CopY weitergeleitet und können hier
die Zn(II)-Ionen ersetzen. Dies bedingt die Ablösung des Dimers von der DNA und somit die
Derepression des cop-Operons. Ein weiterer Regulator der vor allem bei Gram-positiven Bakterien weit verbreiteten CopY-Familie ist TcrY aus Enterococcus faecium (Hasman, 2005).
12
Einleitung
Ein erst kürzlich in Mycobacterium tuberculosis identifizierter Typ Kupfer-spezifischer Regulatoren ist CsoR, ein Vertreter der zuvor uncharakterisierten DUF156-Proteinfamilie (Liu et
al., 2007). In Abwesenheit von Kupfer bindet CsoR an die Promotor-DNA des cso-Operons,
welches für CsoR und den putativen Kupfer-Exporter CtpV kodiert (Palmgren & Axelsen,
1998). Die Bindung von zwei Cu(I)-Ionen pro CsoR-Dimer führt zu einer Konformationsänderung, welche die Loslösung von der DNA und damit die Derepression bewirkt.
4.
Regulation der Taurin-Schwefel-Assimilation
4.1
Taurin als Schwefel-Quelle
Schwefel ist essentiell für biologische Systeme. Als Bestandteil der Aminosäuren Cystein und
Methionin ist er von struktureller Bedeutung. Darüber hinaus ist Schwefel als Komponente
von Eisen-Schwefel-Clustern, Biotin, Coenzym A und anderen Cofaktoren entscheidend für
die katalytische Aktivität von Enzymen. Zusätzlich dient er der Koordinierung von MetallIonen in Proteinen. Folglich ist die Deckung des Schwefel-Bedarfs für ein effektives Wachstum unerlässlich.
Die bevorzugte bakterielle Schwefel-Quelle ist Sulfat (SO42-), welches durch Assimilierung
im Zuge der Cystein-Biosynthese reduziert und in organische Verbindungen eingebaut wird
(Kredich, 1996). Cystein steht daraufhin als intrazellulärer Schwefel-Donor für biosynthetische Reaktionen zur Verfügung.
In Böden macht Sulfat oftmals weniger als 5 % des bioverfügbaren Schwefels aus. Der Großteil des Schwefels liegt hier organisch gebunden vor, wobei neben Aminosäuren und Peptiden
vor allem Sulfonate und Sulfatester die vorherrschenden Verbindungen sind (Kertesz, 2000).
Verschiedene Bakterien können sich diese Sulfonate und Sulfatester als alternative SchwefelQuellen zu nutze machen (Cook & Denger, 2002; Eichhorn & Leisinger, 2001; UriaNickelsen et al., 1993). So kann das weit verbreitete Taurin (2-Aminoethansulfonat) durch
viele Prokaryoten verwertet werden, wobei die Taurin-Aufnahme häufig über einen tauABCkodierten ABC-Transporter erfolgt (Eichhorn et al., 2000; Gorzynska et al., 2006). Die primäre Assimilation des Taurin-Schwefels wird in aeroben Bakterien durch die TaurinDioxygenase TauD katalysiert. Hierbei zerfällt Taurin durch Inkorporation von Sauerstoff zu
Aminoacetaldehyd und Sulfit (Abb. 6 A) (Eichhorn et al., 1997; O´Brien et al., 2003). Bei
Anaerobiern hingegen erfolgt zunächst eine Deaminierungsreaktion von Taurin durch Tpa
13
Einleitung
(Taurin:Pyruvat-Aminotransferase) oder durch TDH (Taurin-Dehydrogenase) (Abb. 6 B)
(Denger et al., 2004b). Tpa katalysiert die Übertragung der Amino-Gruppe von Taurin auf
Pyruvat (Denger et al., 2006). Demgegenüber katalysiert TDH die Freisetzung von Ammonium unter Reduktion von Cytochrom c. Das bei Anaerobiern über beide Reaktionswege gebildete Sulfoacetaldehyd wird im Folgenden durch die Sulfoacetaldehyd-Acetyltransferase Xsc
zu Acetylphosphat und Sulfit umgesetzt (Denger et al., 2004a). Somit steht Schwefel nach der
primären Assimilation aus Taurin in Form von Sulfit für weitere Reaktionen zur Verfügung
und kann u. a. in die Sulfatassimilation eingespeist werden.
A
HSO3-
NH3+
- OS
3
TauD
Taurin
B
- OS
3
HSO3-
Tpa
Taurin
H
TD
NH3
+
NH2
Aminoacetaldehyd
Alanin
NH3+
- OS
3
O
- OS
3
O
O
Xsc
OPO32NH4+
Sulfoacetaldehyd
Acetylphosphat
Abb. 6: Nutzung von Taurin als Schwefel-Quelle. Stark vereinfachte Darstellung des primären Taurin-Abbaus. Bei aeroben Bakterien (A) wird Taurin durch TauD zu Hydroxytaurin umgesetzt, welches
sofort zu Aminoacetaldehyd und Hydrogensulfit (HSO3-) zerfällt. Bei anaeroben Bakterien (B) kann
Taurin durch Tpa oder TDH zu Sulfoacetaldehyd umgesetzt werden. Die Freisetzung des Schwefels in
Form von Hydrogensulfit erfolgt anschließend durch Xsc.
4.2
Regulation der Taurin-Assimilation
In E. coli unterliegen Gene des Schwefel-Metabolismus der Transkriptionskontrolle durch
CysB, einem Regulator der LysR-Familie (Iwanicka-Nowicka et al., 2007). So ist CysB in
Abhängigkeit von der Schwefel-Verfügbarkeit sowohl der Haupt-Aktivator von Genen der
Cystein-Biosynthese und der Sulfonat-Verwertung als auch ein autoregulatorischer Repressor
(van der Ploeg et al., 2001). Ein Maß für den intrazellulären Schwefel-Status stellt dabei NAcetylserin dar (Kredich, 1992). Unter Sulfat-Mangel kommt es zur intrazellulären Akkumulation von O-Acetylserin, welches neben Sulfid für den letzten Schritt der Cystein-Biosyn14
Einleitung
these benötigt wird. O-Acetylserin isomerisiert spontan zu N-Acetylserin, welches als CysB
Induktor dient (Kredich, 1996). N-Acetylserin-Bindung an CysB stimuliert die Anlagerung
der N-terminalen HTH-Domäne an positiv regulierte Promotoren und hemmt zugleich die
Bindung an den negativ regulierten cysB-Promotor (Lochowska et al., 2004; van der Ploeg et
al., 1997).
Neben diesem globalen Regulationsprinzip scheint auch eine spezifische Regulation der Taurin-Verwertung realisiert zu sein. So wurden in Taurin-Genclustern vieler α- und β-Proteobakterien Gene identifiziert, welche für einen putativen Regulator (TauR) kodieren (Brüggemann et al., 2004). TauR weist Homologien zur GntR-Superfamilie bakterieller Transkriptionsregulatoren auf. Es ist jedoch bislang noch ungeklärt, ob und wie TauR-Homologe in die
Regulation der Taurin-Verwertung eingreifen.
5.
Das phototrophe Purpurbakterium Rhodobacter capsulatus
Das phototrophe Nichtschwefel-Purpurbakterium R. capsulatus gehört innerhalb der Gruppe
der α-Proteobakterien der Familie der Rhodospirillaceae an und ist in Süßgewässer wie Seen
und Flüssen weit verbreitet (Weaver et al., 1975). Der Name des stäbchenformigen Gramnegativen Bakteriums beruht auf einer roten Pigmentierung und dem Besitz einer Polysaccharidkapsel. R. capsulatus zählt zu den fakultativen Anaerobiern und verfügt über vielseitige
Stoffwechselleistungen. So kann der Energiebedarf durch anaerobe Respiration, aerobe Respiration oder anoxygene Photosynthese gedeckt werden (Madigan, 1995). Ferner können zur
Deckung des Kohlenstoff-Bedarfs unter photoautotrophen Bedingungen Kohlendioxid und
unter heterotrophen Bedingungen verschiedene Carbonsäuren assimiliert werden (Stahl &
Sojka, 1973). Das Purpurbakterium wächst jedoch bevorzugt unter photoheterotrophen Bedingungen.
Im Zentrum dieser Arbeit stehen vier regulatorische Systeme von R. capsulatus mit Funktionen im Stickstoff-Metabolismus, im Molybdat-Metabolismus, bei der Kupfer-Toleranz und
bei der Taurin-Assimilation.
15
Einleitung
5.1
Stickstoff-Kontrolle durch „cross-talk“ von Zwei-Komponenten-Regulationssystemen
R. capsulatus kann molekularen Luftstickstoff (N2) als einzige Stickstoff-Quelle nutzen. Die
Stickstoff-Fixierung kann über zwei Nitrogenase-Systeme erfolgen, welche sich in der
Metallzusammensetzung ihrer Cofaktoren unterscheiden (Schüddekopf et al., 1993; Schneider
et al., 1997). Die konventionelle nif-kodierte Nitrogenase weist einen FeMoco auf und wird
auch als Molybdän-Nitrogenase bezeichnet. Demgegenüber trägt die anf-kodierte alternative
Nitrogenase einen Eisen-Eisen-Cofaktor (FeFeco) und wird daher auch als Heterometall-freie
Nitrogenase bezeichnet (Schneider et al., 1991). Unter diazotrophen Bedingungen katalysieren die hoch konservierten Nitrogenase-Enzymkomplexe die Reduktion von N2 zu Ammoniak
(NH3), wobei die hierfür erforderlichen Elektronen durch ATP-Hydrolyse zur Verfügung gestellt werden (Rees & Howard, 2000; Rees et al., 2005):
N2 + 8 H+ + 8 e- + 16 ATP
Nitrogenase
2 NH3 + H2 + 16 ADP + 16 Pi
Die Sauerstoffempfindlichkeit beider Nitrogenasen und der hohe Energiebedarf der Stickstoff-Fixierungsreaktion verlangen eine strikte Regulation von Synthese und Aktivität der
Enzymkomplexe. Umweltfaktoren wie Ammonium, Molybdän, Licht und Sauerstoff spielen
hierbei eine Rolle (Masepohl et al., 2004).
Die Stickstoff-abhängige Regulation beider Nitrogenase-Systeme erfolgt auf Ebene der
Transkription durch ein übergeordnetes Regulationsnetzwerk, welches dem enterobakteriellen
Ntr-System homolog ist (Abb. 7). Dieses setzt sich entsprechend aus GlnD, den beiden PII
Proteinen GlnB sowie GlnK und dem TCS NtrB/NtrC zusammen (Cullen et al., 1996; Hübner
et al., 1991; Masepohl et al., 2002a). Im Gegensatz zu E. coli wird die Aktivität der Sensorkinase NtrB hauptsächlich durch GlnB gesteuert (Drepper et al., 2003; Pawlowski et al., 2003).
Zudem bindet R. capsulatus NtrC-P nicht wie andere NtrC-Proteine an σ54-abhängige Promotoren, sondern aktiviert die Transkription seiner Zielgene im Zusammenspiel mit der σ70RNA-Polymerase (Bowman & Kranz, 1998; Foster-Hartnett et al., 1994; Richard et al.,
2003). NtrC-P induziert u. a. die Transkription der Gene nifA1, nifA2 und anfA. Diese kodieren für σ54-abhängige EBPs, die unter Stickstoff-Mangel die Transkription der übrigen nifund anf-Gene aktivieren, wobei sich NifA1 und NifA2 funktionell ersetzen können (Fischer,
1994; Masepohl et al., 1988; Schüddekopf et al., 1993). Die Aktivierung der nifund anfGene führt zur Synthese der beiden Nitrogenase-Komplexe.
16
Einleitung
GlnB
+N
NtrB
NtrC
P
NtrB
-N
NtrC
+
+
nifA1
NifA1
NtrB
P
+
nifA2
NifA2
+
anfA
AnfA
+
nif-Gene
anf-Gene
MoNitrogenase
FeNitrogenase
Abb. 7: Ntr-abhängige Regulation der Stickstoff-Fixierung in R. capsulatus. Unter StickstoffMangel (- N) kommt es zur NtrB abhängigen Phosphorylierung von NtrC. NtrC-P induziert (+) die
Expression der Gene nifA1, nifA2 und anfA. Bei Stickstof-Verfügbarkeit (+ N) interagiert GlnB mit
NtrB und die Dephosphorylierung von NtrC erfolgt. Die dargestellten Promotoren sind entweder σ70abhängig (Kästen) oder σ54-abhängig (Kreise).
Des Weiteren unterliegen verschiedene Gene des Stickstoff-Metabolismus in R. capsulatus
der Kontrolle durch NtrC. Diese kodieren für einen hoch affinen Molybdat-Transporter (siehe
5.2) und für Proteine der Harnstoff-Verwertung (Kutsche et al., 1996; Masepohl et al.,
2001b).
Wie im Fall von nifA liegen auch andere Gene der Stickstoff-Fixierung bei R. capsulatus in
duplizierter Form vor (Masepohl & Klipp, 1996). So sind stromabwärts des nifR3-ntrB-ntrCOperons Gene lokalisiert, welche für ein zweites, putatives TCS bestehend aus NtrY und
NtrX kodieren (Abb. 8). Eine ähnliche Genorganisation liegt auch in anderen Stickstoff- fixierenden Organismen vor, wobei die postulierten Funktionen von NtrX/NtrY bei der StickstoffRegulation differieren (Pawlowski et al., 1991; Ishida et al., 2002). Im Rahmen dieser Arbeit
1 kb
nifR3
ntrB
ntrC
ntrY
ntrX
Abb. 8: Die ntr-Genregion aus R. capsulatus. Die Gene ntrBC und ntrYX kodieren für zwei TCS.
Die Funktion des Genprodukts von nifR3 ist unbekannt.
17
Einleitung
wurde die Rolle von R. capsulatus NtrY näher untersucht (Drepper et al., 2006). Hierbei
konnte NtrY als alternativer Phospho-Donor für NtrC identifiziert und somit ein „cross-talk“
zwischen beiden TCS gezeigt werden.
5.2
Molybdat-abhängige Regulation durch MopA und MopB
Neben der Molybdän-Nitrogenase, welche den FeMoco trägt, kodiert R. capsulatus auch für
Moco-Enzyme wie die Xanthin-Dehydrogenase (Neumann et al., 2006). Diese katalysiert die
Oxidation von Hypoxanthin oder Xanthin zu Harnsäure und leitet dadurch die Nutzung dieser
Verbindungen als alternative Stickstoff-Quelle ein (Leimkühler et al., 1998). Folglich ist die
Deckung des Molybdat-Bedarfs für das Purpurbakterium essentiell. Die Aufnahme von Molybdat erfolgt über den hoch affinen Molybdat-Transporter ModABC (Wang et al., 1993).
Dieser setzt sich als typischer ABC-Transporter aus dem periplasmatischen Molybdat-Bindeprotein ModA, dem Transmembranprotein ModB und der ATPase ModC zusammen. Die
entsprechenden Gene sind in der Reihenfolge modABCD organisiert, wobei die Funktion von
ModD unbekannt ist (Abb. 9). Da der Phänotyp einer modABC Mutante durch Gabe hoher
Molybdat-Konzentrationen aufgehoben werden kann, wird die Existenz eines nieder affinen
Transportsystems für Molybdat angenommen (Wang et al., 1993).
Wie für ModC-Proteine typisch, weist R. capsulatus ModC eine C-terminale mop-Domäne
auf. Solche Molybdat-bindenden Domänen lassen sich in drei weiteren Proteinen von R. capsulatus identifizieren. So kodiert das Purpurbakterium für ein putatives Molybdat-Speicherprotein Mop, welches lediglich aus einer mop-Domäne aufgebaut ist. Im Gegensatz zu E. coli
lassen sich ferner zwei ModE-ähnliche Regulatoren identifizieren, MopA und MopB, welche
jeweils eine C-terminale di-mop-Domäne und eine N-terminale DNA-Bindedomäne mit
wHTH-Motiv aufweisen (Kutsche et al., 1996). Die Gene mopA und mopB sind zusammen
1 kb
mopB
mopA
modA
modB
modC
modD
Abb. 9: Die mop-mod-Genregion aus R. capsulatus. Die Genprodukte von modABC formen einen
hoch affinen Molybdat-ABC-Transporter. Die Gene mopA und mopB kodieren für ModE-homologe
Transkriptionsregulatoren.
18
Einleitung
mit modABCD in einer Genregion organisiert. Die genaue Funktion und Wirkungsweise von
MopA und MopB innerhalb der Molybdat-abhängigen Genregulation wurde im Rahmen dieser Arbeit untersucht (Wiethaus et al., 2006b; Wiethaus et al., eingereicht).
5.3
Regulation der Kupfer-Toleranz durch CutR
In R. capsulatus wurden verschiedene Kupfer-Proteine mit essentiellen Stoffwechsel-Funktionen identifiziert. Die zur Familie der Häm-Kupfer-Oxidasen zählende cbb3 Cytochrom-Oxidase ist z. B. an der aeroben Respiration beteiligt (Öztürk & Mandaci, 2006). Demgegenüber
stehen Kupfer-Toleranzmechanismen, welche dem toxischen Charakter des Schwermetalls
entgegenwirken. Das Purpurbakterium verfügt über die MCO CutO, welche Kupfer-Toleranz
unter aeroben und anaeroben Bedingungen vermittelt (Wiethaus et al., 2006a). Das stromabwärts von cutO lokalisierte cutR-Gen kodiert für einen neuartigen Kupfer-abhängigen Transkriptionsregulator (Abb. 10) (Wiethaus et al., 2006a).
1 kb
orf633
orf635
cutO
cutR
Abb. 10: Die cut-Genregion aus R. capsulatus. Das cutR-Gen kodiert für einen Regulator des
orf635-cutO-cutR-Operons. Das cutO Genprodukt ist eine MCO und vermittelt Kupfer-Toleranz. Den
mit orf gekennzeichneten Genen konnte bisher keine Funktion zugeordnet werden.
5.4
Regulation der Taurin-Assimilation durch TauR
Unter phototrophen Bedingungen kann R. capsulatus Taurin als einzige Schwefel-Quelle
effektiv nutzen (Masepohl et al., 2001a). Die Taurin-Aufnahme erfolgt dabei vermutlich über
den ABC-Transporter TauABC. Unmittelbar stromaufwärts des tauABC-Operons finden sich
drei Gene, welche in entgegengesetzter Richtung transkribiert werden. Zwei der entsprechenden Genprodukte weisen Homologien zu Enzymen der anaeroben Taurin-Assimilation bei
Bilophila wadsworthia und Alcaligenes defragans auf (Abb. 11) (Laue & Cook, 2000; Ruff et
al., 2003). Bei diesen handelt es sich um die Taurin:Pyruvat-Aminotransferase Tpa und die
19
Einleitung
Sulfoacetaldehyd-Acetyltransferase Xsc. Die primäre, anaerobe Taurin-Assimilation bei
R. capsulatus könnte also wie in Abb. 6 B dargestellt über Tpa verlaufen.
Das dritte Gen stromaufwärts von tauABC kodiert für den Regulator TauR. Über Genbanksuchen lassen sich zwar bei verschiedenen Proteobakterien tauR-homologe Gene identifizieren,
diese sind jedoch bisher nicht näher untersucht worden (Brüggemann et al., 2004). Im Rahmen dieser Arbeit war somit die Charakterisierung von TauR als erstem Vertreter dieser
Gruppe von Regulatoren der Taurin-Assimilation von besonderem Interesse (Schubert et al.,
in Vorbereitung).
1 kb
tauC
tauB
tauA
tpa
tauR
xsc
Abb. 11: Die tau-Genregion aus R. capsulatus. Die tau-Genregion kodiert sowohl für einen TaurinTransporter (tauABC) als auch für Enzyme der Taurin-Assimilation (tpa, xsc). TauR ist ein neuartiger
Transkriptionsregulator.
20
Cross-talk towards NtrC in Rhodobacter capsulatus
B
Cross-talk towards the response regulator NtrC
controlling nitrogen metabolism in Rhodobacter capsulatus
Drepper T., Wiethaus J., Giaourakis D., Gross S., Schubert B., Vogt M., Wiencek Y.,
McEwan A. G. and Masepohl B.
2006
FEMS Microbiology Letters 258(2):250-6
21
Cross-talk towards the response regulator NtrC controlling nitrogen
metabolism in Rhodobacter capsulatus
Thomas Drepper1, Jessica Wiethaus2, Daphne Giaourakis2, Silke Groß2, Britta Schubert2, Markus Vogt2,
Yvonne Wiencek2, Alastair G. McEwan3 & Bernd Masepohl2
1
Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Jülich, Germany; 2Lehrstuhl für Biologie der Mikroorganismen,
Ruhr-Universität Bochum, Bochum, Germany; and 3Department of Microbiology, University of Queensland, Brisbane, Qld, Australia
Correspondence: Bernd Masepohl,
Lehrstuhl für Biologie der Mikroorganismen,
Ruhr-Universität Bochum, 44780 Bochum,
Germany. Tel.: 149 0 234 32 25632;
fax: 149 0 234 32 14620; e-mail:
bernd.masepohl@ruhr-uni-bochum.de
Received 6 December 2005; revised 13
February 2006; accepted 6 March 2006.
First published online 3 April 2006.
doi:10.1111/j.1574-6968.2006.00228.x
Editor: Dieter Jahn
Abstract
Rhodobacter capsulatus NtrB/NtrC two-component regulatory system controls
expression of genes involved in nitrogen metabolism including urease and nitrogen
fixation genes. The ntrY–ntrX genes, which are located immediately downstream
of the nifR3–ntrB–ntrC operon, code for a two-component system of unknown
function. Transcription of ntrY starts within the ntrC–ntrY intergenic region as
shown by primer extension analysis, but maximal transcription requires, in
addition, the promoter of the nifR3–ntrB–ntrC operon. While ntrB and ntrY single
mutant strains were able to grow with either urea or N2 as sole nitrogen source, a
ntrB/ntrY double mutant (like a ntrC-deficient strain) was no longer able to use
urea or N2. These findings suggest that the histidine kinases NtrB and NtrY can
substitute for each other as phosphodonors towards the response regulator NtrC.
Keywords
Rhodobacter capsulatus; two-component
regulatory system; NtrB; NtrC; urease;
nitrogenase.
Introduction
Two-component systems (TCS) are widespread regulatory
proteins, found in Bacteria, Archaea and Eukaryotes, involved in signal sensing and transduction. TCS consist of
a sensory histidine kinase (HK) and a response regulator
(RR). Typically, a membrane-associated domain of the
sensor HK monitors an environmental signal, and in
response to this signal, autophosphorylates a conserved His
residue. The His-bound phosphoryl group of the HK is
transferred onto a specific Asp residue on the cognate RR.
The phosphorylated RR, in turn, activates transcription of
genes whose products enable the cells to react to the
environmental signal. In addition to the transphosphorylation between the cognate HK/RR pairs, some RRs receive a
phosphoryl group from noncognate HKs, by a process called
‘cross-talk’ (Chang & Stewart, 1998). Furthermore, small
metabolites such as acetyl phosphate may serve as phosphodonor for some RRs (Pioszak & Ninfa, 2004, and references
therein).
Escherichia coli and many other proteobacteria contain a
TCS, the NtrB/NtrC system, which responds to the nitrogen
status of the cell (Hakenbeck & Stock, 1996; Xu & Hoover,
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
2001; Pioszak & Ninfa, 2004). In contrast to archetypal HKs,
NtrB is not membrane-anchored, and sensing of the nitrogen status involves PII-like signal transduction proteins.
Many of the operons belonging to the NtrC regulon of E.
coli encode transport systems for nitrogen-containing compounds and are activated upon nitrogen starvation (Zimmer
et al., 2000). In addition to its cognate HK NtrB, the
noncognate HK UhpB (mainly involved in control of the
transport of phosphorylated sugars) may serve as a phosphodonor for NtrC as demonstrated by in vitro functional
characterization of all TCS from E. coli (Yamamoto et al.,
2005). In vivo, significant cross-talk towards NtrC was not
detectable in wild-type cells, but cross-talk from UbpB and
PhoR (responding to phosphate limitation) was observed in
a ntrB deletion strain (Verhamme et al., 2002). Furthermore,
in the absence of NtrB, E. coli NtrC can also be rapidly
phosphorylated by acetyl phosphate (Feng et al., 1992;
Verhamme et al., 2002).
In the present study, we focused on the NtrB/NtrC system
from the photosynthetic nonsulfur purple bacterium Rhodobacter capsulatus. In vitro reconstitution of the R. capsulatus NtrB/NtrC system revealed that NtrB-P is an efficient
phosphodonor for NtrC, whereas acetyl phosphate did not
FEMS Microbiol Lett 258 (2006) 250–256
251
serve as a substrate for autophosphorylation of NtrC
(Cullen et al., 1996). Rhodobacter capsulatus NtrC is the
transcriptional activator of a number of genes directly or
indirectly involved in nitrogen fixation and assimilation
(glnB-glnA, glnK-amtB, nifA1, nifA2, anfA and mopA-modABCD; Bowman & Kranz, 1998; Masepohl et al., 2002) and
urea utilization (ureDABCEFG; Masepohl et al., 2001). As
demonstrated by footprint analyses, NtrC binds to tandem
binding sites upstream of the respective transcription units
including the ure operon. Phosphorylation of NtrC by NtrB
increases DNA binding to the ureD promoter by at least
eightfold (Masepohl et al., 2001).
Although R. capsulatus NtrC has long been known to be
absolutely required for nitrogen fixation and urea utilization
as demonstrated by mutational analysis (Kutsche et al.,
1996; Masepohl et al., 2001, 2002; and references therein),
the exact role of NtrB has not been genetically characterized
until now. The results obtained in this study indicate that
NtrB and another putative HK, NtrY, can substitute for each
other as phosphodonors for NtrC.
Materials and methods
Bacterial strains, plasmids, and growth
conditions
The bacterial strains and plasmids used in this study are
listed in Table 1. As a basis for construction of R. capsulatus
ntrB (YWRUB11), ntrY (DG7-I, DG7-II) and ntrX (DG9)
mutant strains, appropriate restriction fragments from the
ntr gene region were cloned into mobilizable vector plasmids, which cannot replicate in R. capsulatus. The respective
ntr genes were disrupted by either a gentamicin (Gm) or a
kanamycin (Km) cassette (Fig. 1a). Methods for conjugational plasmid transfer between Escherichia coli and R.
capsulatus, and the selection of mutants, growth media,
growth conditions, and antibiotic concentrations were as
described previously (Masepohl et al., 1988, 2001; Klipp
et al., 1988).
DNA biochemistry
DNA isolation, restriction enzyme analysis, agarose gel
electrophoresis, and cloning procedures were performed
using standard methods (Sambrook et al., 1989). Restriction
enzymes and T4 DNA ligase were purchased from MBI
Fermentas (St Leon-Rot, Germany), and used as recommended by the supplier.
b-Galactosidase assays
To determine b-galactosidase activities of R. capsulatus wildtype and mutant strains carrying a chromosomal ntrY–lacZ
fusion (based on plasmid pDG10) or the broad host range
plasmid pNIRUB35 (ureDA-lacZ), cultures were grown in
RCV minimal medium containing either 9.5 mM serine
[nitrogen-limiting ( N) conditions] or 20 mM ammonium
[nitrogen-sufficient (1N) conditions] as described previously (Masepohl et al., 2001). Following growth in the
respective media to late exponential phase, b-galactosidase
activities were determined by the sodium dodecyl sulfate
(SDS)-chloroform method (Miller, 1972).
RNA isolation and primer extension
Rhodobacter capsulatus wild-type cultures were grown in
RCV minimal medium containing either 9.5 mM serine
Table 1. Bacterial strains and plasmids used in this study
Strain or plasmid
Strains
Escherichia coli
JM83
S17-1
B10S
PBK2
TD50
Plasmids
pDG7-I
pDG7-II
pDG9
pDG10
pJW39
pNIRUB35
pYWRUB11
Relevant characteristics
Reference or source
Host for cloning and plasmid amplification
RP4-2 (Tc::Mu) (Km::Tn7) integrated into the chromosome
Vieira & Messing (1982)
Simon et al. (1983)
Spontaneous Smr mutant of R. capsulatus B10
ntrC::[O-Kmr]
ntrC::[Gmr 4]
Klipp et al. (1988)
Kutsche et al. (1996)
Masepohl et al. (2001)
pSUP202 derivative carrying ntrY::[Kmr 4], Spr
pSUP202 derivative carrying ntrY::[o Kmr], Spr
pSUP401 derivative carrying ntrX::[Gmr 4], Spr
pSUP202 derivative carrying ntrY–lacZ; Tcr
pK18 derivative carrying the ntrY promoter region, Kmr
pPHU235 derivative carrying ureDA-lacZ, Tcr
Mobilizable pACYC184 derivative carrying ntrB::[Gmr 4], Tcr
This work
This work
This work
This work
This work
Masepohl et al. (2001)
This work
Gmr, gentamicin resistance; Kmr, kanamycin resistance; Smr, streptomycin resistance; Spr, spectinomycin resistance; Tcr, tetracycline resistance.
c
252
T. Drepper et al.
1 kb
(a)
nifR3
ntrB
ntrC
ntrY
Pst I Pst IXhoI Bcl I
YWRUB11 Gm >
TD50 Gm >
DG7-I
ntrX
BamHI
EcoRI
Km >
Gm > DG9
nrfA hflX
DG7-II < Km
PBK2 ΩKm
(b)
+1
ntrC
stop
−35
−10
TAAATGGGCCACAGGTAATTGTGGCAAATCCACCTCACGGGATTCGCCGCGGCGGAGAACCGGGGGACCCGTCGTGTCCATG
ntrY
start
Fig. 1. Mutational analysis of the Rhodobacter capsulatus ntr gene region. (a) The physical map shows only those restriction sites used for mutant
construction. The gentamicin (Gm) and kanamycin (Km) resistance interposons used to disrupt the respective ntr genes are not drawn to scale. The
direction of transcription of the resistance genes is symbolized by arrowheads, indicating polar and nonpolar insertions. (b) The DNA sequence of the
ntrC–ntrY intergenic region is shown, and the experimentally determined transcription start site (Fig. 2) is marked by (11). Putative ( 35) and ( 10)
promoter elements are marked.
( N) or 20 mM ammonium (1N) before isolation of total
RNA using the Micro-to-Midi Total RNA Purification
System (Invitrogen, Karlsruhe, Germany). Primer extension
was carried out as described previously (Babst et al., 1996)
using a synthetic oligonucleotide primer, GSP4 (5 0 CCCCAAAGGCCCGAGCACGAG-3 0 ), to map the transcription start site of ntrY.
Results and discussion
Genetic organization of the Rhodobacter
capsulatus ntr gene region
DNA sequence and genetic analyses suggest that the
R. capsulatus ntr (nitrogen regulation) gene region (Fig. 1a)
consists of three transcriptional units, namely nifR3–ntrB–ntrC (Cullen et al., 1998, and references therein),
ntrY–ntrX (this study), and nrfA–hflX (Drepper et al., 2002).
The gene pairs ntrB–ntrC and ntrY–ntrX code for twocomponent regulatory systems, namely the well-characterized
NtrB/NtrC system controlling nitrogen metabolism, and the
NtrY/NtrX system, the function of which is less certain for R.
capsulatus. In contrast to the cytoplasmatic protein NtrB, the
putative sensor kinase NtrY contains an N-terminal extension, which might act as a membrane anchor.
Similar to R. capsulatus, the ntrB, ntrC, ntrY, and ntrX
genes are also clustered in several other bacteria including
Azorhizobium caulinodans (Pawlowski et al., 1991) and
Azospirillum brasilense (Ishida et al., 2002). Both NtrC and
c
NtrX of A. caulinodans control nifA expression, but neither
of the two proteins is essential for N2 fixation (Pawlowski
et al., 1991). In contrast, mutational analysis revealed that R.
capsulatus NtrC is absolutely required for growth with either
N2 or urea as sole nitrogen source (Foster-Hartnett et al.,
1994; Masepohl et al., 2001, 2002), suggesting that neither
NtrX nor any other transcriptional activator can substitute
for NtrC.
As a basis for genetic analysis of the R. capsulatus ntr gene
region, interposon cassettes carrying gentamicin or kanamycin resistance genes were used to construct appropriate
mutant strains (Fig. 1a; Materials and methods). Both
interposons are known to induce polar or nonpolar mutations depending on their orientation (Hübner et al., 1991;
Schmehl et al., 1993; Masepohl et al., 2001). Therefore, in
ntrB mutant strain YWRUB11 (ntrB::[Gm 4]) transcription
of ntrC is driven by the gentamicin cassette, and in ntrY
mutant DG7-I (ntrY::[Km 4]) expression of ntrX depends
on the Km interposon.
Null mutant strains carrying nonpolar mutations in ntrB
(YWRUB11), ntrC (TD50), or ntrY (DG7-I) formed colonies of wild-type size on rich-medium plates, ruling out that
NtrB, NtrC, or NtrY were essential for viability. Next, we
analyzed growth of these mutant strains in liquid minimal
medium with ammonium as an N source (Fig. 3a). All
mutant strains exhibited growth properties comparable to
those of the wild type, demonstrating that NtrB, NtrC, and
NtrY are dispensable under nitrogen sufficient conditions. It
is worth to note, however, that ntrX mutant strain DG9
253
Table 2. Expression of ntrY–lacZ in Rhodobacter capsulatus wild-type
and ntrC mutant strains
b-Galactosidase activity
A
C
G
T [+N][–N]
A
T (−10)
T
C
G
C
C
G
C
G
G
C (+1)
G
G
A
G
A
A
C
C
G
G
G
G
G
A
w
Strain
1N
N
B10S
TD50
PBK2
Wild-type, ntrY–lacZ
ntrC::[Gm 4], ntrY–lacZ
ntrC::[OKm], ntrY–lacZ
443 4
301 20
260 13
469 36
361 17
283 9
Rhodobacter capsulatus strains contained plasmid pDG10 (ntrY–lacZ)
integrated into the chromosome via single recombination.
Results represent the means and standard deviations of three independent measurements.
1N, nitrogen-sufficient conditions; N, nitrogen-limiting conditions.
w
formed colonies on rich-medium plates which were significantly smaller than wild-type colonies. Similar results
were obtained for ntrY mutant strain DG7-II carrying an
interposon insertion acting polar on ntrX expression. This
apparent growth defect of strain DG7-II compared with
normal growth properties of ntrY mutant DG7-I (carrying a
nonpolar Km cassette) strongly suggests that ntrY and ntrX
form part of a transcriptional unit.
In contrast to mutant strains defective for ntrX (DG9 and
DG7-II), a ntrC mutant strain containing a strictly polar
omega cassette (PBK2, Fig. 1a; Table 1) formed colonies of
wild-type size on rich-medium plates. Furthermore, doubling
times of PBK2 (8.9 h) were comparable to those of the wild
type (9.2 h) in minimal medium with ammonium as an Nsource. This was the first hint, that transcription of the
ntrY–ntrX operon starts from a promoter located downstream
of ntrC. To verify this assumption, we compared expression of
a chromosomal ntrY–lacZ fusion in the wild-type and two
ntrC mutant strains carrying either a polar (PBK2) or a
nonpolar (TD50) interposon cassette (Table 2). Rhodobacter
capsulatus strains were grown phototrophically under either
nitrogen-sufficient conditions [with ammonium as an Nsource (1N)] or under nitrogen-limiting conditions [with
serine as an N-source ( N)] before determination of lacZderived b-galactosidase activity. The results shown in Table 2
may be summarized as follows. (i) A strictly polar mutation in
ntrC (PBK2) did not abolish ntrY–lacZ transcription, confirming that transcription of ntrY starts from an NtrCindependent promoter located downstream of the insertion
site of the O cassette within ntrC. (ii) As ntrY expression was
significantly lower in the mutant strain carrying a polar ntrC
mutation (PBK2) compared with the wild type, one might
speculate that a second promoter upstream of ntrC contributes to maximal expression of ntrY. The most-likely candidate is a promoter upstream of nifR3, which drives expression
of the nifR3–ntrB–ntrC operon with levels of transcript
equivalent under [1N] and [ N] conditions (Cullen et al.,
1998). (iii) Transcription of ntrY–lacZ was not regulated by
the nitrogen source, thus demonstrating that ntrY does not
Fig. 2. Transcription start site mapping of Rhodobacter capsulatus ntrY.
Primer extension was carried out with total RNA from R. capsulatus cells
grown either under nitrogen-limiting [ N] or nitrogen-sufficient [1N]
conditions. Primer GSP4 (binding to the 5 0 region of ntrY mRNA) was
used for reverse transcription. The corresponding sequencing reactions
(A, C, G, T) with plasmid pJW39 carrying the ntrC–ntrY gene region
served as length standard, and the start of the reverse transcript is
marked by (11).
belong to the NtrC regulon. (iv) Expression of ntrY–lacZ was
higher in the mutant strain containing a nonpolar ntrC
mutation (TD50) compared with the mutant carrying a polar
ntrC mutation (PBK2). This finding shows that transcription
initiated from the gentamicin cassette in TD50 was not
terminated downstream of ntrC, but instead contributed
significantly to ntrY expression.
To further confirm the presence of a ntrY-specific promoter downstream of ntrC, we determined the transcription
start of ntrY by primer extension analysis. Two independent
sets of experiments were carried out leading to essentially
the same results, and the result of one experiment is shown
in Fig. 2. Total RNA isolated from R. capsulatus wild-type
cultures grown either under [1N] or [ N] conditions were
used as templates for reverse transcription with primer
GSP4 (Materials and methods), complementary to the 5 0
end of ntrY-mRNA. Reverse transcripts based on RNA
isolated either from [1N] or [ N] cultures were of identical
length and comparable intensity (Fig. 2) confirming that
ntrY transcription is not regulated by the nitrogen source
(Table 2). As expected from ntrY–lacZ expression studies,
the transcription start mapped within the ntrC–ntrY intergenic region (Fig. 1b). Putative 10/ 35 promoter elements were identified at corresponding positions upstream
of the transcription start.
c
254
Optical density at 660 nm
(a)
T. Drepper et al.
2
1.5
wild-type
ntrC
ntrB
ntrY
ntrB /ntrY
1
0.5
0
0
(b)
25
50
75
100
2
1.5
wild-type
ntrC
ntrB
ntrY
ntrB /ntrY
1
0.5
0
0
(c)
25
50
75
100
2
1.5
wild-type
ntrC
ntrB
ntrY
ntrB /ntrY
1
0.5
0
0
25
50
75
100
Fig. 3. Growth of Rhodobacter capsulatus wild-type and selected mutant strains with ammonium (a), urea (b) or N2 (c) as sole nitrogen source.
Rhodobacter capsulatus strains were grown phototrophically in minimal
medium containing either 7.5 mM ammonium or 2 mM urea as an Nsource or under a pure N2 atmosphere. B10S (wild-type), TD50 (ntrC),
YWRUB11 (ntrB), DG7-I (ntrY), YWRUB11/DG7-I (ntrB, ntrY).
NtrB and NtrY can substitute for each other
In contrast to R. capsulatus NtrC, which is essential for N2
fixation and urea utilization (Kutsche et al., 1996; Masepohl
et al., 2001, 2002), nothing was known about the in vivo role
of its cognate HK, NtrB. In this study we analyzed ntrB
mutant strain YWRUB11 (Fig. 1a) for its ability to activate
NtrC-dependent genes. Based on the close proximity of the
ntrC and ntrY genes and clear similarity between the sensor
kinases NtrY and NtrB (28% identity and 50% similarity
over a stretch of 322 amino acids representing more than
90% of the NtrB protein), we included ntrY mutant DG7-I
in our studies on NtrC-dependent gene activation. We first
analyzed growth of ntrB and ntrY mutant strains with urea
c
as sole nitrogen source under phototrophic growth conditions (Fig. 3b). To avoid diazotrophic growth, the gas
atmosphere in the culture tubes was changed against argon.
As expected from earlier studies (Masepohl et al., 2001), the
ntrC mutant strain TD50 did not grow with urea (Fig. 3b).
Surprisingly, although biochemical studies strongly suggest
that NtrB is the cognate phosphodonor for NtrC (Cullen
et al., 1996), the ntrB mutant strain grew like the wild-type
as did the ntrY mutant strain DG7-I (Fig. 3b). In contrast to
these single mutants, the ntrB/ntrY double mutant
YWRUB11/DG7-I did not grow at all with urea as sole Nsource (like the ntrC mutant TD50). These results demonstrate that either NtrB or NtrY must be present for ureasedependent growth, strongly suggesting that NtrB and NtrY
can substitute for each other as phosphodonors for NtrC.
Furthermore, these data indicate that, at least under the
conditions tested in this study, no further HK is involved in
cross-talk towards R. capsulatus NtrC.
Several RRs, such as NtrC and PhoB from Escherichia coli,
are phosphorylated by acetyl phosphate both in vivo and
in vitro (Feng et al., 1992; Wanner & Wilmes-Riesenberg,
1992; McCleary et al., 1993). Acetyl phosphate has also been
discussed to activate expression of R. capsulatus DMSO
reductase by direct phosphorylation of the transcriptional
activator DorR (Kappler et al., 2002). However, since no
growth with urea as sole N-source was observed for the ntrB/
ntrY double mutant, acetyl phosphate does not seem to play
a significant role in phosphorylation of R. capsulatus NtrC,
at least under the conditions tested in this study. This is in
line with the finding that acetyl phosphate did not serve as a
phosphodonor for R. capsulatsu NtrC in vitro (Cullen et al.,
1996).
To rule out that the phenotype of the ntrB/ntrY double
mutant was specific for urease-dependent growth (Fig. 3b),
we examined nitrogenase-dependent diazotrophic growth of
the corresponding mutant strains (Fig. 3c). For this purpose, R. capsulatus wild-type and mutant strains were grown
in minimal medium without a source of fixed nitrogen
under an atmosphere of pure dinitrogen gas. As expected
from earlier studies (Masepohl et al., 2002, and references
therein), the ntrC mutant strain was not able to grow with
N2 as sole nitrogen source. Single mutant strains defective
for either NtrB or NtrY grew diazotrophically albeit at
slightly reduced levels compared with the wild type. Most
importantly, as shown for the ntrC mutant, no diazotrophic
growth at all was observed for the ntrB/ntrY double mutant,
thus prooving that either NtrB or NtrY must be present for
transcriptional activation of NtrC-dependent target genes.
To analyze the role of NtrB and NtrY in transcriptional
activation of NtrC-dependent genes in more detail, we
examined ure gene expression in R. capsulatus wild-type
and selected mutant strains carrying a ureDA-lacZ fusion
(pNIRUB35; Masepohl et al., 2001). For this purpose, R.
255
Table 3. Expression of ureDA-lacZ in Rhodobacter capsulatus ntrB and ntrY mutant strains
b-Galactosidase activityw
Strain
1N
N
B10S
TD50
YWRUB11
DG7-I
YWRUB11/DG7-I
Wild-type, ureDA-lacZ
ntrC, ureDA-lacZ
ntrB, ureDA-lacZ
ntrY, ureDA-lacZ
ntrB, ntrY, ureDA-lacZ
384 35
00
198 33
772 100
00
2971 179
00
391 89
3593 83
00
Rhodobacter capsulatus strains contained the broad host range plasmid pNIRUB35 (ureDA-lacZ).
w
Results represent the means and standard deviations of four independent measurements.
1N, nitrogen-sufficient conditions; N, nitrogen-limiting conditions.
capsulatus strains were grown phototrophically under either
[1N] or [ N] conditions prior to determination of lacZderived b-galactosidase activity. The results shown in Table 3
may be summarized as follows. (i) As described previously
(Masepohl et al., 2001), maximum expression of the ure
genes occured under [ N] conditions. Expression of the
ureDA-lacZ fusion was clearly down-regulated under [1N]
conditions, but significant expression remained in the presence of ammonium. NtrC was essential for ure gene
expression under both [ N] and [1N] conditions. (ii) As
expected from mutational analysis described above (Fig.
2b), no ure gene expression was observed in the ntrB/ntrY
double mutant strain. (iii) Despite the fact that the ntrB
single mutant strain exhibited no growth defect compared
with the wild type with urea as sole N-source (Fig. 3b),
expression of the ureDA-lacZ fusion was strongly reduced in
this mutant background. Therefore, although NtrB was not
essential for ure gene expression, it was required for maximal NtrC-mediated transcriptional activation underlining
its role as the cognate phosphodonor for NtrC. However,
since lower levels of ure gene expression in the ntrB mutant
did not affect growth with urea as sole nitrogen source, one
has to assume that the basal urease activity was still high
enough to produce sufficient amounts of NH3 from urea
allowing the ntrB mutant to grow at wild-type rates. (iv)
Interestingly, expression of ureDA-lacZ was enhanced in
ntrY mutant strain DG7-I, indicating that NtrY, in addition
to its role as a phosphodonor for NtrC, may counteract
phosphorylation of NtrC. This might be explained by a role
of NtrY in dephosphorylation of NtrC, or alternatively, of
NtrB, thereby indirectly affecting phosphotransfer to NtrC.
Conclusions
In summary, genetic data obtained in this study strongly
suggest cross-talk towards the R. capsulatus RR NtrC, with
the HKs NtrB and NtrY being able to substitute for each
other as phosphodonors. NtrB perceives the nitrogen status
of the cell via direct interaction with the PII-like signal
transduction protein GlnB as previously shown by yeast
two-hybrid studies (Pawlowski et al., 2003). In contrast, the
signal perceived by NtrY is not known. As ure gene expression in the ntrB mutant strain was still regulated by
ammonium availability (Table 3), one has to assume that
NtrY – like NtrB – is able to respond to the N status of the
cell. At present it remains unknown, how NtrY perceived the
N status and whether GlnB is involved in signal transduction
towards NtrY.
Acknowledgements
The authors thank Franz Narberhaus (Ruhr-Universität
Bochum) and Ulrike Kappler (University of Queensland)
for critically reading the manuscript. This work was supported by financial grants from the Fonds der Chemischen
Industrie and Deutsche Forschungsgemeinschaft, Germany.
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Molybdenum regulation in Rhodobacter
C
Overlapping and specialized functions of the
molybdenum-dependent regulators MopA and MopB in
Wiethaus J., Wirsing A., Narberhaus F. and Masepohl B.
2006b
Journal of Bacteriology 188(24):8441-51
29
JOURNAL OF BACTERIOLOGY, Dec. 2006, p. 8441–8451
0021-9193/06/$08.00⫹0 doi:10.1128/JB.01188-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 188, No. 24
Overlapping and Specialized Functions of the Molybdenum-Dependent
Regulators MopA and MopB in Rhodobacter capsulatus䌤
Jessica Wiethaus, Andrea Wirsing, Franz Narberhaus, and Bernd Masepohl*
Lehrstuhl für Biologie der Mikroorganismen, Fakultät für Biologie, Ruhr-Universität Bochum, 44780 Bochum, Germany
Received 1 August 2006/Accepted 26 September 2006
The phototrophic purple bacterium Rhodobacter capsulatus encodes two similar but functionally not identical
molybdenum-dependent regulator proteins (MopA and MopB), which are known to replace each other in
repression of the modABC genes (coding for an ABC-type high-affinity Mo transport system) and anfA (coding
for the transcriptional activator of Fe-nitrogenase genes). We identified further Mo-regulated (mor) genes
coding for a putative ABC-type transport system of unknown function (MorABC) and a putative Mo-binding
protein (Mop). The genes coding for MopA and the ModABC transporter form part of a single transcriptional
unit, mopA-modABCD, as shown by reverse transcriptase PCR. Immediately upstream of mopA and transcribed
in the opposite direction is mopB. The genes coding for the putative MorABC transporter belong to two
divergently transcribed operons, morAB and morC. Expression studies based on lacZ reporter gene fusions in
mutant strains defective for either MopA, MopB, or both revealed that the regulators substitute for each other
in Mo-dependent repression of morAB and morC. Specific Mo-dependent activation of the mop gene by MopA,
but not MopB, was found to control the putative Mo-binding protein. Both MopA and MopB are thought to
bind to conserved DNA sequences with dyad symmetry in the promoter regions of all target genes. The
positions of these so-called Mo boxes relative to the transcription start sites (as determined by primer
extension analyses) differed between Mo-repressed genes and the Mo-activated mop gene. DNA mobility shift
assays showed that MopA and MopB require molybdenum to bind to their target sites with high affinity.
tors are widespread in bacteria. In addition to E. coli, these
proteins have been characterized in greater detail for
Anabaena variabilis (30), Azotobacter vinelandii (20), Bradyrhizobium japonicum (6), Rhodobacter capsulatus (29), and Staphylococcus carnosus (21).
Molybdate binding involves a conserved domain of about 70
amino acids, the Mop domain (for a review, see reference 22).
Mop domains occur in three classes of cytoplasmic proteins
with distinct functions. Molbindins (Mop proteins), which are
implicated in Mo homeostasis within the cell, consist solely of
Mop domains present either as single Mop domains or tandem
Mop repeats. The Mo-dependent regulatory protein ModE
contains a C-terminal tandem Mop repeat, and a single Mop
domain occurs in the C-terminal domain of ModC.
While E. coli contains only a single copy of modE, the phototrophic purple bacterium Rhodobacter capsulatus codes for
two ModE-like regulator proteins (MopA and MopB) (29). In
addition, R. capsulatus can synthesize a Mo-dependent nitrogenase (Mo-nitrogenase) and an alternative iron-only nitrogenase (Fe-nitrogenase), the latter being expressed only in the
absence of molybdenum (for a review, see reference 17).
MopA and MopB replace each other in Mo repression of anfA
(which codes for the transcriptional activator of Fe-nitrogenase genes) and the mopA-modABCD genes (13).
In the present study, we compared expression of known
Mo-controlled genes (anfA and mopA-modABCD) and newly
identified Mo-regulated genes (morABC, coding for a putative
ABC-type transporter, and mop, coding for a putative Mo
homeostasis protein) in R. capsulatus by genetic means. Modependent transcription control depends on direct interaction
of MopA and MopB with their respective target promoters as
shown by DNA mobility shift assays. While MopA and MopB
Molybdenum serves as a cofactor for many redox enzymes
catalyzing basic reactions in the nitrogen, sulfur, and carbon
cycles. There are two distinct types of molybdoenzymes. Monitrogenase, which catalyzes the reduction of N2 to ammonia,
has a unique molybdenum-iron-sulfur cofactor called FeMoco. All other molybdoenzymes, such as nitrate reductase, dimethyl sulfoxide (DMSO) reductase, and xanthine dehydrogenase, contain a cofactor (called Mo-co) in which a mononuclear
Mo atom is coordinated to the sulfur atoms of a pterin.
Many bacteria actively take up molybdate by use of a highaffinity ABC-type transport system comprising three proteins
(25). ModA, the molybdate-binding protein, is localized in the
periplasm in gram-negative bacteria or attached to the outer
side of the cytoplasmic membrane in gram-positive bacteria.
ModB is the membrane integral channel protein, and ModC is
the cytoplasmic ATPase. In Escherichia coli, the modABC
genes constitute a single operon, whose expression requires
Mo starvation (8). When the intracellular Mo content is
high, a molybdate-dependent regulator, ModE, binds to the
modABC promoter and represses transcription of the Mo
transport operon. ModE consists of two functionally distinct
domains, an N-terminal DNA-binding domain and a C-terminal molybdate-binding domain (9). In the presence of molybdenum, ModE binds to a region of the modABC promoter with
a dyad symmetric element (the so-called Mo box) that overlaps
the transcription start (3).
ModABC-like Mo transport systems and ModE-like regula* Corresponding author. Mailing address: Ruhr-Universität Bochum,
Fakultät für Biologie, Lehrstuhl für Biologie der Mikroorganismen,
44780 Bochum, Germany. Phone: 49 (0) 234 32 25632. Fax: 49 (0) 234
32 14620. E-mail: bernd.masepohl@rub.de.
䌤
Published ahead of print on 6 October 2006.
8441
8442
WIETHAUS ET AL.
J. BACTERIOL.
TABLE 1. Bacterial strains and plasmids
Relevant characteristicsa
Strain or plasmid
Source or reference
E. coli
DH5␣
BL21(DE3)
S17-1
Host for plasmid amplification
Host for expression of MopAHis and MopBHis
RP4-2 (Tc::Mu) (Km::Tn7) integrated in the chromosome
10
Novagen, Darmstadt, Germany
27
R. capsulatus
B10S
KS94A
R423AI
R423BI
R423CI
R438II
Spontaneous Smr mutant of R. capsulatus B10
anfA::关Sp兴 insertion mutant of B10S
mopA::关Gm⬎兴 insertion mutant of B10S
mopB::关Gm⬎兴 insertion mutant of B10S
⌬(mopA mopB)::关Gm兴 deletion mutant of B10S
mopA::关⬍Gm兴 polar insertion mutant of B10S
12
29
13
13
13
29
Plasmids
pAW12
pBluescript KS
pBSL86
pET22b(⫹)
pJW32
pJW33
pJW42
pJW45
pJW59
pKS131A
pML5
pMOT15
pMOT16
pPHU236
pSL21I
pSL21II
pWKR459
pBluescript KS derivative carrying ⌬morABC::关Km兴
High-copy expression vector; Ap
Km cassette flanked by polylinker; Ap
High-copy His tag expression vector; Ap
pET22b(⫹) derivative carrying mopAhis
pET22b(⫹) derivative carrying mopBhis
pUC18 derivative carrying a mop promoter fragment
pUC18 derivative carrying the morA-morC intergenic region
pML5 derivative carrying a mop-lacZ transcriptional fusion
pPHU236 derivative carrying an anfA-lacZ translational fusion
Mobilizable lac fusion broad-host-range vector; Tc
pML5 derivative carrying a morA-lacZ transcriptional fusion
pML5 derivative carrying a morC-lacZ transcriptional fusion
Mobilizable lac fusion broad-host-range vector; Tc
pML5 derivative carrying a modA-lacZ transcriptional fusion
pML5 derivative carrying a mopB-lacZ transcriptional fusion
mob Tc Km
This study
Stratagene, Amsterdam, The Netherlands
1
This study
This study
This study
This study
This study
13
14
This study
This study
11
This study
This study
7
a
Ap, ampicillin; Gm, gentamicin; Km, kanamycin; Tc, tetracycline; Sm, streptomycin; Sp, spectinomycin.
replaced each other in repressing transcription of anfA, mopAmodABCD, morAB, and morC, only MopA was required for
activation of mop gene expression.
MATERIALS AND METHODS
Strains, plasmids, and growth conditions. The bacterial strains and plasmids
used in this study are listed in Table 1. Methods for conjugational plasmid
transfer between E. coli S17-1 and R. capsulatus, the selection of mutants, rich
medium (PY), molybdenum-free minimal medium (AK-NL), growth conditions,
and antibiotic concentrations were as previously described (see reference 26 and
references therein).
Construction of R. capsulatus morABC mutant strain AW12. A 3.5-kb DNA
fragment carrying the R. capsulatus mor gene region (see Fig. 2) was PCR
amplified using primer pair PAW3-U/PAW3-L (Table 2). A 2.5-kb BamHI-XhoI
fragment from the PCR amplification product was cloned into vector plasmid
pBluescript KS. Subsequently, a 1.5-kb SmaI fragment encompassing the entire
morA gene, the morA-morC intergenic region, and large parts of morB and morC
was replaced by a 1.2-kb SmaI kanamycin cartridge from pBSL86. Finally, insertion of a 8.8-kb XhoI fragment (containing a tetracycline resistance gene and
the mob locus of PR4) from pWKR459 led to the mobilizable hybrid plasmid
pAW12. Conjugational transfer of pAW12 from E. coli S17-1 into R. capsulatus
and selection for marker rescue were carried out as described earlier (12, 29).
␤-Galactosidase assays. R. capsulatus strains carrying reporter fusions between Mo-regulated genes and the promoterless E. coli lacZ gene, namely,
anfA-lacZ, mopA-modA-lacZ, morA-lacZ, morC-lacZ, and mop-lacZ (Table 1),
were grown in Mo-free AK-NL minimal medium containing either 9.5 mM serine
(nitrogen-limiting conditions) or 20 mM ammonium (nitrogen-sufficient conditions). When required, 10 ␮M Na2MoO4 was added. Following growth to late
exponential phase, ␤-galactosidase activities were determined by the sodium
dodecyl sulfate-chloroform method (19).
RNA isolation, transcriptional analysis by RT-PCR, and primer extension. R.
capsulatus wild-type cultures were grown in Mo-free AK-NL minimal medium
containing 9.5 mM serine as the sole nitrogen source. When required, 10 ␮M
Na2MoO4 was added. Total RNA was isolated using the Micro-to-Midi total
RNA purification system according to the instructions of the manufacturer
(Invitrogen, Karlsruhe, Germany). Specific transcripts were analyzed with the
ThermoScript reverse transcriptase PCR (RT-PCR) system (Invitrogen). To
analyze transcription of the mopA-modABCD, morAB, morC, and mop operons,
the primers shown in Table 2 were used for cDNA synthesis and/or secondstrand synthesis and subsequent PCR amplification steps. Primer extension was
carried out as described previously (4) using synthetic oligonucleotide primers
(Table 2) to map the transcription start sites of morC and mop, respectively.
Overexpression of His-tagged R. capsulatus MopA and MopB proteins in E.
coli. The mopA and mopB coding regions were PCR amplified with primer pairs
PJW27-U/PJW27-L and PJW28-U/PJW28-L (encompassing recognition sites for
NdeI and XhoI, respectively) (Table 2), using R. capsulatus chromosomal DNA
as a template. Subsequently, the 0.8-kb NdeI-XhoI fragments with either mopA
or mopB were cloned into expression vector pET22b(⫹), resulting in hybrid
plasmids pJW32 (mopAhis) and pJW33 (mopBhis), respectively. The plasmids
were transformed into E. coli strain BL21(DE3), which served as a host for
overexpression of the tagged R. capsulatus MopA and MopB proteins (MopAHis
and MopBHis). Purification of the recombinant proteins was carried out as
described previously (23).
DNA mobility shift assays. DNA fragments encompassing Mo-regulated promoters were obtained by PCR amplification with appropriate primer pairs (Table
2), using chromosomal DNA as a template. Amplification products were purified
using a NucleoSpin Extract II kit (Macherey-Nagel, Düren, Germany) prior to
32
P labeling of 5⬘ ends with T4 polynucleotide kinase (Fermentas, St. Leon-Rot,
Germany). Different amounts (up to 150 pmol) of either MopAHis or MopBHis
in buffer B (40 mM NaH2PO4 [pH 8.0], 500 mM NaCl) in a total volume of 16
␮l were preincubated at room temperature. When required, 250 nmol Na2MO4
was added at the beginning of the preincubation phase. After 10 min, a mixture
consisting of 1 ␮l 32P-labeled DNA (5 fmol/␮l), 1 ␮l poly(dI-dC) (1 ␮g/␮l), and
2 ␮l binding buffer (25 mM HEPES [pH 8.0], 50 mM K-glutamate, 50 mM
MgSO4, 1 mM dithiothreitol, 0.1 mM EDTA, 0.05% Igepal CA-630) was added
to the protein samples. After incubation at 30°C for 20 min, samples were
MOLYBDENUM REGULATION IN RHODOBACTER
VOL. 188, 2006
8443
TABLE 2. Primers used for RT-PCR and PCR amplification of selected DNA fragments
Primer
Oligonucleotide sequences (5⬘33⬘)
PAW3-U
PAW3-L
ACGGGGAAGCGCGGGGGAAAGAGG
GCGCGACAGAAAGCCGAACAGC
Amplification of mor gene region, 3,548 bp
PJW1-U
PJW1-L
CACCGTTGCACCGCCCACAGT
TGCCCCCACCGACACCACGATTCT
RT-PCR (modC-modD), 694 bp (fragment 1 in Fig. 1)
PJW2-U
PJW2-L
GGTGATCTGCCGCCCCTCCTG
GGTCGTCGGCTCGGTCATCTATTC
RT-PCR (modB-modC), 606 bp (fragment 2 in Fig. 1)
PJW3-U
PJW3-L
CCAGCCCCGCGAAGGTGAAGGA
TGACAAGGGCGCGGTGCTGAAAAC
RT-PCR (modA-modB), 688 bp (fragment 3 in Fig. 1)
PJW4-U
PJW4-L
GACGCATCGGCCGAAAGAAAGAC
CGCGCCGGAAAAAGCCCTCAAC
RT-PCR (mopA-modA), 757 bp (fragment 4 in Fig. 1)
PJW5-U
PJW5-L
GCGCCGTGCCATTGAAA
GGCGCTTGATCCCGACACC
RT-PCR (morC-orf1281), 639 bp (fragment 1 in Fig. 2)
PJW6-U
PJW6-L
ACGGCAAGGCGGGGCGGCAGTAT
CCAGCACGATCGGCGGAAACACCA
RT-PCR (morA-morB), 519 bp (fragment 3 in Fig. 2)
PJW7-U
PJW7-L
GCTTGGCGCGGGGCTCTT
CGGGGCTGACGCAAATCC
RT-PCR (morB-orf1277), 672 bp (fragment 4 in Fig. 2)
PJW8-U
PJW8-L
CGGTCTGGTGCGGATGGGGTCTTC
TCGGCGGCGGCTTCGTTGGTGAT
RT-PCR (orf413-mop), 624 bp (fragment 1 in Fig. 3)
PJW9-U
PJW9-L
CAATATTTGGCGGGCAAGGTCAC
GCGCGAAGCAAGGCAGGAGA
RT-PCR (mop-orf411), 559 bp (fragment 3 in Fig. 3)
PJW9-U
PJW8-L
CAATATTTGGCGGGCAAGGTCAC
RT-PCR (mop), 117 bp (fragment 2 in Fig. 3)
PJW12-U
PJW12-L
GGGCGGCCGTTCCTGTTCCT
mop promoter fragment (509 bp) in pJW42
PJW18
GAAGGCCCCGTCAGCACCAGAAAT
Primer extension (morC)
PJW19
Primer extension (mop)
PJW27-U
PJW27-L
AACATATGAACGAACAGCCCCTCATCG
TTCTCGAGGGGCATCGCCAGGATGACATG
Amplification of mopA coding region
PJW28-U
PJW28-L
AACATATGACGGACGGTGTGCGCGGGG
TTCTCGAGGGGCAGGGCCAGGATCACATG
Amplification of mopB coding region
PJW29-U
PJW29-L
CCTCGGCGGTCTCGTGGCTTGTCATCA
ACTGCCGCCCCGCCTTGCCGTAAAT
morA-morC intergenic region (1,139 bp) in pJW45
PJW36-U
PJW36-L
CCTCGGCGGTCTCGTGGCTTGTCATC
CGCGGTCGCTGGGCTTTGTCTTTCA
RT-PCR (morC), 363 bp (fragment 2 in Fig. 2)
PJW49-U
PJW49-L
GGCACTGACCGACCTTTTGACC
AGAATATTGCGTGCGCTGAGTTT
DNA mobility shift, 222 bp (mop promoter)
PJW52-U
PJW52-L
ACGGGCAGGCGCGGGGTTCT
CGGTAAAGCGTCGGCAGCAGGTTCA
DNA mobility shift, 236 bp (anfA promoter)
PJW53-U
PJW53-L
GCATCCCAGGCGGTCTTGTAGG
ATGAGGCCGCGGGTGATAACG
DNA mobility shift, 272 bp (mopA promoter)
PJW54-U
PJW54-L
CAGCCCGACATCGAGCGTGAAC
CGGCAGAGGCGGAAAGGAGAAGA
DNA mobility shift, 244 bp (morC/A promoter)
PJW55-U
PJW55-L
ACTGCGCCGCGATCCCCGAGAC
CGCCGCAATCACCCGCACATCA
DNA mobility shift, 251 bp (anfA internal fragment)
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WIETHAUS ET AL.
FIG. 1. Transcriptional analysis of the R. capsulatus mopAmodABCD gene region. (A) Physical and genetic maps of the mopmod gene region. The physical map is given for BamHI, EcoRI, and
HindIII (B, E, and H, respectively). Black bars below the genetic map
indicate DNA fragments 1 to 4 emerging from RT-PCR (see Materials
and Methods and panel B). The corresponding primer pairs used for
RT-PCR are listed in Table 2. Mutant strains defective for either
mopA (R423AI), mopB (R423BI), mopA and mopB (R423CI), or
modABCD (R438II) contain gentamicin resistance cassettes, with the
directions of transcription of the Gm resistance gene symbolized by
arrows. Hybrid plasmids pSL21I and pSL21II, carrying transcriptional
modA-lacZ and mopB-lacZ fusions, respectively, are based on the
mobilizable broad-host-range plasmid pML5. In these reporter plasmids, the BamHI sites were destroyed (indicated by B⫺) by cutting
with BamHI, filling in protruding ends, and blunt-end religation, leading to a frameshift within the mopA coding region. Neither the Gm
cassette nor the lacZ gene is drawn to scale. (B) Transcriptional analysis of the mopA-modABCD operon by RT-PCR. Total RNA was
isolated from R. capsulatus cells grown under Mo-limiting conditions.
Either RNA samples were treated with reverse transcriptase to synthesize cDNA (⫹) or, as a negative control, reverse transcriptase was
omitted (⫺). A 50-bp DNA ladder (Fermentas, St. Leon-Rot, Germany) was used as a length standard.
separated on 6% polyacrylamide gels before 32P-labeled bands were documented
using a Hyperscreen X-ray film (Fuji Photo Film Europe, Düsseldorf, Germany).
RESULTS AND DISCUSSION
Genetic organization of selected molybdenum-regulated
genes in R. capsulatus. Genes coding for two ModE-like Modependent regulators (mopA and mopB) and a Mo transport
system (modABC) were previously identified downstream of
the structural genes of Mo-nitrogenase, nifHDK (29). These
genes are organized in two divergently transcribed operons,
mopA-modABCD and mopB. Cotranscription of mopA-
J. BACTERIOL.
FIG. 2. Transcriptional analysis of the R. capsulatus mor gene region. (A) Physical and genetic maps of the mor gene region. The
physical map is given for BamHI and SmaI (B and M, respectively).
Black bars below the genetic map indicate DNA fragments 1 to 4
emerging from RT-PCR (see Materials and Methods and panel B).
The corresponding primer pairs used for RT-PCR are listed in Table
2. The morABC deletion mutant AW12 contains a kanamycin resistance cassette (not drawn to scale). Hybrid plasmids pMOT15 and
pMOT16, carrying transcriptional morA-lacZ or morC-lacZ fusions,
respectively, are based on the mobilizable broad-host-range plasmid
pML5. (B) Transcriptional analysis of the morAB and morC operons
by RT-PCR. Total RNA was isolated from R. capsulatus cells grown
under Mo-limiting conditions. Either RNA samples were treated with
reverse transcriptase to synthesize cDNA (⫹) or, as a negative control,
reverse transcriptase was omitted (⫺). A 50-bp DNA ladder (Fermentas,
St. Leon-Rot, Germany) was used as a length standard.
modABCD was demonstrated by RT-PCR (see Materials and
methods) (Fig. 1). Total RNA was isolated from R. capsulatus
wild-type cells grown in Mo-free minimal medium. After reverse transcription, selected primer pairs (Table 2; Fig. 1) were
used to PCR amplify DNA fragments overlapping the gene
borders of mopA-modA, modA-modB, modB-modC, and
modC-modD. The presence of amplification products was completely dependent on the addition of reverse transcriptase to
the reaction mixtures, indicating that the RNA was not contaminated with DNA. The presence of PCR products based on
all four primer pairs strongly suggested that mopA-modABCD
comprise a single transcription unit.
Using the ModABC proteins as query to screen the R. capsulatus genome database (www.ergo-light.com), we identified a
related ABC-type transport system encoded by open reading
frames Rc1279, Rc2331, and Rc1280. Since this study revealed
that expression of these genes is repressed by molybdenum
(see below), we propose new designations, namely, morA,
morB, and morC (for Mo-regulated genes) (Fig. 2). In addition,
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8445
TABLE 3. Expression of Mo-controlled lacZ reporter fusions in R. capsulatus wild-type and mutant strains
Strain(plasmid)
Genetic
background
Reporter
fusion
B10S(pKS131A)
R423AI(pKS131A)
R423BI(pKS131A)
R423CI(pKS131A)
Wild type
mopA
mopB
⌬(mopAB)
B10S(pSL21I)
R423AI(pSL21I)
R423BI(pSL21I)
R423CI(pSL21I)
␤-Galactosidase activitya
⫹Mo/⫺N
⫺Mo/⫺N
⫹Mo/⫹N
⫺Mo/⫹N
anfA-lacZ
anfA-lacZ
anfA-lacZ
anfA-lacZ
1⫾1
1⫾1
0⫾0
72 ⫾ 18
63 ⫾ 10
73 ⫾ 11
64 ⫾ 10
67 ⫾ 9
1⫾1
1⫾1
0⫾0
2⫾2
0⫾0
0⫾0
0⫾0
1⫾1
Wild type
mopA
mopB
⌬(mopAB)
modA-lacZ
modA-lacZ
modA-lacZ
modA-lacZ
11 ⫾ 1
7⫾9
6⫾5
87 ⫾ 9
70 ⫾ 7
88 ⫾ 5
78 ⫾ 11
89 ⫾ 13
6⫾2
7⫾2
13 ⫾ 6
0⫾0
4⫾3
3⫾2
6⫾0
2⫾2
B10S(pSL21II)
R423AI(pSL21II)
R423BI(pSL21II)
R423CI(pSL21II)
Wild type
mopA
mopB
⌬(mopAB)
mopB-lacZ
mopB-lacZ
mopB-lacZ
mopB-lacZ
21 ⫾ 3
18 ⫾ 3
16 ⫾ 1
17 ⫾ 4
22 ⫾ 2
18 ⫾ 4
22 ⫾ 1
20 ⫾ 4
18 ⫾ 2
18 ⫾ 4
16 ⫾ 2
21 ⫾ 2
18 ⫾ 1
23 ⫾ 7
18 ⫾ 4
20 ⫾ 2
B10S(pMOT15)
R423AI(pMOT15)
R423BI(pMOT15)
R423CI(pMOT15)
Wild type
mopA
mopB
⌬(mopAB)
morA-lacZ
morA-lacZ
morA-lacZ
morA-lacZ
43 ⫾ 3
51 ⫾ 4
120 ⫾ 19
452 ⫾ 66
369 ⫾ 28
441 ⫾ 52
408 ⫾ 44
435 ⫾ 63
37 ⫾ 3
48 ⫾ 3
70 ⫾ 11
175 ⫾ 19
117 ⫾ 3
67 ⫾ 27
171 ⫾ 7
202 ⫾ 21
B10S(pMOT16)
R423AI(pMOT16)
R423BI(pMOT16)
R423CI(pMOT16)
Wild type
mopA
mopB
⌬(mopAB)
morC-lacZ
morC-lacZ
morC-lacZ
morC-lacZ
438 ⫾ 27
411 ⫾ 48
454 ⫾ 32
891 ⫾ 63
733 ⫾ 76
791 ⫾ 84
785 ⫾ 61
862 ⫾ 56
148 ⫾ 14
147 ⫾ 13
196 ⫾ 18
388 ⫾ 36
387 ⫾ 39
368 ⫾ 10
456 ⫾ 34
494 ⫾ 41
B10S(pJW59)
R423AI(pJW59)
R423BI(pJW59)
R423CI(pJW59)
Wild type
mopA
mopB
⌬(mopAB)
mop-lacZ
mop-lacZ
mop-lacZ
mop-lacZ
551 ⫾ 51
24 ⫾ 3
382 ⫾ 18
44 ⫾ 13
40 ⫾ 15
25 ⫾ 20
18 ⫾ 4
36 ⫾ 11
316 ⫾ 40
41 ⫾ 5
333 ⫾ 22
57 ⫾ 17
64 ⫾ 5
43 ⫾ 3
22 ⫾ 2
43 ⫾ 5
a
R. capsulatus strains were grown under phototrophic conditions in AK-NL minimal medium either without addition of Mo (⫺Mo) or in the presence of 10 ␮M
Na2MoO4 (⫹Mo). N-sufficient or N-limiting conditions were achieved by addition of either 15 mM (NH4)2SO4 (⫹N) or 9.5 mM serine (⫺N), respectively.
␤-Galactosidase activity is given in Miller units (19). Results represent the means and standard deviations of three independent measurements.
a mop-like gene (Rc412) coding for a small protein similar to
the Mop domain of ModC was identified 2.3 kb downstream of
nifB1 coding for a protein involved in biosynthesis of the cofactor of Mo-nitrogenase, FeMo-co. As was the case for anfA
and the mopA-modABCD operon (13), putative Mo boxes
(binding sites for MopA and/or MopB) were identified in the
intergenic region between morAB and morC as well as in the
mop promoter region (Fig. 5A), giving a first hint for Mo
regulation of both the putative transporter MorABC and the
putative Mo homeostasis protein Mop.
The genetic organization of the mor and mop gene regions
was analyzed by RT-PCR essentially as described above for the
mopA-modABCD operon. These studies revealed that morAB
formed part of a bicistronic operon, while morC formed a
monocistronic transcription unit (Fig. 2). While mod and mor
gene expression was repressed by molybdenum, transcription
of the mop gene was Mo activated (Table 3) (see below).
Therefore, in contrast to studies on mod and mor, for analysis
of mop gene organization, total RNA from cultures grown in
the presence of molybdenum was used. While mop expression
studies clearly demonstrated the presence of a Mo-activated
promoter within the orf413-mop intergenic region (Table 3)
(see below), RT-PCR studies identified an amplification product overlapping the gene border of orf413-mop (Fig. 3). These
findings are most likely explained by the presence of two promoters driving expression of the mop gene, one immediately
upstream of the mop coding region and the second either
upstream of or within the coding region of orf413.
Regulation of mod, mor, mop, and anf transcription by
molybdenum. To analyze Mo regulation of selected genes, R.
capsulatus reporter strains carrying fusions between these
genes and the promoterless E. coli lacZ gene were used (Table
1). In detail, we examined expression of lac fusions with anfA
(pKS131A), mopA-modA (pSL21I), mopB (pSL21II), morA
(pMOT15), morC (pMOT16), and mop (pJW59). The lac fusions were introduced into wild-type R. capsulatus (B10S) and
mutant strains defective for either MopA (R423AI), MopB
(R423BI) or both (R423CI). These mutant strains contain a
gentamicin resistance (Gm) cassette (Fig. 1A), which drives
expression of downstream genes reading in the same direction
as the Gm gene (29). Therefore, although mopA is the first
gene of the mopA-modABCD operon, expression of the Mo
transport system is not abolished in mutant strains R423AI
(mopA) and R423CI (mopA mopB). In agreement with previous studies (13), transcription of modA and anfA was not only
repressed by molybdenum but also inhibited by ammonium. To
analyze whether other Mo-regulated genes were also controlled by the N source, R. capsulatus reporter strains were
cultivated under four different growth conditions, namely, in
the presence or absence of molybdenum in combination with
either nitrogen-sufficient conditions (ammonium as the N
source) or nitrogen-limiting conditions (serine as the N
8446
WIETHAUS ET AL.
J. BACTERIOL.
FIG. 4. Effect of increasing molybdate concentrations on the activity of Mo-nitrogenase. R. capsulatus strains were grown in AK-NL
minimal medium containing the indicated Mo concentrations and 9.5
mM serine as the sole nitrogen source (nitrogenase-derepressing conditions). The activity of Mo-nitrogenase was determined by the reduction
of acetylene to ethylene, as assayed by gas chromatography, and is expressed as a percentage of the maximal value obtained in Mo-sufficient
medium (100% corresponds to 662 nmol ethylene produced ⫻ h⫺1 ⫻ mg
protein⫺1). R. capsulatus strains KS94A (anfA), KS94A-AW12 (anfA
morABC), KS94A-R438II (anfA modABC), and KS94A-R438II-AW12
(anfA morABC modABC) were used.
FIG. 3. Transcriptional analysis of the R. capsulatus mop gene region. (A) Physical and genetic maps of the mop gene region. The
physical map is given for EcoRI (E). Black bars below the genetic map
indicate DNA fragments 1 to 3 emerging from RT-PCR (see Materials
and Methods and panel B). The corresponding primer pairs used for
RT-PCR are listed in Table 2. Hybrid plasmid pJW59, carrying a
transcriptional mop-lacZ fusion, is based on the mobilizable broadhost-range plasmid pML5. (B) Transcriptional analysis of the mop
gene region by RT-PCR. Total RNA was isolated from R. capsulatus
cells grown in the presence of 10 ␮M Na2MoO4. Either RNA samples
were treated with reverse transcriptase to synthesize cDNA (⫹) or, as
a negative control, reverse transcriptase was omitted (⫺). Amplification products corresponding to DNA fragments 1 and 2 are marked by
arrows. A 50-bp DNA ladder (Fermentas, St. Leon-Rot, Germany)
was used as a length standard.
source), prior to determination of ␤-galactosidase activities. R.
capsulatus can efficiently use serine as the sole nitrogen source,
but, in contrast to ammonium, serine does not inhibit synthesis
of the ModABC transport system or nitrogenase (13).
The results of expression studies on Mo-regulated genes
(Table 3) may be summarized as follows. (i) In the wild-type
background significant expression of both anfA and mopAmodA occurred only under Mo- and N-limiting conditions (13;
this study). (ii) Mo repression was mediated by either MopA or
MopB, which are able to replace each other in repression of
mopA-modA and anfA. In other words, MopA autoregulates its
own expression. (iii) Like the anfA and mopA-modA genes,
morA and morC were repressed by MopA or MopB in the
presence of Mo. However, Mo repression of morC was less
pronounced compared to that of anfA, mopA-modA, and
morA. Significant expression of MorC in the presence of Mo
might suggest that the protein has, in addition to its energizer
function of the putative ABC transporter in the absence of Mo,
another yet-unknown function. (iv) While mopA-modA (like
anfA) was strongly inhibited by ammonium, the N source had
only a minor influence on expression of morA and morC. (v) In
contrast to that of mopA, expression of mopB was not regulated by molybdenum or ammonium. As a consequence, the
cellular MopA/MopB ratio should strongly differ in response
to Mo and N availability, if expression data reflect the actual
amounts of MopA and MopB protein. (vi) In contrast to the
Mod and Mor systems and the Fe-nitrogenase, which were Mo
repressed, expression of the putative Mo homeostasis protein
Mop was activated by molybdenum. (vii) Interestingly, Mo
activation specifically required the MopA protein, whereas
MopB had little influence on mop transcription. Like R. capsulatus MopA, E. coli ModE can act as both a repressor and an
activator (2). It represses the modABC operon and activates
transcription of genes involved in Mo-co biosynthesis. It is
worth noting, however, that expression of R. capsulatus Mo-co
biosynthesis genes moeA and moeB is not Mo regulated (15)
(data not shown). (viii) In contrast to that of mopA-modA and
anfA, expression of mop was almost unaffected by ammonium.
This finding might be explained by a role of the postulated Mo
homeostasis protein as an Mo donor not only for Mo-nitrogenase, which is expressed exclusively under nitrogen-limiting
conditions, but also for other Mo-containing enzymes such as
DMSO reductase and xanthine dehydrogenase (16).
The morABC genes are not required for Mo-nitrogenase
activity. R. capsulatus mutant strains defective for the modABC
genes are impaired in high-affinity Mo uptake as estimated
from the Mo-nitrogenase activity (29). While the parental
strain exhibited full Mo-nitrogenase activity at Mo concentrations of as low as 100 nM, a modABC mutant strain required at
least 100-fold-higher Mo concentrations for maximum Monitrogenase activity (Fig. 4) (29). A second, yet-uncharacterized low-affinity transport system has been discussed as being
responsible for Mo uptake at concentrations of above 10 ␮M
(29). Since the morAB and morC genes were shown to be
repressed by molybdenum, we asked whether the putative
MorABC transporter was involved in low-affinity Mo uptake.
For this purpose, R. capsulatus mutant strains defective for
VOL. 188, 2006
8447
FIG. 5. DNA sequence comparison of Mo-regulated promoters (A) and transcription start site mapping of the morC (B) and mop (C) genes.
DNA sequences of Mo boxes are compared to the consensus as defined by Kutsche et al. (13). The morA-morC intergenic region contains a single
Mo box, which is thought to control expression of the divergently transcribed mor operons (Fig. 2). For clarity, two complementary sequences
(morA and morC) of the same Mo box from this region are shown. The transcription start sites of anfA and the mopA-modABCD operon were
taken from Kutsche et al. (13). To determine the transcription start sites of the other genes, primer extension was carried out with total RNA from
R. capsulatus cells grown either under Mo-limiting conditions (⫺Mo) or in the presence of 10 ␮M Na2MoO4 (⫹Mo). Primers PJW18 and PJW19
(binding to the 5⬘ regions of morC and mop, respectively) were used for reverse transcription. The corresponding sequencing reactions (A, C, G,
and T) with plasmids pJW45 (morC) and pJW42 (mop) served as length standards. No transcription start site was mapped for morA.
either ModABC (R438II) (Fig. 1), MorABC (AW12) (Fig. 2),
or both (R438II-AW12) were assayed for their Mo-nitrogenase
activities at different Mo concentrations (Fig. 4). To rule out
any interference with Fe-nitrogenase, which does not require
molybdenum for activity, Mo-nitrogenase activity was measured in an anfA mutant background (KS94A), thus preventing
transcription of Fe-nitrogenase genes. Based on Mo-nitrogenase activity, the morABC mutant strains were indistinguishable from their parental strains (Fig. 4), strongly suggesting
that MorABC is not the previously postulated low-affinity Mo
uptake system.
Transcription start sites of Mo-regulated genes. Typically,
repressor binding sites either overlap or are located downstream of the transcription start site of the respective target
genes. This has previously been demonstrated for the Mo
boxes implicated in binding of MopA and MopB upstream of
anfA and the mopA-modABCD operon (13). The situation is
more complex for the divergently transcribed morAB and
morC operons, which are expected to share a single Mo box
located in the intergenic region between the two operons (Fig.
2). The transcription start site of the morC gene was deter-
mined by primer extension analysis (Fig. 5B). For this purpose,
total RNAs isolated from R. capsulatus wild-type cultures
grown in either the presence or absence of Mo were used as
templates for reverse transcription with primer PJW18, complementary to the 5⬘ ends of morC mRNA (see Materials and
Methods) (Table 2). Reverse transcripts based on RNA isolated from cultures grown in either the presence or absence of
Mo were identical in length and of comparable intensity. This
finding was in line with morC-lacZ expression studies showing
significant expression under both conditions (Table 3). The
transcription start site of morC mapped upstream of the putative Mo box (Fig. 5A), suggesting that binding of MopA and
MopB to this Mo box interferes with transcription. Despite
several attempts using three different primers complementary
to the 5⬘ ends of morA mRNA, no transcription start site could
be determined for morA.
In parallel, we determined the transcription start site of the
mop gene (Fig. 5C). Reverse transcripts based on RNA isolated from cultures grown in either the presence or absence of
Mo were identical in length but clearly differed in intensity,
which was in line with mop-lacZ expression studies showing
8448
WIETHAUS ET AL.
J. BACTERIOL.
FIG. 6. DNA mobility shift assays with Mo-regulated promoter fragments and purified recombinant MopA and MopB proteins. DNA
fragments encompassing the promoters of anfA (PanfA), mopA-modABCD (PmopA), and mop (Pmop) and the intergenic region between the
divergently transcribed mor operons (Pmor) were generated by PCR amplification using appropriate primer pairs (Table 2) prior to 32P labeling (see
Materials and Methods). Incubation of increasing amounts of MopA and MopB (0, 0.03, 0.06, 0.12, 0.23, 0.47, 0.94, 1.88, 3.75, and 7.5 ␮M) with
labeled DNA fragments was carried out either in the absence (⫺Mo) or presence (⫹Mo) of molybdenum. All reactions were performed with 5
fmol 32P-labeled DNA fragment probes.
that maximal mop expression occurred in the presence of Mo
(Table 3). The mop transcription start site was mapped downstream of the putative Mo box (Fig. 5A). As activator binding
sites are typically located upstream of the transcription start
site, this finding is consistent with MopA-dependent mop gene
activation.
Binding of MopA and MopB to target promoters. As shown
above by expression studies, MopA and MopB regulate tran-
scription of their target genes in response to Mo availability.
The presence of conserved Mo boxes in the promoter regions
of all target genes suggested that MopA and MopB specifically
bind to these promoters. DNA mobility shift assays were carried out to verify this assumption. For this purpose, MopA and
MopB were overexpressed and purified as C-terminally Histagged recombinant proteins from E. coli (see Materials and
Methods). DNA fragments, ranging from 222 to 272 bp, en-
VOL. 188, 2006
8449
FIG. 7. Specificity controls for binding of MopA and MopB to anfA promoter DNA. (A) Use of an internal region of anfA (anfAintern) as
negative control for DNA mobility shift assays. The control DNA fragment (anfAintern) (Table 2) was PCR amplified, 32P labeled, and incubated
with either 7.5 ␮M MopA or MopB in the absence (⫺Mo) or presence (⫹Mo) of molybdenum. (B) Use of unlabeled anfA promoter fragments
as specific competitor DNA. MopA or MopB (2.5 ␮M) and 32P-labeled anfA promoter fragments were mixed with a 400-, 800-, or 1,600-fold excess
of unlabeled competitor DNA (compared to the labeled probe). (C) Use of anfAintern in competition assays. MopA or MopB (2.5 ␮M) and
32
P-labeled anfA promoter fragments were mixed with a 550-, 1,100-, or 2,200-fold excess of unlabeled anfA internal fragments (compared to the
labeled probe). All reactions were performed with 5 fmol 32P-labeled PanfA probes.
compassing selected promoters were PCR amplified using
appropriate primer pairs (Table 2) and radioactively labeled
at their 5⬘ ends (see Materials and Methods). All binding
assays were performed in the presence of poly(dI-dC) as
competitor DNA.
The results of DNA mobility shift assays with increasing
amounts of either MopAHis or MopBHis are shown in Fig. 6.
Both MopA and MopB bound to the anfA promoter (PanfA).
Binding of both regulators occurred in the absence of Mo
but was clearly improved in the presence of Mo. In contrast
to binding to the anfA promoter, neither MopA nor MopB
bound to a control DNA fragment derived from an internal
region of anfA (Fig. 7A), thus corroborating binding specificity. Binding of MopA and MopB to radioactively labeled
anfA promoter fragments could be reversed by addition of
increasing amounts of nonlabeled anfA promoter DNA (Fig.
7B) but not by the internal anfA fragment (Fig. 7C). The
importance of the Mo box upstream of anfA as a cis-regulatory element has been demonstrated by analysis of mutant
promoters carrying small deletions within this element (13).
Taken together, these findings strongly suggest that MopA
and MopB control anfA expression by binding to this Mo
box overlapping the transcription start site (Fig. 5A). Although in vitro binding of MopA and MopB to the anfA
promoter implies that no additional proteins are required
for Mo repression of anfA, fine-tuning by other protein
factors in vivo cannot be excluded.
Under comparable conditions, binding of MopA to the
mopA-modABCD promoter (PmopA) and the intergenic region
between the divergently transcribed mor operons (Pmor) was
much weaker than binding of MopB, and binding of MopA to
PmopA and Pmor was not detectable at all in the absence of Mo
(Fig. 6). Most interestingly, only MopA (and not MopB) bound
to the promoter of the mop gene (Pmop), coding for a putative
Mo homeostasis protein (Fig. 6). Binding of MopA in the
absence of Mo was barely detectable. More efficient binding
occurred in the presence of Mo. As one would expect for a
cis-regulatory element serving exclusively as a binding site for
MopA, the mop-specific Mo box differs at three positions from
all the other Mo boxes, which serve as binding sites for both
MopA and MopB (Fig. 5A).
Compared to binding to the anfA, mopA, and mor promoters, binding of MopA to the mop promoter was fairly weak
(even in the presence of Mo). Generally, binding of activator
proteins to their target promoters is believed to be much
weaker than binding of repressor proteins to operator sequences (5). The suggested role of MopA acting either as a
repressor (for anfA, mopA-modABCD, morAB, and morC expression) or as an activator (for mop regulation) perfectly
agrees with this general observation.
Conclusions. MopA and MopB have overlapping functions,
as they can substitute for each other in Mo repression of anfA,
mopA-modABCD, morAB, and morC (13; this study). In addition to its role as a repressor, MopA serves as an activator of
transcription of the mop gene (this study), which is the first
example of a specialized function of MopA in Mo regulation.
8450
WIETHAUS ET AL.
It is worth noting that MopB has been described to be
essential for activity of DMSO reductase (dor encoded) in R.
capsulatus strain 37b4 (28). A putative Mo box has been
identified upstream of the dorX gene, consistent with the
view that the dorX gene is the target for MopB-dependent
Mo regulation in strain 37b4 (18). DorX, in turn, has been
suggested to activate transcription of an operon whose products are required for Mo-co biosynthesis and, hence, DMSO
reductase activity. R. capsulatus strain B10S, which was used
in this study, also has the capacity to synthesize DMSO
reductase. However, in contrast to strain 37b4, strain B10S
does not contain a dorX-like gene at the equivalent position
relative to the other dor genes (data not shown) or elsewhere in the chromosome (www.ergo-light.com), suggesting
that regulation of DMSO reductase activity differs in the
two R. capsulatus strains.
The current model for Mo regulation in R. capsulatus
suggests that the Mo-dependent regulators, MopA and
MopB, are involved in regulation of the internal Mo concentration by repressing transcription of the modABCD
transport operon (13; this study) and thus limiting the
amount of the transporter at high Mo concentrations. Mutant strains defective for ModABC express Fe-nitrogenase
at Mo concentrations of up to 1 ␮M, while synthesis of
Fe-nitrogenase is repressed at much lower concentrations in
the parental strain (29), suggesting that the putative
MorABC transporter does not substitute for the ModABC
system. Although expression of the morAB and morC genes
is controlled by molybdenum, at present it remains unknown
whether the gene products are involved in Mo uptake at all.
The presence of the high-affinity Mo transporter ModABC,
which provides sufficient Mo for the Mo-nitrogenase at low
Mo concentrations, is physiologically favorable, as Mo-nitrogenase is more efficient than Fe-nitrogenase with respect
to N2 reduction rates (24).
MopA-dependent mop gene activation occurred only in the
presence of Mo, thus ensuring that the putative Mo homeostasis protein is not expressed under Mo-limiting conditions.
However, since expression of mopA (as part of the mopAmodABCD operon) is down-regulated in response to increasing Mo concentrations, MopA-dependent synthesis of the Mop
protein is expected to decrease when Mo is abundant.
ACKNOWLEDGMENTS
We thank Thomas Drepper for helpful discussions, Corinna
Hasecke and Britta Schubert for analysis of the DMSO reductase gene
region from R. capsulatus strain B10S, Silke Leimkühler for construction of plasmids pSL21I and pSL21II, and Andrea Kreuz for construction of plasmids pMOT15 and pMOT16.
This work was supported by a financial grant from Deutsche
Forschungsgemeinschaft (Ma 1814/3-1).
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Interaction between MopA, MopB and Mop
D
Protein-protein interactions between MopA, MopB,
and Mop from Rhodobacter capsulatus
Wiethaus J., Narberhaus F. and Masepohl B.
Journal of Bacteriology; zur Publikation eingereicht
41
ABSTRACT
The phototrophic purple bacterium Rhodobacter capsulatus encodes two ModE-like
transcriptional regulators, MopA and MopB, with overlapping and specific functions in
molybdate-dependent gene regulation. We demonstrate via yeast two-hybrid studies,
glutaraldehyde crosslinking, gel filtration chromatography, and copurification experiments
that both proteins form homodimers in the presence and absence of molybdate. Heteromer
formation between MopA and MopB also was independent of molybdate availability.
Interestingly, MopB, but not MopA, was able to interact with the Mop protein. Mop is a
putative molbindin protein that might function as intracellular storage protein for molybdate.
Like other molbindins, the Mop protein formed hexamers, which were stabilized by
molybdate.
INTRODUCTION
Molybdenum is an essential trace element due to its role as cofactor for a number of enzymes
(10). Molybdenum nitrogenase has a unique iron-molybdenum cofactor called FeMoco,
whereas all other molybdoenzymes such as xanthine dehydrogenase contain a molybdopterin
cofactor called Moco.
Many bacteria actively take up the oxyanion molybdate (MoO42-) by a high affinity ABC-type
transport system (19). Components of this system are the periplasmic substrate binding
protein ModA, the integral membrane protein ModB, and the cytoplasmic ATPase ModC. In
Escherichia coli, expression of the modABC operon is repressed by the Mo-dependent
regulatory protein ModE (6). ModE functions as a homodimer with two distinct domains, an
N-terminal DNA-binding domain and a C-terminal Mo-binding domain (8). The latter
consists of two so-called mop domains per monomer. Mop domains of about 70 amino acids
are responsible for cytoplasmic Mo binding and are found in three classes of proteins with
distinct functions (15). Molbindins, thought to be involved in Mo storage and Mo
homeostasis, solely consist of either a mono-mop domain or a di-mop domain. C-terminal
mop domains combined with other modules exist in ModC (mono-mop domain) and in ModE
(di-mop domain).
Under appropriate conditions, the phototrophic purple bacterium Rhodobacter capsulatus
synthesizes proteins containing either the FeMoco (molybdenum nitrogenase) or the Moco
42
(xanthine dehydrogenase) (Fig. 1). Mo incorporation during biosynthesis of these cofactors is
thought to be carried out by NifQ (FeMoco) and MogA or MoeA (Moco) (12, 14).
R. capsulatus synthesizes two ModE-like proteins, MopA and MopB. These regulators can
replace each other in Mo-dependent repression of several genes including modABC and
morABC, the latter one coding for a putative ABC-type transport system of unknown function
(22). In the presence of Mo, MopA (but not MopB) activates transcription of the mop gene,
coding for a putative mono-mop molbindin. While mopA expression is repressed by Mo, the
mopB gene is transcribed independent of Mo-availability. As a consequence, the
MopA/MopB-ratio changes in response to the intracellular Mo level.
In this study we examined the oligomerisation properties of the regulatory proteins MopA and
MopB, and the putative Mo-storage protein Mop by different approaches. Our results show
that MopA and MopB form both homodimers and heteromers independent of Mo availability.
Furthermore, MopA and MopB differ with regard to interaction with Mop. Mop forms a
hexamer that is stabilized by Mo.
Strains, plasmids, and growth conditions. The bacterial strains, yeast strains, and plasmids
used in this study are listed in Table 1. Media, growth conditions, and antibiotic
concentrations were as previously described (11, 16).
Yeast two-hybrid studies. The R. capsulatus genes mop, modC, morC, moeA, mogA, and
nifQ were PCR-amplified using appropriate oligonucleotides designed for amplification of
full-length genes flanked by MunI restriction sites (Table 2). Cloning of MunI fragments into
the EcoRI site of the Escherichia coli / yeast shuttle vectors pEG202 (lexA-DBD) and pJG4-5
(B42-AD) generated in-frame fusions with either the DNA-binding domain (DBD) or the
activation domain (AD) (Table 1). In addition, XhoI-EcoRI-fragments containing either mopA
or mopB from plasmids pAB4II and pAB5II were cloned into pEG202 and pJG4-5.
Subsequently, DBD and AD fusion plasmids were cotransformed into yeast strain EGY48
(pSH18-34) containing a lacZ reporter gene controlled by the LexA operator by the
polyethylene glycol-lithium acetate method (3). ß-Galactosidase activities of yeast reporter
strains were determined by the sodium dodecyl sulfate-chloroform method (16).
43
Construction of mopA, mopB, and mop expression plasmids. Construction of hybrid
plasmids pJW32 (mopAhis) and pJW33 (mopBhis) has been described earlier (22). ApoI
fragments with either mopAhis or mopBhis from plasmids pJW32 and pJW33 were cloned into
the EcoRI site of plasmid pSUP401, which carries a kanamycin resistance gene for selection.
The mop coding region was PCR-amplified with primers encompassing recognition sites for
NdeI and XhoI, respectively (Table 2). Subsequently, the NdeI-XhoI fragment was cloned
into expression vector pET22b(+) to create a mophis fusion (Table 1). In addition, mopA,
mopB, and mop coding regions were PCR-amplified with primers carrying recognition sites
for EcoRI and SalI, respectively. Subsequently, the EcoRI-SalI fragments were cloned into
expression vector pASK-IBA3 to create mopAstrep, mopBstrep, and mopstrep fusions.
Overexpression and purification of His-tagged proteins. Plasmids for overexpression of
His-tagged proteins were transformed into E. coli strain BL21(DE3). Overexpression and
purification of recombinant proteins was carried out as described previously (22).
Copurification of His- and Strep-tagged proteins. Plasmids for overexpression of His- and
Strep-tagged proteins were cotransformed into E. coli strain BL21(DE3). For overexpression
of the recombinant proteins 200 ml of selective LB medium were inoculated with 2 ml from
an overnight culture of BL21(DE3) carrying the respective hybrid plasmids and cultivated at
37°C to an OD580 of 0.7. Protein expression was induced by the addition of
anhydrotetracycline (for Strep-tagged proteins) and IPTG (for His-tagged proteins). After
incubation for further 2.5 hours, cells were harvested by centrifugation and resuspended in 20
ml of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole). After disruption in a
French press cell at 2000 psi the lysate was centrifuged at 22,548 g for 30 min. When required
the supernatant was adjusted to 100 µM Na2MoO4. Crude extracts were loaded onto Ni-NTAagarose columns. After washing with buffer (50 mM NaH2PO4, 300 mM NaCl) with
increasing imidazole concentrations (10 – 30 mM imidazole) His-tagged proteins were eluted
by raising the imidazole concentration to 250 mM. When required washing and elution
buffers were adjusted to 10 µM Na2MoO4. Aliquots of crude extracts and elution fractions
were analyzed by SDS-PAGE and Western blots using either the Penta-His HRP conjugate
(Qiagen, Hilden, Germany) or Strep-Tactin HRP conjugate (IBA, Göttingen, Germany).
Crosslinking experiments. His-tagged proteins were incubated with either 0.01 % (MopAHis,
MopBHis) or 0.05 % (MopHis) glutaraldehyde in a total volume of 15 µl at room temperature.
44
When needed reaction mixtures were adjusted to 10 mM Na2MoO4. Reactions were stopped
with 2.5 µl 1 M Tris (pH8), before samples were analyzed by SDS-PAGE and Western blots.
Gel filtration chromatography. Purified MopAHis, MopBHis, and MopHis proteins in elution
buffer were loaded on a Superdex 75 HR 10/30 gel filtration column (Amersham Biosciences,
Freiburg, Germany) pre-equilibrated with 100 mM NaCl, 50 mM NaH2PO4 at pH 8.
Separation was performed at 4°C at a flow rate of 0.3 ml/min. The following standards were
used to calibrate the column: albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A
(25 kDa), and ribonuclease A (13.7 kDa).
RESULTS
Interaction profile of proteins involved in Mo-metabolism. To analyze protein-protein
interactions between proteins involved in R. capsulatus Mo-metabolism (Fig. 1), yeast twohybrid studies were carried out. For this purpose, appropriate DBD and AD fusions were
constructed (Materials and Methods, Table 1). All DBD fusions were tested for self-activation
properties. Only DBD-NifQ showed significant background activity, and was therefore not
suitable for yeast two-hybrid studies.
The results of yeast two-hybrid studies shown in Fig. 2 may be summarized as follows. (i)
Homomer formation was found for the regulatory proteins MopA and MopB, the putative
Mo-storage protein Mop, and the Moco biosynthesis proteins MogA and MoeA. (ii)
Heteromer formation was observed for the following protein pairs: MopA-MopB, MopBMopA, MopB-Mop, Mop-MopB, MopB-MogA, ModC-MogA, and MogA-MopA. (iii) MopB
interacted with MogA, but not with the other Mo-cofactor biosynthesis proteins, MoeA and
NifQ. (iv) The Mo-transporter component ModC interacted with MogA but not with MoeA or
NifQ. (v) No interaction at all was observed for the putative transport protein MorC.
Mop forms stable oligomers in the presence of Mo. To study oligomer formation
biochemically, MopA, MopB, and Mop were overexpressed and purified as C-terminal Histagged proteins by Ni-NTA affinity chromatography. Migration of the purified proteins
during SDS-PAGE was in agreement with their calculated molecular masses of 28.2 kDa
(MopAHis), 27.9 kDa (MopBHis), and 7.9 kDa (MopHis) (Fig. 3 A). Surprisingly, when MopHis
was purified in the presence of Mo, an additional band appeared at approximately 35 kDa
45
(Fig. 3 B). This band most likely corresponds to the hexameric form of Mop as indicated by
crosslinking experiments (see below). This oligomer was stable even under the harsh
conditions during SDS-PAGE. To address the question whether Mo led to formation or
stabilisation of the oligomer, further experiments were carried out (see below).
MopA and MopB form homodimers and Mop forms hexamers. As shown above by yeast
two-hybrid studies, MopA, MopB, and Mop formed homomeric structures. To analyze
whether these interactions were due to the formation of dimers or higher oligomers,
glutaraldehyde crosslinking with either MopAHis, MopBHis, or MopHis was performed (Fig. 4).
MopA and MopB showed comparable crosslinking profiles in the presence and absence of
Mo. In addition to the monomeric forms, a second band corresponding to MopAHis dimers
(56.4 kDa) or MopBHis dimers (55.8 kDa) appeared after crosslinking. MopB formed an
higher-order complex, presumably a homotetramer (111.6 kDa).
The size of crosslinked MopHis complexes increased with increasing incubation time in the
absence of Mo (Fig. 4). After 20 min, MopHis particles ranging from the monomer up to small
amounts of the hexamer (47.4 kDa) occurred. In the presence of Mo, the hexameric form
appeared even without crosslinking. Intermediate complexes were not detected and after 3
min of incubation with glutaraldehyde all monomers had disappeared.
MopA, MopB, and Mop oligomers are formed in the absence of Mo. To validate
crosslinking data and to test the influence of Mo on oligomerisation in more detail, size
exclusion chromatography with purified His-tagged proteins was performed. Gel filtration
profiles for MopAHis, MopBHis, and MopHis in the presence of Mo are presented in Fig. 5.
MopAHis and MopBHis eluted as complexes of about 61 kDa, which correlates well with
deduced sizes of MopAHis homodimers (56.4 kDa) and MopBHis homodimers (55.8 kDa).
Since peaks corresponding to monomeric or higher oligomeric forms were missing, it seems
likely that both MopA and MopB exist exclusively in the dimeric state in solution. In contrast
to MopAHis and MopBHis, MopHis exhibited a more complex elution profile. Four peaks were
detected with calculated masses of about 92 kDa, 46 kDa, 16 kDa, and 10 kDa. The larger
peaks most likely represent the MopHis dodecamer (94.8 kDa) and the hexamer (47.4 kDa),
while the smaller peaks may correspond to the dimer (15.8 kDa) and the monomer (7.9 kDa),
indicating that Mop forms mainly dodecamers and hexamers in solution.
In the absence of Mo, the elution profiles of MopAHis, MopBHis, and MopHis were very similar
to those obtained in the presence of Mo (data not shown). Thus, these findings confirm
46
crosslinking data showing oligomer formation independent of the Mo-status. However, this
does not necessarily preclude further stabilization by Mo.
MopB forms heteromers with MopA and Mop. Copurification experiments were performed
to confirm MopA-MopB and MopB-Mop heteromer formation as determined by yeast twohybrid studies. Suitable combinations of His- and Strep-tagged MopA, MopB, and Mop
proteins were coexpressed in E. coli and subsequently purified by Ni-NTA chromatography.
After elution from the Ni-NTA column, His-tagged proteins and attached Strep-tagged
proteins were detected by SDS-PAGE and Western blot analyses with His- or Strep-specific
HRP-conjugates. Data obtained in the presence of Mo (Figs. 6 and 7) were essentially the
same as determined in the absence of Mo (data not shown). As controls, crude extracts with
the individual His-tagged or Strep-tagged proteins were loaded on Ni-NTA columns. As
expected, His-tagged proteins were retained by the Ni-NTA columns, whereas neither of the
Strep-tagged proteins bound unspecifically (Fig. 6). Both MopHis and MopStrep (9 kDa) formed
hexamers (54 kDa in case of MopStrep) (Figs. 6B and 6C).
Copurification experiments showed binding of MopAStrep to MopAHis as well as binding of
MopBStrep to MopBHis verifying homomer formation (Fig. 7). Heteromer formation of the
regulatory proteins was tested in both possible combinations. Consistently, binding of
MopAStrep to MopBHis as well as binding of MopBStrep to MopAHis was observed.
MopStrep copurified with MopBHis but did not interact with MopAHis (Fig. 7). In line with the
yeast two-hybrid studies, these findings strongly suggest that both regulatory proteins differ
with regard to interaction with Mop.
DISCUSSION
In E. coli Mo-dependent gene regulation is mediated by ModE consisting of an N-terminal
DNA-binding and a C-terminal Mo-binding di-mop domain (8). The crystal structure of
ligand-bound ModE revealed a homodimeric complex with two molybdate anions bound per
dimer. In contrast to E. coli, the phototrophic purple bacterium R. capsulatus encodes two
ModE-like regulators, MopA and MopB, which regulate transcription of several genes in a
Mo-dependent manner (22). Regulation involves direct binding of either MopA or MopB to
the promoters of their target genes. In addition to its role as a repressor, MopA activates the
47
transcription of mop, which codes for a putative member of the molbindin family, if
molybdate is abundant.
In the present study we analyzed oligomer formation of MopA and MopB by different
approaches (Fig. 8). As determined by yeast two-hybrid studies and confirmed by
crosslinking experiments, gel filtration chromatography, and copurification assays, MopA and
MopB form homodimers independent of Mo-availability. MopB tetramers observed in
crosslinking experiments might be unspecific as the crystal structure of E. coli ModE revealed
that it forms dimers both in the apo- and ligand-bound states (18). Upon Mo-binding, the
ModE dimer undergoes extensive conformational rearrangements not only in the Mo-binding
domain, but also in the DNA-binding domain. These changes are thought to improve DNAbinding, and therefore, might serve as a mechanism to adapt gene expression to the Mo-status
of the cell (5, 18). We suggest a similar mechanism for homodimeric MopA and MopB
proteins, as in vitro binding of both proteins to their target DNAs was clearly enhanced by Mo
(22).
Formation of MopA-MopB heteromers was detected by copurification assays in the presence
and absence of Mo, and by yeast two-hybrid studies. The oligomeric state of these heteromers
remains unclear. Crosslinking experiments and gel filtration chromatography with a mixture
of MopA and MopB resulted in dimers indistinguishable from those obtained with either
MopA or MopB alone (data not shown). It is reasonable to assume that heteromers formed
under these conditions are also heterodimers since no higher-ordered structures were
identified. It is plausible, that MopA-MopB heterodimers play a role in Mo-dependent gene
regulation in R. capsulatus under certain conditions. As mopA is repressed by Mo, while
mopB is Mo-constitutively expressed, the MopA/MopB-ratio changes dependent on Mo
availability. At present, we do not know whether MopA-MopB heterodimers differ from the
respective homodimers with regard to affinity to target promoters.
Yeast two-hybrid studies with selected proteins involved in Mo-metabolism revealed MogA
as potential interaction partner of MopA and MopB. MogA as well as MoeA are thought to be
responsible for Mo incorporation during Moco biosynthesis (12). Our findings imply that
apart from their roles in gene regulation, MopA and MopB might provide Mo for Moco
biosynthesis. An additional, more direct route from ModC to MogA is suggested by
interaction of the corresponding protein pair in the yeast two-hybrid system. In contrast, the
Mo source for FeMoco biosynthesis remains unclear as no interaction of MopA, MopB, Mop,
or ModC with the FeMoco biosynthesis protein NifQ was detected in the yeast two-hybrid
system.
48
The R. capsulatus molbindin-like protein Mop solely consists of a mono-mop domain. The
physiological role of molbindins remains unclear but they have been implicated in Mo
homeostasis and Mo storage (7). The putative R. capsulatus Mo-storage protein Mop showed
no apparent interaction with the transporter proteins ModC and MorC, or the cofactor
biosynthesis proteins NifQ, MoeA, and MogA in the yeast two-hybrid system. Interestingly,
several lines of evidence indicated that Mop contacts MopB suggesting that Mop exchanges
Mo with MopB (but not with MopA). Expression of the mop gene is activated by MopA in
the presence of Mo. As mopA expression itself is repressed by Mo, mop transcription
decreases when Mo becomes abundant. Thus, Mop will be synthesized only in a tight range of
relatively high Mo-concentrations suggesting a role of Mop as cytoplasmic Mo-buffer system.
When MopA and MopB are Mo-saturated and fully active as transcriptional regulators, Mop
might bind surplus Mo. Under these conditions, transcription of mopA will be repressed. As
the intracellular Mo-content decreases, Mop might deliver Mo to MopB, which in turn might
pass it onto MogA.
Structural data for several molbindins are available (1, 17, 20). Mono-mop molbindins like
MopII from Clostridium pasteurianum and Mop from Sporomusa ovata are arranged as a
trimer of dimers. The di-mop molbindin ModG from Azotobacter vinelandii is trimeric.
Consistent with these structures, R. capsulatus Mop formed hexamers as shown by
crosslinking experiments. Gel filtration chromatography demonstrated formation of Mop
dodecamers, hexamers and dimers. These findings suggest that Mop is composed of dimeric
building blocks like other mono-mop molbindins. It is likely that the hexamer is the native
form while dodecamer formation might be unspecific due to unphysiologically high protein
concentrations during gel filtration chromatography. Molbindins bind Mo with a
stoichiometry of 8 mol Mo per hexamer (1, 13, 17, 20). The crystal structure of apomolbindin also revealed a hexameric complex (17). In line with these findings, R. capsulatus
Mop oligomerized even in the absence of Mo, but binding of Mo stabilized the R. capsulatus
Mop hexamer.
49
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Golemis, E. A., J. Gyuris, and R. Brent. 1994. p. 13.14.1–13.14.17. In F. M. Ausubel,
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5.
Gourley, D. G., A. W. Schüttelkopf, L. A. Anderson, N. C. Price, D. H. Boxer, and
W. N. Hunter. 2001. Oxyanion binding alters conformation and quaternary structure of
the c-terminal domain of the transcriptional regulator mode. Implications for molybdatedependent regulation, signaling, storage, and transport. J. Biol. Chem. 276:20641-7.
6.
Grunden, A. M., R. M. Ray, J. K. Rosentel, F. G. Healy, and K. T. Shanmugam.
1996. Repression of the Escherichia coli modABCD (molybdate transport) operon by
ModE. J. Bacteriol. 178:735-44.
7.
Grunden, A. M., and K. T. Shanmugam. 1997. Molybdate transport and regulation in
bacteria. Arch. Microbiol. 168:345-54.
8.
Hall, D. R., D. G. Gourley, G. A. Leonard, E. M. Duke, L. A. Anderson, D. H.
Boxer, and W. N. Hunter. 1999. The high-resolution crystal structure of the
molybdate-dependent transcriptional regulator (ModE) from Escherichia coli: a novel
combination of domain folds. EMBO J. 18:1435-46.
9.
Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol.
Biol. 166:557-580.
10.
Kisker, C., H. Schindelin, and D. C. Rees. 1997. Molybdenum-cofactor-containing
enzymes: structure and mechanism. Annu. Rev. Biochem. 66:233-67.
11.
Klipp, W., B. Masepohl, and A. Pühler. 1988. Identification and mapping of nitrogen
fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region. J. Bacteriol.
170:693-699.
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12.
Leimkühler, S., S. Angermüller, G. Schwarz, R. R. Mendel, and W. Klipp. 1999.
Activity of the molybdopterin-containing xanthine dehydrogenase of Rhodobacter
capsulatus can be restored by high molybdenum concentrations in a moeA mutant
defective in molybdenum cofactor biosynthesis. J. Bacteriol. 181:5930-9.
13.
Masters, S. L., G. J. Howlett, and R. N. Pau. 2005. The molybdate binding protein
Mop from Haemophilus influenzae--biochemical and thermodynamic characterisation.
Arch. Biochem. Biophys. 439:105-12.
14.
Moreno-Vivian, C., S. Hennecke, A. Pühler, and W. Klipp. 1989. Open reading
frame 5 (ORF5), encoding a ferredoxinlike protein, and nifQ are cotranscribed with
nifE, nifN, nifX, and ORF4 in Rhodobacter capsulatus. J. Bacteriol. 171:2591-8.
15.
Pau, R. N. 2004. Molybdenum uptake and homeostasis, p. 225-256. In W. Klipp, B.
Masepohl, J. R. Gallon, and W. E. Newton (ed.), Genetics and regulation of nitrogen
fixation in free-living bacteria
Kluwer Academic Publishers, Dordrecht, The
Netherlands.
16.
Pawlowski, A., K.-U. Riedel, W. Klipp, P. Dreiskemper, S. Groß, H. Bierhoff, T.
Drepper, and B. Masepohl. 2003. Yeast two-hybrid studies on interaction of proteins
involved in regulation of nitrogen fixation in the phototrophic bacterium Rhodobacter
capsulatus. J. Bacteriol. 185:5240-7.
17.
Schüttelkopf, A. W., J. A. Harrison, D. H. Boxer, and W. N. Hunter. 2002. Passive
acquisition of ligand by the MopII molbindin from Clostridium pasteurianum: structures
of apo and oxyanion-bound forms. J. Biol. Chem. 277:15013-20.
18.
Schüttelkopf, A.W., D. H. Boxer, and W. N. Hunter. 2003. Crystal structure of
activated ModE reveals conformational changes involving both oxyanion and DNAbinding domains. J. Mol. Biol. 326:761-7.
19.
Self, W. T., A. M. Grunden, A. Hasona, and K. T. Shanmugam. 2001. Molybdate
transport. Res. Microbiol. 152:311-321.
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Simon, R., U. Priefer, and A. Pühler. 1983. A broad host range mobilization system
for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria.
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Wagner, U. G., E. Stupperich, and C. Kratky. 2000. Structure of the
molybdate/tungstate binding protein Mop from Sporomusa ovata. Structure 8:1127-36.
22.
Wiethaus, J., A. Wirsing, F. Narberhaus, and B. Masepohl. 2006. Overlapping and
specialized functions of the molybdenum-dependent regulators MopA and MopB in
Rhodobacter capsulatus. J. Bacteriol. 188:8441-51.
51
ACKNOWLEDGEMENTS
We thank Silke Leimkühler and Meina Neumann for helpful discussions and Antonios Baslis
for constructing plasmids pAB4II and pAB5II. This work was supported by a financial grant
from Deutsche Forschungsgemeinschaft (Ma 1814/3-1).
52
Table 1: Bacterial strains, yeast strains and plasmids
Strain or plasmid
Relevant characteristics a
Source or reference
DH5α
BL21(DE3)
Host for plasmid amplification
Host for expression of recombinant proteins
9
Novagen, Darmstadt,
Germany
Spontaneous Smr mutant of R. capsulatus
B10
11
E. coli
R. capsulatus
B10S
S. cerevisiae
EGY48
Plasmids
pAB4II
URA3 TRP1 HIS3 6op-LEU2
2
pUC18 derivative carrying mopA
pAB5II
pUC18 derivative carrying mopB
pASK-IBA3
high-copy strep-tag expression vector, Ap
pAW2
pEG202
pET22b(+)
pASK-IBA3 derivative carrying mopBstrep
lexA-DBD HIS3 Ap
high-copy his-tag expression vector, Ap
pJG4-5
pJW26
pJW32
pJW33
pJW50
pJW52
pJW63
pJW64
pJW65
pJW66
pJW67
pJW68
pJW69
pJW70
pJW80
pJW81
pJW82
pJW83
pJW84
pJW85
pJW86
pJW88
pJW89
pJW90
pSH18-34
pSUP401
PGAL1-B42-AD TRP1, Ap
pASK-IBA3 derivative carrying mopAstrep
pET22b(+) derivative carrying mopAhis
pET22b(+) derivative carrying mopBhis
pSUP401 derivative carrying mopAhis
pSUP401 derivative carrying mopBhis
pEG202 derivative containing DBD-nifQ
pJG4-5 derivative containing AD-nifQ
pEG202 derivative containing DBD-moeA
pJG4-5 derivative containing AD-moeA
pEG202 derivative containing DBD-mogA
pJG4-5 derivative containing AD-mogA
pEG202 derivative containing DBD-modC
pEG202 derivative containing DBD-modC
pEG202 derivative containing DBD-mop
pEG202 derivative containing DBD-mopA
pJG4-5 derivative containing AD-mopA
pEG202 derivative containing DBD-mopB
pJG4-5 derivative containing AD-mopB
pEG202 derivative containing DBD-morC
pJG4-5 derivative containing AD-morC
pJG4-5 derivative containing AD-mop
pET22b(+) derivative carrying mophis
pASK-IBA3 derivative carrying mopstrep
URA3 8op-lacZ, Ap
Km
A. Baslis & B.
Masepohl, Bochum
A. Baslis & B.
Masepohl, Bochum
IBA, Göttingen,
Germany
This study
4
Novagen, Darmstadt,
Germany
4
This study
22
22
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
4
20
(a)
Ap, ampicillin; Km, kanamycin; Sm, streptomycin.
53
Table 2: Primers used for PCR amplification of selected DNA fragments
Primer
Oligonucleotide sequences (5´→3´)
mopB-up
mopB-down
CGAATTCCCGGTTTGCGCCACAATGGCGGC
GCAGGTCGACGGGCAGGGCCAGGATCACATGGC
mopB coding region
(purification of MopBStrep)
UP-mopA
LP-mopA
GAATTCCTATATAACGATCCACCT
GTCGACGGGCATCGCCAGGATGAC
mopA coding region
(purification of MopAStrep)
PJW20-U
PJW20-L
GCCCAATTGATGCTGCACTTTCCTGAT
CGGCAATTGTCAATCGGGGGCAAAGCA
nifQ coding region (Y2H)
PJW21-U
PJW21-L
GAACAATTGATGCCCGCACTCGATCAC
GCTCAATTGTCAGCGCGCCCCGGCCTC
moeA coding region (Y2H)
PJW22-U
PJW22-L
GGCCAATTGATGACGGCACGGGTTGCC
CGCCAATTGTTACTTCGTCTTCGGGCG
mogA coding region (Y2H)
PJW23-U
PJW23-L
GGCCAATTGATGATCTCGGCGCGGTTC
GAACAATTGCTACCCTCCGGTTTGCGC
modC coding region (Y2H)
PJW24-U
PJW24-L
ACACAATTGGTGCCAACCGCCGCCCCC
TCCCAATTGTCAGACACGCACGAGGCG
morC coding region (Y2H)
PJW56-U
PJW56-L
GACCAATTGATGAAACTCAGCGCACGC
GTCCAATTGTCAGTTCTTGCCGACGAT
mop coding region (Y2H)
PJW66-U
PJW66-L
GCAGAATTCGACTCAATCGTTCCGGGA
GTTGTCGACGTTCTTGCCGACGATGAC
(purification of MopHis)
PJW67-U
PJW67-L
ACCCATATGAAACTCAGCGCACGCAAT
CGACTCGAGGTTCTTGCCGACGATGAC
mop coding region
(purification of MopStrep)
mop coding region
54
Transport
Regulation
ModA
MorA
Mo
?
Periplasm
ModB
MorB
Membrane
ModC
MorC
MopB
MopA
Storage ?
Mop
FeS
Cofactor
biosynthesis
Mo-enzyme
Cytoplasm
MPT
NifQ
MoeA
MogA
FeMoco
Moco
Mo-N2ase
XDH
Fig. 1. Proteins involved in Mo-metabolism in R. capsulatus.
Functional categories are indicated to the left Transported substrates are either molybdate
(Mo) or unknown (?). Mop domains in ModC, MopA, and MopB are shown as grey boxes.
Abbreviations: FeS, iron-sulfur cluster; FeMoco, iron-molybdenum cofactor; MPT, molybdopterin; Moco, molybdopterin cofactor; Mo-N2ase, Mo nitrogenase; XDH, xanthine
dehydrogenase.
55
40000
30000
6000
DBD-MopA
vs.
10000
1500
0
0
45000
DBD-MopB
vs.
1200
30000
800
15000
400
0
4000
3000
DBD-MogA
vs.
0
DBD-Mop
vs.
300
2000
200
1000
100
0
800
0
600
DBD-MoeA
vs.
3000
20000
ß-Galactosidase activity
4500
DBD-ModC
vs.
1500
200
500
0
0
DBD vs.
AD
AD
if Q
-N
AD ogA
-M
AD oeA
-M
AD p
o
-M
AD opB
-M
AD opA
-M
AD odC
-M
AD orC
-M
AD
AD
1000
if Q
-N
AD ogA
-M
AD oeA
-M
AD p
o
-M
AD opB
-M
AD opA
-M
AD odC
-M
AD orC
-M
400
DBD-MorC
vs.
Fig. 2. Protein-protein interactions identified by yeast two-hybrid studies.
DBD fusion proteins are quoted in each diagram, while AD fusion proteins are given on the xaxis. The corresponding ß-galactosidase activities of three independent yeast transformands
are given in Miller units. Note the different scales for each panel.
56
Fig. 3. Purification of MopAHis, MopBHis, and MopHis.
His-tagged proteins were overproduced in E. coli BL21(DE3) and purified by Ni-NTA
chromatography in the absence (A) or presence (B) of Mo. Aliquots of MopAHis, MopBHis,
and MopHis were analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining.
MopHis protein bands are emphasized by dashed ovals. The PageRuler prestained protein
ladder (Fermentas, St. Leon-Rot, Germany) was used as a molecular weight standard.
57
Fig. 4. Homomer formation of MopAHis, MopBHis, and MopHis.
Purified proteins were crosslinked by incubation with glutaraldehyde. The reactions were
carried out either in the absence (- Mo) or presence (+ Mo) of Mo for the indicated time
intervals. Proteins incubated without glutaraldehyde served as controls (C). Homomer
formation was analysed by SDS-PAGE followed by Western blot and detection of His-tagged
proteins. The oligomeric state ranging from monomer (1 x) to hexamer (6 x) was calculated
using the PageRuler prestained ladder (Fermentas, St. Leon-Rot, Germany).
58
2x
Absorbance 280 nm
MopAHis
MopBHis
MopHis
2x
12 x
6x
2x
1x
0
0
5
10
15
Elution volume (ml)
Fig. 5. Gel filtration profiles of MopAHis, MopBHis, and MopHis.
Purified proteins were analyzed by size exclusion chromatography on a Superdex HR 10/30
column. The proteins were detected by absorbance at 280 nm. The oligomeric states are
indicated.
59
Fig. 6. Binding properties of His- and Strep-tagged proteins to Ni-NTA agarose.
As a control for copurification experiments (Fig. 7) His- and Strep-tagged versions of MopA,
MopB and Mop were used for Ni-NTA affinity chromatography in the presence of Mo. Crude
extracts of soluble proteins (S) and eluates (E) were analysed by SDS-PAGE (A) and Western
blot (B, C). Either His-tagged (B) or Strep-tagged (C) proteins were detected. The His-tagged
proteins in (A) are marked by dashed ovals.
60
Fig. 7. Oligomer formation of MopA, MopB, and Mop.
Copurification experiments were performed with His- and Strep-tagged MopA, MopB, and
Mop proteins coexpressed in E. coli BL21(DE3). The respective combinations of coexpressed
proteins are indicated above the gels. After Ni-NTA chromatography in the presence of Mo,
crude extracts of soluble proteins (S) and eluates (E) were analysed by SDS-PAGE (A) and
Western blot (B, C). Either His-tagged (B) or Strep-tagged (C) proteins were detected. Bands
in (C) corresponding to copurified Strep-tagged proteins are labelled.
61
dimer
Y2H, CL,
GF, CP
Y2H, CP
MopA
Y2
H,
CP
MopB
dimer
Y2H, CL,
GF, CP
CP
,
H
Y2
MOP
Y2H, CL,
GF, CP
hexamer
Fig. 8. Interaction map of MopA, MopB, and Mop.
Interactions were determined by yeast two-hybrid studies (Y2H), crosslinking experiments
(CL), gel filtration chromatography (GF), and copurification experiments (CP). Homo- and
heteromer formation is shown by black arrows, and oligomeric states (dimer or hexamer) are
indicated. No interaction was detected between MopA and Mop, as indicated by a crossed
arrow.
62
Rhodobacter multicopper oxidase
E
The multicopper oxidase CutO confers copper
tolerance to Rhodobacter capsulatus
Wiethaus J., Wildner G. F. and Masepohl B.
2006a
FEMS Microbiology Letters 256(1):67-74
63
The multicopper oxidase CutO confers copper tolerance to
Jessica Wiethaus1, Günter F. Wildner2 & Bernd Masepohl1
1
Lehrstuhl für Biologie der Mikroorganismen, Fakultät für Biologie, Ruhr-Universität Bochum, Bochum, Germany and 2Lehrstuhl für Biochemie der
Pflanzen, Bochum, Germany
Correspondence: Bernd Masepohl,
Lehrstuhl für Biologie der Mikroorganismen,
Fakultät für Biologie, Ruhr-Universität
Bochum, D-44780 Bochum, Germany.
Tel.:149 0 234 32 25632; fax: 149 0 234 32
14620; e-mail: bernd.masepohl@rub.de
Received 27 October 2005; revised 2 December
2005; accepted 2 December 2005.
First published online 10 January 2006.
doi:10.1111/j.1574-6968.2005.00094.x
Abstract
The cutO gene of the photosynthetic purple bacterium Rhodobacter capsulatus
codes for a multicopper oxidase as demonstrated by the ability of the recombinant
Strep-tagged protein to oxidize several mono- and diphenolic compounds known
as substrates of Escherichia coli CueO and multicopper oxidases from other
organisms. The R. capsulatus cutO gene was shown to form part of a tri-cistronic
operon, orf635–cutO–cutR. Expression of the cutO operon was repressed under low
copper conditions by the product of the cutR gene. CutO conferred copper
tolerance not only under aerobic conditions, as described for the well-characterized E. coli multicopper oxidase CueO, but also under anaerobic conditions.
Editor: Karl Forchhammer
Keywords
gene regulation; photosynthetic bacterium;
Rhodobacter capsulatus; copper tolerance;
multicopper oxidase; laccase.
Introduction
Multicopper oxidases (MCOs) couple the one-electron
oxidation of substrate(s) to full reduction of molecular
oxygen to water by employing a functional unit formed by
three types of copper-binding sites with different spectroscopic and functional properties (Solomon et al., 1996;
Rensing & Grass, 2003). Type 1 ‘blue’ copper (T1) is the
primary electron acceptor for the substrate, while a trinuclear cluster formed by type 2 ‘normal’ copper (T2) and
‘binuclear’ type 3 copper (T3) is the oxygen-binding and
oxygen-reduction site.
A large subfamily of MCOs are the laccases, which
function in diverse pathways, such as lignin degradation,
pigmentation and pathogenesis in fungi, as well as in cell
wall formation in plants (Henson et al., 1999). Fungal
laccases have long been the subject of biotechnological
applications such as pulp delignification, textile dye bleaching, and removal of phenolics from wines. Laccase-like
enzymes have recently also been detected in many bacteria
by in silico searches of complete and unfinished microbial
genome databases (Alexandre & Zhulin, 2000; Claus, 2003).
Despite the abundance of putative bacterial laccases, only
few enzymes including Escherichia coli CueO and Bacillus
subtilis CotA have been studied in greater detail.
Both CueO and CotA exhibit in vitro phenoloxidase
activity (Enguita et al., 2003; Rensing & Grass, 2003). In
vivo, CueO is a periplasmic protein involved in copper
homeostasis in E. coli and it mediates copper tolerance
under aerobic conditions (Outten et al., 2001). A functional
role for CueO in protection against copper toxicity includes
the removal of cuprous ions (Singh et al., 2004). In addition,
CueO-mediated protection from copper toxicity involves
oxidation of enterobactin, the catechol iron siderophore of
E. coli, in the presence of copper (Grass et al., 2004). A
different in vivo function is discussed for B. subtilis CotA,
which is an abundant component of the outer coat layer of
endospores. CotA is implicated in the endospore differentiation by protecting the spore coat against UV and hydrogen
peroxide (Enguita et al., 2003).
In the present study we analyzed Rhodobacter capsulatus
CutO, which is the first laccase-like multicopper oxidase
described from purple nonsulfur photosynthetic bacteria.
Members of this group of alphaproteobacteria display exceptional metabolic versatility and are capable of distinctly
different modes of growth. R. capsulatus CutO is of special
interest, as it clearly differs from the well-characterized
c
68
J. Wiethaus et al.
E. coli MCO CueO, with regard to copper-induced expression and its role for copper tolerance under anaerobic
growth conditions.
Materials and methods
Bacterial strains and plasmids
The bacterial strains and plasmids used in this study are
listed in Table 1. Methods for conjugational plasmid transfer
between Escherichia coli and Rhodobacter capsulatus and the
procedures for selection of mutants, growth conditions, and
antibiotic concentrations have been described earlier (Weaver et al., 1975; Klipp et al., 1988; Masepohl et al., 1988;
Moreno-Vivian et al., 1989a, b).
Construction of Rhodobacter capsulatus cutO
mutant strains JW12-I and JW12-II
A 1515 bp DNA fragment carrying the R. capsulatus cutO
gene region was PCR-amplified using synthetic primers (5 0 GGATCCCAGGTTCCCATTCTTGAA-3 0 and 5 0 -CTGCAG
GGCGCTGACGACGAATT-3 0 ). The PCR fragment was
blunt-end cloned into the SmaI site of pSVB10 resulting in
hybrid plasmid pJW8. To disrupt the cutO gene, a 1.2 kb SalI
kanamycin cartridge from pBSL15 was inserted into the
XhoI site of pJW8 resulting in hybrid plasmids pJW11-I and
pJW11-II. Finally, a 7.4 kb PstI fragment (containing a
tetracycline resistance gene and the mop locus of RP4) from
pWKR459 was inserted into pJW11-I and pJW11-II leading
to the mobilizable hybrid plasmids pJW12-I and pJW12-II,
respectively (Fig. 2). Conjugational transfer of pJW12-I and
pJW12-II from E. coli S17-1 into R. capsulatus and selection
for marker rescue were carried out as described earlier
(Klipp et al., 1988; Masepohl et al., 1988). The resulting
R. capsulatus cutO mutant strains JW12-I and JW12-II are
distinguished from each other by the orientation of the
kanamycin cassette.
Construction of hybrid plasmid pJW13
(cutO--lacZ) and b-galactosidase assays
A 1535 bp DNA fragment carrying the putative R. capsulatus
cutO promoter region was PCR-amplified using synthetic
primers (5 0 -CCAGGGCGGCGCGGTAGAAC-3 0 and 5 0 ATGGGCGTGGCGGAATGGTC-3 0 ). The PCR fragment
was cloned into the SmaI site of vector plasmid pIC19H
resulting in hybrid plasmid pJW7. Subsequently, a 1.6 kb
HindIII fragment from pJW7 was cloned into reporter
plasmid pHH1 (containing the promoterless E. coli lacZ
gene) leading to hybrid plasmid pJW13, with a transcriptional cutO–lacZ fusion. Finally, hybrid plasmid pJW13
was introduced into R. capsulatus wild-type and cutO
mutant strains as described earlier (Klipp et al., 1988;
Masepohl et al., 1988). Selection for the vector-encoded
Table 1. Bacterial strains and plasmids used in this study
Strain or plasmid
Strains
Escherichia coli
BL21(DE3)
DH5a
S17-1
B10S
B10S:pJW13
JW12-I/II
JW12-I/II:pJW13
Plasmids
pASK-IBA3
pBSL15
pHH1
pIC19H
pJW7
pJW8
pJW11-I/II
pJW12-I/II
pJW13
pJW14
pSVB10
pWKR459
Reference or source
Host for overexpression of CutOStrep
Host for pUC plasmids
RP4-2 (Tc<Mu) (Km<Tn7) integrated in the chromosome
Vieira & Messing (1982)
Simon et al. (1983)
Spontaneous Smr mutant of Rhodobacter capsulatus B10
Chromosomal cutO–lacZ fusion in wild-type background
cutO<[Km] (transconjugant of B10S with pJW12-I or pJW12-II)
Chromosomal cutO–lacZ fusion in cutO mutant background
Klipp et al. (1988)
This study
This study
This study
Strep-tag, tet p/o, tetR, Ampr
pUC derivative, Ampr, Kmr
pSUP401 derivative, mob, lacZYA, Cmr, Tcr
pUC derivative, lacZa, Ampr
1.5-kb PCR fragment (cutO promoter) in pIC19H
1.5-kb PCR fragment (cutO) in pSVB10
1.2-kb SalI fragment (Kmr) from pBSL15 in pJW8
7.4-kb PstI fragment (mob, Tcr) from pWKR459 in pJW11-I or pJW11-II
1.6-kb HindIII fragment (cutO promoter) from pJW7 in pHH1
1.5-kb BamHI–PstI fragment (cutO) from pJW8 in pASK-IBA3
pUC derivative, lacZa, Ampr
pK18 derivative, Ampr, mob, Tcr
IBA, Göttingen, Germany
Alexeyev (1995)
B. Masepohl, Bochum
Marsh et al. (1984)
This study
This study
This study
This study
This study
This study
Arnold & Pühler (1988)
Drepper et al. (2002)
Amp, ampicillin; Cm, chloramphenicol; Km, kanamycin; Tc, tetracycline; tet p/o, tetracycline promoter/operator; tetR, tetracycline repressor gene; Sm,
streptomycin.
c
69
tetracycline resistance lead to isolation of R. capsulatus
mutant strains B10S:pJW13, JW12-I:pJW13, and JW12II:pJW13, respectively, carrying pJW13 integrated in the
chromosome.
To determine the b-galactosidase activity of R. capsulatus
reporter strains carrying the cutO–lacZ reporter fusion,
cultures were grown in RCV minimal medium (Weaver
et al., 1975) with tetracycline under photoheterotrophic
conditions. When required, CuSO4 was added at 3 mM final
concentration. Following growth to the late-exponential
phase, LacZ-mediated b-galactosidase activities were determined by the sodium dodecyl sulfate–chloroform method
(Miller, 1972; Hübner et al., 1991).
and accumulated continually (data not shown). After 4 h of
incubation at 30 1C, the cells were harvested by centrifugation at 13 689 g for 10 min and washed with 0.1 M Tris–HCl
buffer, pH 8.0. The pellet was rapidly frozen in liquid
nitrogen and stored at 20 1C. Frozen cells were suspended
in the same buffer and subsequently disrupted in a French
press cell (at 2000 psi). The lysate was centrifuged at 22 548 g
for 15 min and the supernatant was adjusted to 2 mM
CuSO4. The protein was purified by chromatography on a
Strep-tag binding column (IBA) and after the first washing
steps with 0.1 M Tris–HCl buffer, pH 8.0, plus 2 mM CuSO4,
copper was omitted, and, finally, the protein was eluted by
2.5 mM desthiobiotin.
Isolation of total RNA from Rhodobacter
capsulatus and transcriptional analysis by RT-PCR
Protein gel electrophoresis, enzymograms, and
CutO enzyme assays
Rhodobacter capsulatus cultures were grown in RCV minimal medium until late logarithmic phase. RNA of these
cultures was isolated using the Micro-to-Midi Total RNA
Purification System (Invitrogen, Karlsruhe, Germany) following the instructions of the manufacturer. Subsequently,
specific transcripts were analyzed with the ThermoScriptTM
RT-PCR System (Invitrogen). To analyze transcription of
the orf635–cutO–cutR operon, the following primers/primer
pairs (also see, Fig. 2) were used for cDNA synthesis and/or
second-strand synthesis and subsequent PCR amplification
steps: UP1/LP1 (5 0 -TCGCCCAAGACCACCAC-3 0 /5 0 -GACCG
GCCCTGCGCATCCAAAAG-3 0 ) and UP2/LP3 (5 0 -TCGACG
GGCGCAGCTGGGATAAC-30 /5 0 -GGATGCCGCTTTGCCCC
TTGAG-3 0 ).
Nondenaturing and SDS polyacrylamide gel electrophoresis
was carried out with the Laemmli system (Laemmli, 1970).
For nondenaturing gels, SDS was omitted in stacking gels
(4%) and running gels (10 %) as well as in all buffers to
avoid protein denaturation. Protein samples (2 mg) were
diluted at a ratio of 1 to 2 with sample buffer (65.5 mM
Tris–HCl, pH 6.8, 10% glycerol, and 0.1% bromophenol
blue). For denaturing gels, 5% b-mercaptoethanol and 3%
SDS were added to the sample buffer. After the completion
of electrophoresis, nondenaturing gels were incubated for
5 min at room temperature in 0.1 M sodium phosphate, pH
5.0, and subsequently with the same buffer containing 3 mM
2,6-dimethoxyphenol (Aldrich, Taufkirchen, Germany) and
0.5 mM CuSO4 until a yellowish-orange band appeared. For
total protein detection, similarly prepared gels were stained
with Coomassie brilliant blue.
Oxidase activity was determined by oxygen consumption
analysis in an oxygraph (Rank Brothers, Cambridge, UK) at
25 1C. The standard reaction mixture contained in
2 mL : 0.1 M Tris–HCl buffer, pH 6.7, 1 mM CuSO4, 1 mM
CutO protein, and substrates (2,3-dihydroxybenzoic acid, 4hydroxy-3,5-dimethoxybenzoic acid (syringic acid), 2,6dimethoxyphenol, or Fe21, Sigma) in a concentration range
from 0.1 to 2 mM. A pH range from 4 to 9 was examined to
determine the pH optimum of CutO.
Overexpression of Strep-tagged Rhodobacter
capsulatus CutO protein in Escherichia coli
As a basis for overexpression of R. capsulatus CutO in E. coli,
hybrid plasmid pJW14 was constructed. For this purpose, a
1.5 kb BamHI–PstI fragment from pJW8 was cloned into
expression vector pASK-IBA3 (Table 1; IBA, Göttingen,
Germany) resulting in hybrid plasmid pJW14, carrying an
in-frame cutO–Strep–TagII fusion (cutOStrep). Plasmid
pJW14 was transformed into E. coli strain BL21(DE3),
which served as a host for overexpression of the tagged R.
capsulatus CutO protein (CutOStrep).
Escherichia coli BL21(DE3)(pJW14) was grown at 37 1C in
selective LB medium until an OD580 of 0.6 was reached. The
culture was cooled down to 30 1C and protein synthesis was
induced by the addition of anhydrotetracycline (AHT).
Aliquots of the culture were withdrawn over a 4 h time
period, and accumulation of CutOStrep was followed by
polyacrylamide gel electrophoresis and Western blot analysis
using a tag-specific Strep-tactin–alkaline-phosphatase conjugate. A protein of the expected size (53 kDa), which
reacted with the conjugate, came up after AHT induction
Results and discussion
Genetic organization of the Rhodobacter
capsulatus cutO gene region
The deduced product of Rhodobacter capsulatus ORF orf636
(accession number AAC16140; Alexandre & Zhulin, 2000)
exhibits significant similarity (21% identity over the entire
length of the protein) to the MCO CueO from Escherichia coli.
The motifs typical of periplasmatic MCOs are present in
Orf636. These are the four histidine-rich copper-binding
c
70
J. Wiethaus et al.
Regulatory copper
T2
T3
T3
T3
5
5
5
Blue copper
5
T1
T2
T3
T3
T1
T3
T1
T1
101
139
355
439
497
CueO
H W H G
W F H P H
M D P M L D
D M M L H P F H I H G
M A H C H L L E H E D T G M
CutO
H W H G
W M H S H
A Q P M V M
S M M A H P M H L H G
M L H C H H M G H L A T G M
CotA
H L H G
W Y H D H
A G T Q D E
T R G T H P I H L H L
V W H C H I L E H E D Y D M
Domain I
Domain II
Domain III
Domain IV
Fig. 1. Alignment of copper-binding domains of selected multicopper oxidases. The copper-binding domains I–IV of MCOs as defined by Kim et al.
(2001) are shown for Escherichia coli CueO (accession number P36649), Rhodobacter capsulatus CutO (accession number AAC16140), and Bacillus
subtilis CotA (accession number P07788). The numbers on top of the alignment refer to the sequence positions in the E. coli protein. One copper atom
of the MCOs is bound by the T1 ‘blue’ copper site, and three other copper atoms are coordinated by a trinuclear cluster consisting of the T2 and T3 sites
(Roberts et al., 2002, and references therein). The motif involved in binding of a fifth regulatory copper as defined for E. coli CueO (corresponding amino
acid residues are marked with ‘5’; Roberts et al., 2003) is not conserved in CutO and CotA.
motifs (Fig. 1) and an N-terminal signal peptide containing a
twin-arginine motif, suggesting that transport of Orf636 into
the periplasm involves the TAT system. Unlike E. coli CueO, R.
capsulatus Orf636 does not contain the methionine-rich motif
known to coordinate a fifth regulatory copper atom (Roberts
et al., 2003). Since this study revealed distinct genetical and
functional features of Orf636 relative to the known bacterial
MCOs, we propose a new designation for R. capsulatus
Orf636, namely CutO (for Cu tolerance-mediating oxidase).
The R. capsulatus cutO gene is flanked by two open
reading frames (orf635 and orf637) oriented in the same
direction as cutO (Fig. 2a). In the course of this study,
Orf637 was identified as a copper-dependent repressor of
cutO expression. Therefore, we also propose a new designation for R. capsulatus Orf637, namely CutR (for cut regulatory protein). In contrast to CutO and CutR, no function
could be assigned to the orf635 gene product.
The orf635, cutO, and cutR coding regions are separated
by only 40 and 16 bp, respectively, indicating that these three
genes comprise a single transcription unit. This assumption
was corroborated by reverse transcriptase (RT)-PCR (Materials and methods; Fig. 2). Total RNA was isolated from R.
capsulatus wild-type cells grown either in the presence of
2 mM CuSO4 or without copper added. After reverse transcription, selected primer pairs (Materials and methods;
Fig. 2) were used to PCR amplify DNA fragments overlapping the gene borders of either orf635–cutO (UP1/LP1)
or cutO–cutR (UP2/LP3). The presence of PCR products
based on primer pairs UP1/LP1 (leading to amplification of
a 405 bp DNA fragment; Fig. 2b) and UP2/LP3 (leading to
amplification of a 547 bp DNA fragment; Fig. 2b) strongly
suggested that orf635–cutO–cutR belong to the same tran2005 Federation of European Microbiological Societies
c
scription unit. In both cases, PCR products were obtained
only with RNA derived from cultures grown in the presence
of copper, suggesting that transcription of the orf635–
cutO–cutR operon is positively regulated by copper.
Rhodobacter capsulatus CutO confers copper
tolerance under both aerobic and anaerobic
conditions
To determine the role of CutO in copper homeostasis in the
facultative anaerobic bacterium R. capsulatus, mutant
strains JW12-I and JW12-II were constructed (Materials
and methods; Fig. 2a). Both mutant strains are defective for
cutO since they contain a kanamycin resistance (Km)
cassette within the cutO coding region. The two mutant
strains are distinguished from each other by the orientation
of the resistance gene relative to cutO. The Km cassette was
previously shown to induce polar or nonpolar mutations
depending on its orientation (Schmehl et al., 1993; Hübner
et al., 1993), and, therefore, expression of the cutR gene (as
part of the orf635–cutO–cutR operon) was expected to be
either constitutive (driven by the Km promoter in JW12-I)
or silent (in JW12-II, with the Km gene reading in the
opposite direction).
To analyze the role of CutO in copper tolerance qualitatively, R. capsulatus wild-type (B10S) and the cutO mutant
strains (JW12-I, JW12-II) were grown on RCV minimal
medium plates with a disc, soaked with 2.5 mM CuSO4,
placed in the middle of the plates, under either phototrophic
conditions (anaerobic, in the light; Fig. 3a) or under aerobic
conditions in the dark (data not shown). For any R.
capsulatus strain tested, inhibition zones produced under
71
orf635
SphI
orf636
(cutO)
UP1 / LP1
UP2 / LP3 SphI
XhoI
Km >
JW12-I
< Km
JW12-II
pJW13
UP1 / LP1
M
UP2 / LP3
–
+
+
+
–
–
+
–
1
2
3
4
M
–
+
+
+
–
–
+
–
5
6
7
8
(b)
JW12-II
2.0
1.0
0.5
Wild-type, – O2
cutO, –O2
Wild-type, +O2
cutO, +O2
0.1
0.1
1
10
100 200
Copper sulfate [µM]
M
0.5 kb
0.2 kb
Fig. 2. Mutational and transcriptional analysis of the Rhodobacter
capsulatus orf635–cutO–cutR operon. (a) Physical and genetic map of
the cutO gene region. The localizations of genes and open reading
frames are given by arrows carrying their respective gene designations.
Black bars below the physical map indicate DNA fragments emerging
from RT-PCR (Materials and methods; b) with primer pairs UP1/LP1 and
UP2/LP3, respectively. A kanamycin resistance cassette (Km) was used to
create cutO mutant strains JW12-I and JW12-II. The directions of
transcription of the Km resistance gene are symbolized by arrowheads,
indicating polar and nonpolar insertions. Hybrid plasmid pJW13 carrying
a transcriptional cutO–lacZ fusion is based on the mobilizable narrowhost-range plasmid pSUP401. Neither the Km cassette nor the lacZ gene
are drawn to scale. (b) Transcriptional analysis of the orf635–cutO–cutR
operon by RT-PCR. Total RNA was isolated from R. capsulatus wild-type
cells grown either in the absence ( Cu; lanes 1, 3, 5, and 7) or presence
of copper (1Cu; lanes 2, 4, 6, and 8). RNA samples were either treated
with reverse transcriptase to synthesize cDNA (1RT; lanes 1, 2, 5, and 6)
or, as a negative control, reverse transcriptase was omitted ( RT; lanes
3, 4, 7, and 8). M, 50 bp DNA ladder (Fermentas, St Leon-Rot, Germany).
anaerobic conditions were larger than those formed under
aerobic conditions suggesting that copper was more toxic in
the absence of oxygen. A higher toxicity for copper under
anaerobic conditions, compared with aerobic conditions has
also been described for E. coli (Outten et al., 2001).
Remarkably, growth of both R. capsulatus cutO mutant
strains was inhibited more severely than the wild-type both
in the presence and absence of oxygen. These findings
JW12-I
orf637
(cutR)
lacZ
Cu
RT
cutO
B10S
orf633
(b)
Wild-type
(a)
1 kb
Final optical density at 660 nm
(a)
Fig. 3. Copper tolerance of Rhodobacter capsulatus wild-type and cutO
mutant strains. (a) Liquid cultures of R. capsulatus wild-type strain B10S
and cutO mutant strains JW12-I and JW12-II were plated onto RCV
minimal medium plates, prior to placement of discs soaked with 2.5 mM
CuSO4 on the center of the plates. Inhibition zones around the discs were
documented after 2 days of incubation under anaerobic conditions in
light. (b) R. capsulatus wild type and cutO mutant strain JW12-I were
inoculated in RCV minimal medium with the indicated copper concentrations at an optical density of 0.1 (at 660 nm). Final optical densities
were recorded after two days of incubation under either anaerobic
( O2) or aerobic (1O2) conditions.
strongly suggest that CutO is important for copper tolerance
under both aerobic and anaerobic conditions. In contrast to
R. capsulatus CutO, the E. coli counterpart, CueO, is not
involved in copper tolerance under anaerobic conditions
(Outten et al., 2001).
To examine the role of CutO in copper tolerance quantitatively, R. capsulatus wild-type and cutO mutant strains
(JW12-I, JW12-II) were grown in liquid RCV medium in
the presence of different concentrations of CuSO4 prior to
estimation of final cell densities (Fig. 3b). As expected from
the qualitative studies, strains defective for CutO were
significantly more sensitive towards copper than the wild
type under both aerobic and anaerobic conditions. As
mentioned above, expression of the cutR gene (as part of
the orf635–cutO–cutR operon) was expected to be either
constitutive (JW12-I) or silent (JW12-II). However, since
both cutO mutants, JW12-I and JW12-II, exhibited identical
phenotypes (Fig. 3b; data not shown), it is unlikely that the
cutR gene product directly influences copper tolerance or
regulates expression of genes that mediate copper tolerance,
other than cutO.
In a parallel approach, we asked whether a cutO mutant
was, in addition to copper, also influenced by other metals.
For this purpose, we analyzed growth of R. capsulatus wild2005 Federation of European Microbiological Societies
c
72
J. Wiethaus et al.
Table 2. Expression of cutO–lacZ in Rhodobacter capsulatus wild-type and cutO mutant background
Strain
Relevant
characteristics
Expected expression
of cutR
CuSO4 (mM)w
b-galactosidase
activityz
B10S
B10S
JW12-I
JW12-I
JW12-II
JW12-II
Wild-type
Wild-type
cutO<[Km 4 ]
cutO<[Km 4 ]
cutO<[ o Km]
cutO<[ o Km]
Cu-regulated
Cu-regulated
Constitutive‰
Constitutive‰
Silentz
Silentz
0
3
0
3
0
3
110.5 5.9
364.3 22.3
91.3 13.7
106.5 21.5
352.6 37.7
388.0 24.6
All strains contained hybrid plasmid pJW13 (transcriptional cutO-lacZ fusion) integrated in the chromosome via single cross-over.
w
Cultures were grown in RCV minimal medium under photoheterotrophic conditions.
b-Galactosidase activities (in Miller units; Miller, 1972) were determined in late-exponential-phase cultures. Miller units and standard deviations were
calculated from three independent assays for each strain.
‰
Expression of cutR is driven by the constitutive kanamycin resistance promoter.
z
Expression of cutR is silent due to polarity of the cutO mutation.
z
type and cutO mutant strain JW12-I in liquid RCV medium
containing different concentrations of either AgNO3,
ZnSO4, or NiSO4, respectively, under both aerobic and
anaerobic conditions. Under any given condition, R. capsulatus wild-type and the cutO mutant exhibited identical
sensitivity towards silver, zinc, and nickel (data not shown).
Therefore, the proposed role for CutO in metal tolerance
seems to be specific for copper.
CutR acts as a repressor of cutO expression under
low copper concentrations
As shown above, the orf635–cutO–cutR genes form part of a
transcriptional unit, which was activated by copper. To
analyze the expression of cutO in more detail, hybrid plasmid
pJW13 carrying a transcriptional cutO–lacZ fusion was
constructed (Materials and methods; Fig. 2a). Plasmid
pJW13 was introduced into R. capsulatus wild-type and both
cutO mutant strains, JW12-I and JW12-II, via conjugation.
Since pJW13 does not replicate in R. capsulatus, selection for
the vector-encoded tetracycline resistance yielded strains
carrying the entire plasmid integrated in the chromosome
via single cross-over. In the resulting strains, called
B10S:pJW13, JW12-I:pJW13 and JW12-II:pJW13 (Table 1),
transcription of the cutO-lacZ fusion is under control of the
natural promoter. As mentioned above, expression of cutR
was expected to be constitutive in cutO mutant JW12-I
(driven by the Km promoter) or silent in JW12-II (with the
Km gene reading in the opposite direction relative to cutR).
Since integration of hybrid plasmid pJW13 into the chromosome of JW12-I or JW12-II did not involve the DNA region
‘downstream’ of the XhoI site within the cutO coding region
(Fig. 2a), expression of cutR should also be constitutive in
JW12-I:pJW13 and silent in JW12-II:pJW13.
Rhodobacter capsulatus cutO–lacZ reporter strains were
grown in RCV minimal medium either in the presence of
c
3 mM CuSO4 or without copper added under photoheterotrophic (anaerobic) conditions until late-exponential phase
prior to determination of LacZ-mediated b-galactosidase
activities. The results shown in Table 2 may be summarized
as follows. (i) In the wild type, expression of cutO–lacZ was
enhanced by copper more than threefold. (ii) In JW12-I, in
which cutR expression is driven by the constitutive kanamycin promoter, cutO expression was low both in the presence
and absence of copper. Therefore, overexpression of cutR
seems to abolish copper induction of cutO transcription.
(iii) Similarly, no copper induction was observed in JW12II, in which cutR was expected to be silent. In contrast to the
situation in JW12-I, expression of cutO was high even in the
absence of copper. These observations are consistent with a
regulatory role of CutR, which acts as a repressor of
orf635–cutO–cutR transcription in the absence of copper,
whereas repression is relieved upon addition of copper. As
mentioned above, however, repression was not relieved in
the strain overexpressing cutR by addition of 3 mM CuSO4. It
is conceivable that higher amounts of copper are required to
restore copper regulation in this strain. This assumption was
not tested, since copper concentrations above 3 mM inhibited growth under anaerobic conditions (Fig. 3).
Analysis of the promoter region of the orf635–cutO–cutR
operon revealed the presence of an almost perfect 11 bp
palindromic DNA sequence (CGGTCAGCACC–N12–GG
GGCTGACCG) located 15 bp upstream of the putative
translational start of orf635. Since repressor proteins typically recognize and bind to palindromic DNA sequences
close to or overlapping the transcription start site of their
respective target genes, it seems reasonable that the palindromic sequence upstream of orf635 acts as the binding site
for CutR.
In summary, expression of R. capsulatus cutO is, like E.
coli cueO (Outten et al., 2000; Kim et al., 2001), up-regulated
upon additon of copper. However, up-regulation is put into
73
action by different regulatory mechanisms in these two
organisms. R. capsulatus CutR acts as a repressor of cutO
transcription in the absence of copper, whereas E. coli CueR
acts as an activator of cueO expression when copper is added
to the medium.
Enzyme properties of Rhodobacter capsulatus
CutO
Escherichia coli CueO and other MCOs are characterized by
the ability to oxidize artificial mono- and diphenolic substrates including 2,6-dimethoxyphenol (DMP), syringic acid
(SA), and 2,3-dihydroxybenzoic acid (DHB). In line with
the assumption that cutO codes for a copper-inducible
MCO, crude extracts prepared from R. capsulatus wild-type
B10S grown in the presence of 2 mM CuSO4 exhibited DMP
oxidase activity (data not shown). In contrast to the wildtype strains, both R. capsulatus cutO mutant strains (JW12I, JW12-II) lost DMP oxidase activity, suggesting that
oxidation of DMP was exclusively mediated by CutO. No
DMP oxidase activity was detected when B10S cells were
grown in the absence of copper (no CuSO4 added).
To test oxidase activity of CutO in more detail, R.
capsulatus CutO was overexpressed and purified as a recombinant Strep-tagged protein from E. coli (CutOStrep; Materials and methods). Binding of CutOStrep to and elution from
a Strep-tag-binding column could easily be followed due to
the intense blue color of the protein, indicating the presence
of T1 copper (Roberts et al., 2002, and references therein). It
is worth mentioning that 3.6 0.4 and 4.13 0.08 copper
atoms per molecule CutO were determined by atomic
absorption spectroscopy (AAS) and total reflexion X-ray
fluorescence spectrometry (TXRF), respectively.
To analyze purity and enzymatic activity of CutOStrep,
about 2 mg protein were loaded on a nondenaturing poly(a)
(b)
M
M
75 kDa
75 kDa
CutO
DMPO
50 kDa
50 kDa
37 kDa
37 kDa
Fig. 4. Electrophoretic analysis of the 2,6-dimethoxyphenol oxidase
activity of Rhodobacter capsulatus CutO. Two 2 mg aliquots of recombinant R. capsulatus CutOStrep protein purified from Escherichia coli were
loaded onto a nondenaturing gel next to prestained molecular markers
(BioRad, München, Germany). After electrophoresis was completed, the
gel was cut into two halves, one of which was stained with Coomassie
brilliant blue (a), whereas the other was stained for DMP (2,6-dimethoxy
phenol) oxidase activity as described in Materials and methods (b).
acrylamide gel (Materials and methods; Fig. 4). A single band
appeared in the Coomassie brilliant blue-stained gel (Fig. 4a),
and DMP oxidase activity was detected at a corresponding
position (Fig. 4b), suggesting that the CutO preparation was
sufficiently pure and active. Further in vitro enzyme reactions
with DMP, SA, and DHB as substrates were followed by
oxygen consumption measurements (Materials and methods). The KM values were determined for the substrates DMP
(1.02 0.11 mM), SA (0.59 0.05 mM), and DHB (0.15 0.01 mM) at the pH optimum of 6.7, which was identical for
all three substrates. This pH optimum is in the physiological
range for periplasmatic enzymes. In summary, with respect to
substrate oxidation, R. capsulatus CutO has a broad substrate
specificity like E. coli CueO.
The physiological substrate for R. capsulatus CutO is
unknown. Recently, however, it was shown for E. coli CueO
that Fe21 served as an electron donor for the enzyme (Singh
et al., 2004). The reoxidation of Fe21 is especially important
as protection against the formation of oxygen radicals
(ROS). A similar role for CutO can be envisaged, since this
enzyme also can oxidize Fe21. However, the ferroxidase
activity of CutO (kcat = 17 min1; this study) was considerably lower than the value for E. coli CueO (kcat = 215 min1;
Singh et al., 2004). Furthermore, E. coli CueO can also
oxidize the catechol siderophore enterobactin, which complexes iron ions (Grass et al., 2004). The existence and nature
of a siderophore in R. capsulatus, however, is still obscure.
As described above, R. capsulatus CutO confers copper
tolerance not only under aerobic conditions, as is the case
for E. coli CueO, but also under anaerobic conditions. Most
likely, copper tolerance in the presence or absence of oxygen
is based on different mechanisms. Oxidation of the yet
unknown physiological substrate with oxygen as terminal
acceptor may account for copper tolerance under aerobic
conditions. Since CueO-mediated protection from copper
toxicity involves oxidation of enterobactin in the presence of
copper (Grass et al., 2004), one might speculate that the
physiological substrate for R. capsulatus CutO may be a yet
unidentified siderophore. On the other hand, the question
how CutO mediates copper tolerance in the absence of
oxygen remains open.
Acknowledgements
We thank Norbert Jakubowski and Jürgen Messerschmidt
(Institut für Spektrochemie und Angewandte Spektroskopie, Dortmund) for spectroscopic analyses (TXRF and AAS)
to determine copper content of CutO, Satish K. Singh
(University of Arizona, Tucson) for determination of CutO
ferroxidase activity, Franz Narberhaus (Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität Bochum) for
critically reading the manuscript, and Ursula Hilp (Lehrstuhl für Biochemie der Pflanzen, Ruhr-Universität
c
74
Bochum) for technical assistance. This work was supported
by financial grants from Fonds der Chemischen Industrie.
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Taurine regulation in Rhodobacter
F
The GntR-like regulator TauR activates expression of
taurine utilization genes in Rhodobacter capsulatus
Schubert B., Wiethaus J., Pfänder Y., Narberhaus F. and Masepohl B.
Manuskript in Vorbereitung
72
ABSTRACT
Rhodobacter capsulatus uses taurine as an alternative sulfur source and the products of the
tpa-tauR-xsc gene region are essential for this process. The tauR gene codes for a MocR-like
member of the GntR superfamily of transcriptional regulators. TauR is essential for activation
of tpa transcription as shown by analysis of wild-type and tauR mutant strains carrying a tpalacZ reporter fusion. Activation of the tpa promoter requires taurine but is not inhibited by
sulfate, which is the preferred sulfur source of many bacteria including R. capsulatus. TauR
directly binds to the tpa promoter as demonstrated by DNA mobility shift assays. As expected
for a transcriptional activator, the TauR binding site is located upstream of the transcription
start site, which has been determined by primer extension. Analysis of site-directed promoter
mutants suggests that TauR binds to direct repeats as shown for another member of the MocR
subfamily, GabR from Bacillus subtilis. In contrast, all other members of the GntR family
analyzed so far bind to inverted repeats.
INTRODUCTION
Nonsulfur purple bacteria (Rhodospirillaceae) and especially Rhodobacter species have long
been used as model organisms for analyses of anoxygenic photosynthesis, carbon
assimilation, hydrogen metabolism, and nitrogen fixation (6, 11, and references therein). As
shown for many other bacteria, sulfate is a preferred sulfur source for Rhodobacter species.
Alternatively,
Rhodobacter
capsulatus
can
efficiently
grow
with
taurine
(2-
aminoethanesulfonate) as sole sulfur source under phototrophic (anaerobic) conditions (12).
Two divergently transcribed gene clusters, tauABC and tpa-tauR-xsc (formerly orf459-484590), are involved in taurine sulfur utilization by R. capsulatus as shown by mutational
analysis (Fig. 1; [12]). The tauABC gene products are predicted to form an ABC transport
system mediating taurine uptake. The tpa and xsc gene products exhibit strong similarity to
taurine:pyruvate aminotransferase (Tpa) from Bilophila wadsworthia (catalyzing the initial
transamination of taurine to 2-sulfoacetaldehyde during anaerobic taurine degradation; [9])
and sulfoacetaldehyde acetyltransferase (Xsc) from Alcaligenes defragrans (converting 2sulfoacetaldehyde into sulfite and acetyl phosphate; [17]). Genes similar to R. capsulatus tauR
were previously identified in close proximity to xsc genes in the genomes of Paracoccus
denitrificans and other proteobacteria by database searches (4, 17), but none of these genes
73
has been experimentally characterized so far. As shown in this study, TauR is a transcriptional
activator, which is essential for tpa expression.
R. capsulatus TauR belongs to the GntR superfamily of bacterial transcription regulation
proteins that bind DNA through a helix-turn-helix motif. The N-terminal DNA-binding
domain is well-conserved for all members of the GntR family, whereas the C-terminal
effector-binding and oligomerization domain is more variable, and therefore, was used to
define six GntR subfamilies, namely the FadR, HutC, MocR, YtrA, AraR, and PlmA families
(10, 15). Highest similarity of R. capsulatus TauR was found with the MocR subfamily
named after Rhizobium meliloti MocR (16).
Several well-characterized members of the FadR, HutC, and YtrA subfamilies bind to
inverted repeats in the promoter regions of their target genes, while MocR-like proteins have
earlier been predicted to bind to direct repeats (15). To date, this hypothesis has been proven
for only one MocR-like protein, GabR from Bacillus subtilis (2, 3).
In this study we analyzed the role of R. capsulatus TauR by genetic and biochemical means.
TauR was shown to be essential for tpa expression in a taurine-dependent manner. Sitedirected mutagenesis of the tpa promoter and DNA-mobility shift assays suggest binding of
TauR to direct repeats.
Strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this
study are listed in Table 1. Methods for conjugational plasmid transfer between E. coli and R.
capsulatus, procedures for selection of mutants and growth conditions have been described
earlier (12, and references therein).
Construction of Ptpa-lacZ reporter plasmid pBSRUB60 and β-galactosidase assays. A
461-bp DNA fragment (fragment A, Fig. 3) encompassing the tpa promoter (Ptpa) was PCRamplified with primer pair UP-tpa-1/LP-tpa-2 (Table 2) using R. capsulatus chromosomal
DNA as a template. The PCR product was blunt-end cloned into the SmaI site of vector
plasmid pBluescript KS leading to a construct (pBSRUB48) in which Ptpa is flanked by
BamHI and HindIII restriction sites. Subsequently, the BamHI-HindIII fragment from
pBSRUB48 carrying Ptpa was cloned into the mobilizable broad-host-range plasmid
pBBR1MCS. Finally, the promoter-less E. coli lacZ gene was inserted at the HindIII site
74
resulting in reporter plasmid pBSRUB60 (tpa-lacZ). R. capsulatus reporter strains carrying
pBSRUB60 (tpa-lacZ) were grown in RCV minimal medium with taurine or sulfate as sulfur
source before β-galactosidase activities were determined as described earlier (12).
RNA isolation and primer extension. R. capsulatus cultures were grown in RCV minimal
medium with taurine as sulfur source until late logarithmic phase. RNA of these cultures was
isolated using the Micro-to-Midi Total RNA Purification System (Invitrogen, Karlsruhe,
Germany) following the instructions of the manufacturer. Primer extension was carried out as
decribed previously (1) using oligonucleotide LP-tpa-2 (Table 2) to map the transcription start
site of tpa.
Overexpression of Strep-tagged R. capsulatus TauR protein in E. coli. The tauR coding
region was PCR-amplified with primer pair UP-tauR/LP-tauR (encompassing recognition
sites for SacII and SalI, respectively; Table 2) using R. capsulatus chromosomal DNA as a
template. Subsequently, the 1.5-kb SacII-SalI DNA fragment with tauR was cloned into
expression vector pASK-IBA3 (Table 1) resulting in hybrid plasmid pBSRUB29, carrying an
in-frame tauR-Strep-TagII fusion (tauRStrep). Plasmid pBSRUB29 was transformed into E.
coli strain BL21(DE3) which served as a host for overexpression of the tagged R. capsulatus
TauR protein (TauRStrep). Purification of the recombinant protein was carried out as decribed
previously (20).
DNA mobility shift assays and site-directed mutagenesis. A 461-bp DNA fragment
encompassing the tpa promoter (Ptpa) was obtained by PCR amplification with primer pair
UP-tpa-1/LP-tpa-2 using plasmid pBSRUB48 as a template (Table 2). Ptpa subfragments were
generated by cutting the 461-bp amplification product with appropriate restriction enzymes
(Fig. 3). Pre-incubation of promoter fragments with TauRStrep protein, and agarose gel
electrophoresis of DNA-protein complexes were carried out as described previously (14).
Point mutations within the tpa promoter region (designated mut-1 to mut-4, Fig. 4) were
generated by overlap extension PCR (19) with appropriate primers (Table 2) and pBSRUB48
as a template. PCR products were blunt-end cloned into the SmaI site of Bluescript KS
leading to hybrid plasmids pBSRUB94 (mut-1), pBSRUB95 (mut-2), pBSRUB96 (mut-3),
pBSRUB97 (mut-4), and pBSRUB110 (mut-3/4). These plasmids served as templates for
PCR amplification of 269-bp mutant tpa promoter fragments using primer pair PJW72U/PJW73-L (Table 2). Purification of amplification products, 32P end-labeling, pre-incubation
75
of labeled promoter DNA with TauRStrep protein, and polyacrylamide gel electrophoresis of
DNA-protein complexes were carried out as described earlier (21).
RESULTS AND DISCUSSION
Mapping of the tpa transcription start site. Genetic analyses indicate that the tpa, tauR, and
xsc genes comprise a single transcription unit ([12]; this study; Fig. 1). To assign the exact
transcription start site of the tpa-tauR-xsc operon, primer extension experiments were
performed (see Materials and Methods; Fig. 2). For this purpose, total RNA isolated from an
R. capsulatus wild-type culture grown with taurine as sole sulfur source was used as template
for reverse transcription with primer LP-tpa-2 complementary to the 5´ end of tpa mRNA (see
Materials and Methods, Table 2). The resulting start site mapped between the TauR binding
site (see below; Fig. 3) and the AUG start codon for the tpa gene.
TauR activates tpa expression. To analyze expression of the tpa-tauR-xsc operon, plasmid
pBSRUB60 carrying a transcriptional tpa-lacZ fusion was introduced into R. capsulatus wildtype and tpa, tauR, and xsc mutant strains (see Materials and Methods, Table 3). The resulting
reporter strains were grown in RCV minimal medium with taurine or sulfate as sulfur source
prior to determination of lacZ-mediated β-galactosidase activity as described earlier (12). The
results of the expression studies shown in Table 3 can be summarized as follows. (i)
Expression of the tpa–lacZ fusion was taurine-inducible as expected from earlier studies (12).
(ii) Transcription from the tpa promoter was not inhibited by sulfate. Instead, sulfate had a
somehow stimulatory effect onto tpa transcription when both taurine and sulfate were
supplied at the same time. (iii) Taurine-dependent induction of tpa expression strictly required
TauR. (iv) In contrast to TauR, neither Tpa nor Xsc were essential for tpa expression. (v) A
tpa mutation, which is polar regarding to tauR transcription, prevented tpa-lacZ expression,
suggesting that tauR expression strictly depends on the tpa promoter. Thus, this finding
confirms co-transcription of tpa and tauR.
TauR binds to the tpa promoter. Since R. capsulatus TauR was essential for tpa-lacZ
expression (Table 3), it seemed likely that TauR binds to the tpa promoter. Interaction of
TauR with the tpa promoter was analyzed by DNA mobility shift assays (see Materials and
Methods). For this purpose, TauR protein was overexpressed and purified as C-terminally
76
Strep-tagged recombinant protein from E. coli (see Materials and Methods). TauRStrep was
pre-incubated either with a 461-bp PCR fragment encompassing the tpa promoter or with
subfragments created by digestion of the PCR product with different restriction enzymes.
Protein-DNA complexes were analyzed by agarose gel electrophoresis followed by ethidium
bromide staining (Fig. 3). Since only one out of two restriction fragments in the respective
assays was retarded, the other fragment served as an internal control for TauR binding
specificity. Comparison of retarded DNA fragments suggests that the TauR binding site is
confined to a 88-bp BglI-TaqI fragment within the tpa promoter region. As one would expect
for the binding site of a transcriptional activator, the TauR binding site is located upstream of
the transcription start site (Fig. 3).
Since tpa-lacZ expression was taurine-inducible (Table 3), we asked whether taurine might
influence binding of TauR to the tpa promoter. The tpa promoter region was PCR-amplified
using primer pair PJW72-U/PJW73-L (Table 2) prior to radioactive 5´ end-labeling of the
269-bp PCR product (marked in Fig. 4). TauRStrep was pre-incubated with the radioactively
labeled tpa promoter fragment either in the presence or absence of taurine. Analysis of
protein-DNA complexes by polyacrylamide gel electrophoresis revealed, however, no
differences between assays performed in the presence or absence of taurine (Fig. 5A, 5B; see
Conclusions). Binding of TauR to the radioactively labeled tpa promoter fragment could be
reversed by addition of increasing amounts of nonlabed tpa promoter DNA (Fig. 5C) but not
by mutant tpa promoter DNA (Fig. 5D; see below) thus demonstrating TauR binding
specificity.
TauR binds to direct repeats. The 88-bp BglI-TaqI fragment (encompassing the TauR
binding site) contains two pairs of almost perfect direct repeats (DR-1a/DR-1b, DR-2a/DR2b; Fig. 4). To examine the role of these sequences for TauR binding, site-directed mutations
were created by overlap extension PCR (see Materials and Methods; Fig. 4). As described
above for the wild-type tpa promoter, DNA fragments carrying mutations mut-1 to mut-4
were PCR-amplified using primer pair PJW72-U/PJW73-L (Table 2), radioactively labeled,
and incubated with TauRStrep. All binding assays were performed in the presence of poly(dIdC) as competitor DNA.
The results of DNA mobility shift assays with increasing amounts of TauRStrep are shown in
Fig. 6. Binding of TauR to the tpa promoter was not influenced by mutations mut-1 and mut-2
as compared to the wild-type promoter. These finding suggest that direct repeats DR-1a and
DR-1b are dispensable for TauR binding. However, these sequences may serve as a binding
77
site for another yet unknown regulatory protein (see Conclusions). In contrast, binding of
TauR was clearly diminished by mutations mut-3 and mut-4. It remains unknown, however,
why the effect of mut-4 was less pronounced as compared to mut-3. Combination of both
mutations (mut-3/4) completely abolished binding of TauR to the tpa promoter strongly
suggesting that direct repeats DR-2a and DR-2b are essential for TauR binding.
Conclusions. R. capsulatus TauR was shown to be essential for tpa expression. Since tauR is
co-transcribed with the tpa gene, TauR appears to be autoregulatory. Interestingly, in vivo tpa
expression was clearly enhanced by taurine, but taurine did not influence binding of TauR to
the tpa promoter in vitro. This discrepancy may be explained by requirement of an unknown
co-activator protein responding to taurine availability. At present, one may only speculate
whether direct repeats DR-1a/DR-1b immediately upstream of the TauR binding site serve as
a binding site for the putative co-activator. Alternatively, one might assume that TauR
interacts with a degradation product of taurine rather than with taurine itself. However, tpa
expression was not altered in a mutant defective for Tpa (taurine:pyruvate aminotransferase),
which is thought to catalyze the initial step of taurine degradation. Since R. capsulatus has the
capacity to synthesize different aminotransferases (www.ergo-light.com), we cannot rule out
that another aminotransferase may partially substitute for Tpa in catalyzing the intial
transamination of taurine, and thus provides sufficient amounts of the putative inducer.
R. capsulatus TauR is a MocR-like member of the GntR superfamily of transcription
regulators (4, 15, this study). This paper presents experimental evidence that TauR binds to
direct repeats and not to inverted repeats, as is the case for members of the FadR, HutC, and
YtrA subfamilies. Binding of TauR to the tpa promotor involves direct repeats located in a
DNA region ranging from – 77 to – 45 upstream of the transcription start site. Due to the
close proximity between the TauR binding site and the putative – 35/– 10 promoter , one
would expect direct interaction between TauR and RNA polymerase, without the necessity of
DNA bending.
78
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Masepohl, B., F. Führer, and W. Klipp. 2001. Genetic analysis of a Rhodobacter
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80
Table 1: Bacterial strains and plasmids used in this study
Strain or plasmid
Escherichia coli
Relevant characteristics (a)
BL21(DE3)
Host for overexpression of TauRStrep
DH5α
S17-1
Host for plasmid construction
Donor providing transfer functions
Reference of source
Novagen, Darmstadt,
Germany
5
18
B10S
FFRUB33
FFRUB47
FFRUB48
FFRUB53
FFRUB54
FFRUB57
Spontaneous Sm resistant mutant of R.
capsulatus B10
Δ(tpa-tauR-xsc)::[<Gm]
tpa::[<Gm]
tpa::[Gm>]
tauR::[Gm>]
tauR::[<Gm]
xsc::[<Gm]
7
12
12
12
12
12
12
Plasmids
pBBR1MCS
Strep-tag expression vector, tet p/o;
tetR, Ap
mobilizable broad-host-range vector
pBluescript KS
high-copy vector, Ap
pASK-IBA3
pBSRUB29
pBSRUB48
pBSRUB60
pBSRUB94
pBSRUB95
pBSRUB96
pBSRUB97
pBSRUB110
(a)
pASK-IBA3 derivative carrying
tauRStrep
pBluescript KS derivative carrying a
461-bp PCR product with Ptpa
pBBR1MCS derivative carrying a
Ptpa-lacZ fusion; Tc
pBluescript KS derivative carrying
Ptpa variant mut-1
Ptpa variant mut-2
Ptpa variant mut-3
Ptpa variant mut-4
Ptpa variant mut-3/4
IBA, Göttingen,
Germany
8
Stratagene, Amsterdam,
The Netherlands
This study
This study
This study
This study
This study
This study
This study
This study
Ap, ampicillin; Gm, gentamicin; Km, Kanamycin; Tc, tetracycline; tet p/o, tetracycline
promoter/operator; tetR, tetracycline repressor gene; Sm, streptomycin
81
Table 2: Synthetic oligonucleotides used in this study
Oligonucleotide
DNA sequence (5´→3´) (a)
UP-tauR
LP-tauR
CCCGCGGAAGCTGCGAGGTCAAGATGGCC
GCAGGTCGACACGCAGGGCGCGGGCGATG
amplification of tauR
coding region, 1485 bp
UP-tpa-1
LP-tpa-2
GCGCGGCACGTTTCAGGGCTTTGGTCAT
CGGATCGCTGGTTTCATAGGGCTTGTGCT
tpa promoter amplification,
461 bp; primer extension
UP-mut-1
LP-mut-1
GGCGGGGGAAGAATATTCCCGTCAGGGTGG
CCACCCTGACGGGAATATTCTTCCCCCGCC
site-directed mutagenesis
of the tpa promoter
UP-mut-2
LP-mut-2
CCCGTCAGGGTCGACAAAACGGCGCTCTGG
CCAGAGCGCCGTTTTGTCGACCCTGACGGG
of the tpa promoter
UP-mut-3
LP-mut-3
CGGCGCTCTGCAGTTAAGGCTTCGGGG
CCCCGAAGCCTTAACTGCAGAGCGCCG
of the tpa promoter
UP-mut-4
LP-mut-4
GGCTTCGGGGAAACTGCAGCTAATCGAAGCGGGG
CCCCGCTTCGATTAGCTGCAGTTTCCCCGAAGCC
of the tpa promoter
PJW72-U
PJW73-L
TCAGAGGTGGCGGAATGTT
CCCCCGCTTCGATTAGGTCCAGT
tpa promoter amplification,
269 bp
(a)
Underlined sequences mark recognition sites for PstI, SacII, SalI, and SspI,
respectively.
82
Table 3: Taurine-dependent expression of tpa-lacZ in R. capsulatus
Strain (a)
B10S
Relevant
characteristics
wild-type
FFRUB33 Δ(tpa-tauR-xsc)
β-Galactosidase activity (b)
sulfate
taurine
taurine + sulfate
178 ± 19
2,837 ± 79
4,700 ± 408
104 ± 23
0 ± 0
122 ± 36
76 ± 32
FFRUB47
tpa, polar
66 ± 10
133 ± 47
FFRUB48
tpa
182 ± 78
5,433 ± 386
FFRUB54
tauR, polar
196 ± 140
190 ± 190
0 ± 0
FFRUB53
tauR
129 ± 58
0 ± 0
59 ± 31
FFRUB57
xsc
95 ± 12
1,485 ± 9
(a)
12,391 ± 94
4,888 ± 348
All R. capsulatus strains contained plasmid pBSRUB60 (tpa-lacZ) based on the broadhost-range vector pBBR1MCS.
(b)
R. capsulatus strains were grown in minimal medium with the indicated sulfur
sources. β-Galactosidase activity is given in Miller units (13). Results represent the
means and standard deviations of three independent measurements.
83
FFRUB33
1 kb
FFRUB48
FFRUB53
FFRUB47
FFRUB54
BglII
tauC
tauB
tauA
tpa
HindIII
FFRUB57
BamHI
tauR
xsc
orf408
pBSRUB60
lacZ
Figure 1: Organization of the R. capsulatus tpa-tauR-xsc gene region.
Mutant strains defective for tpa, tauR, and xsc contain gentamicin resistance cassettes, with
the directions of transcription of the Gm resistance gene symbolized by arrows. Hybrid
plasmid pBSRUB60 carrying a transcriptional fusion of lacZ to the tpa promoter is based on
the mobilizable broad-host-range plasmid pBBR1MCS. Neither the Gm cassette nor the lacZ
gene is drawn to scale.
84
A
C
G
T
PE
C
C
A (-10)
T
C
C
C
A
C
G
C
T
C
A
A (+1)
C
C
A
G RBS
A
G
G
C
C
C
C
C
A
T Tpa
G
Figure 2: Transcription start site mapping of the tpa promoter.
Primer extension was carried out with total RNA from R. capsulatus cells grown with taurine
as sole sulfur source. Primer LP-tpa-2 (binding to the 5´ region of tpa; Table 2) was used for
reverse transcription. The resulting primer extension product is shown in lane PE. The
corresponding sequencing reactions (A, C, G, and T) with plasmid pBSRUB48 served as
length standard.
85
+1
< tauA
tpa >
1
SacII
EcoRI
BglI
TaqI
461 bp
A
B
C
D
E
TauR
461 bp
uncut
SacII
EcoRI
BglI
TaqI
–
+
–
+
–
+
–
+
–
+
1
2
3
4
5
6
7
8
9
10
M
0.5 kb
A
B
E
C
D
0.2 kb
Figure 3: DNA mobility shift assays narrowing down the TauR binding site.
The transcription start site of the tpa gene (labelled +1) is indicated above the physical map.
DNA fragment A (461 bp) encompassing the tauA-tpa intergenic region was generated by
PCR amplification using primer pair UP-tpa-1/LP-tpa-2 (Table 2). In addition, subfragments
were generated by cutting fragment A with the indicated restriction enzymes. DNA fragments
were either pre-incubated with purified TauStrep protein (lanes 2, 4, 6, 8, and 10) or not (lanes
1, 3, 5, 7, and 9) prior to agarose gel electrophoresis. DNA fragments retarded by TauR are
marked (A to E). A 50-bp DNA ladder (Fermentas, St. Leon-Rot, Germany) was used as a
length standard (M).
86
+1
< tauA
tpa >
1
SacII
>>>
>>>>>> >
DR-1a
EcoRI
>>>
BglI
>>>>>> >
DR-1b
TaqI
>>>>>> >>>
DR-2a
461 bp
>>>>>> >>>
DR-2b
GCCGGTCCGGCGGGGGAAGAAAAATCCCGTCAGGGTGGAAAAAACGGCGCTCTGGACTTAAGGCTTCGGGGAAACTGGACCTAATCGA
↓ ↓
↓ ↓
↓ ↓
↓ ↓
AATATT
GTCGAC
CTGCAG
CTGCAG
SspI
SalI
PstI
PstI
mut-1
mut-2
mut-3
mut-4
Figure 4: Site-directed mutagenesis of the tpa promoter.
The heavy line below the physical map depicts a 269-bp PCR fragment (amplified using
primer pair PJW72-U/PJW73-L; Table 2) used for DNA mobility shift assays (Fig. 5). Sitedirected mutations (mut-1, mut-2, mut-3, and mut-4) within direct repeats (DR-1a, DR-1b,
DR-2a, and DR-2b) were created by overlap extension PCR.
87
C
1.17
0.44
0.58
0.29
0.22
0.15
0
0.02
0.04
A
0.07
P-wt
– taurine
bound
probe
bound
probe
free
probe
D
1.17
0.58
0.44
0.29
0.22
0.15
0.04
0.07
0
B
0.02
free
probe
P-mut
+ taurine
free
probe
bound
probe
bound
probe
free
probe
Figure 5: Influence of taurine and specificity controls for TauR binding to the tpa
promoter.
A 269-bp DNA fragment (marked in Fig. 4) carrying the TauR binding site was
32
P end-
labeled prior to incubation with increasing amounts of TauRStrep (0, 0.02, 0.04, 0.07, 0.15,
0.22, 0.29, 0.44, 0.58, and 1.17 µM) either in the absence (A) or presence (B) of 1 µM
taurine. Unlabeled wild-type tpa promoter fragments (P-wt) were used as specific competitor
DNA (C). Mutant tpa promoter fragments (P-mut carrying mutations mut-3 and mut-4; Fig. 4)
served as non-specific competitor DNA (D). 0.58 µM TauR and radioactively labeled wildtype tpa promoter fragments were mixed with a 80-, 160-, 320-, 640-, 1280-, 2560-, 5120-, or
10240-fold excess of unlabeled competitor or non-competitor DNA. All reactions were
performed with 0.6 fmol 32P-labeled DNA.
88
A
1.17
0.44
0.58
0.29
0.22
0.15
0.07
0.04
0.02
0
bound
probe
free
probe
B
bound
probe
free
probe
C
bound
probe
free
probe
D
bound
probe
free
probe
E
bound
probe
free
probe
F
free
probe
Figure 6: DNA mobility shift assays with wild-type and mutant tpa promoter fragments.
Binding of TauRStrep to the wild-type tpa promoter (A) was compared to binding to promoter
variants mut-1 (B), mut-2 (C), mut-3 (D), mut-4 (E), and mut-3/4 (F). 269-bp DNA fragments
encompassing the wild-type and mutant promoters were generated by PCR amplification
using primer pair PJW72-U/PJW73-L (also see Figs. 4 and 5). Incubation of increasing
amounts of TauR (up to 1.17 µM) with
32
P-labeled DNA fragments was carried out in the
presence of 1 µM taurine.
89
Diskussion
G
Diskussion
1.
Stickstoff-Kontrolle durch „cross-talk“ von NtrY und NtrC
1.1
NtrY ist eine bifunktionelle Sensorkinase für NtrC
Zwei-Komponenten-Regulationssysteme (TCS, two-component regulatory system) stellen
das bei Bakterien dominierende System zur Signaltransduktion dar. Eine Möglichkeit zur Erhöhung des Informationsinputs solcher Systeme bietet der „cross-talk“. Hierbei kommt es zur
Phosphorylierung eines Responseregulators durch die Sensorkinase eines anderen TCS (Ninfa
et al., 1988; Wei et al., 2007; Yamamoto et al., 2005). Im Rahmen dieser Arbeit konnte eine
solche Kommunikation zwischen den TCS NtrY/NtrX und NtrB/NtrC aus R. capsulatus gezeigt werden (Drepper et al., 2006). Die Sensorkinase NtrC ist essentiell für die Expression
von Genen der Stickstoff-Fixierung (nif-Gene) und der Harnstoff-Verwertung (ure-Gene).
Dennoch konnten ntrB- und ntrY-Mutanten wie der Wildtyp sowohl molekularen Stickstoff
als auch Harnstoff als Stickstoff-Quelle nutzen. Demnach wird NtrC in beiden Mutanten weiterhin phosphoryliert. Eine ntrB/ntrY-Doppelmutante hingegen konnte weder N2 noch Harnstoff nutzen, was darauf hindeutet, dass die Phosphorylierung von NtrC nicht erfolgt. Somit
ist NtrY der einzige zu NtrB alternative Phospho-Donor für den Responseregulator NtrC.
Diese Beobachtung wird durch Sequenz- und Strukturvergleiche der beteiligten Proteine gestützt. So weisen die Kinasedomänen von NtrY und NtrB eine Identität von 29 % und eine
Ähnlichkeit von 46 % auf. Auch NtrX und NtrC zeigen mit einer Identität von 28 % und einer
Ähnlichkeit von 43 % klare Sequenzhomologien. Ferner zählen beide Responseregulatoren
auf Grund ihrer identischen Domänenstruktur zu den Transkriptionsregulatoren der NtrCFamilie. Folglich sollten die bei der Signaltransduktion involvierten Kontaktregionen der Sensorkinasen und Responseregulatoren strukturell ähnlich sein. Die Kommunikation zwischen
NtrY und NtrC wäre demnach prinzipiell möglich.
Die Interaktion zwischen NtrY und NtrC scheint nicht auf die Phosphorylierung des Responseregulators durch NtrY beschränkt zu sein. So war die Expression des ure-Operons in einer
ntrB-Mutante im Vergleich zum Wildtyp verringert und in einer ntrY-Mutante erhöht (Drepper et al., 2006). Anscheinend katalysiert NtrY als bifunktionelle Sensorkinase neben der
Phosphorylierung vornehmlich die Dephosphorylierung von NtrC. Dies würde zu einer Akkumulierung von NtrC in einer ntrB-Mutante und von NtrC-P in einer ntrY-Mutante führen
90
Diskussion
und die unterschiedlichen Expressionsprofile erklären. Strukturanalysen zeigen zudem, dass
NtrY aus Azorhizobium caulinodans zu den bifunktionellen Sensorkinasen zählt (Alves &
Savageau, 2003). Interessanterweise wird das im Rahmen eines „cross-talks“ eingehende Signal einer bifunktionellen Sensorkinase besser amplifiziert, dass einer monofunktionellen Sensorkinase besser unterdrückt (Alves & Savageau, 2003). Die Bifunktionalität der vermittelnden Sensorkinase deutet somit auf einen physiologischen „cross-talk“ hin. Dies unterstreicht
die Relevanz der Kommunikation zwischen R. capsulauts NtrY und NtrC und schließt aus,
dass es sich um eine unphysiologische Interferenz handelt. NtrY kann NtrB bei der Modifikation von NtrC allerdings nicht voll ersetzen. Für die maximale Induktion des ure-Operons
unter Stickstoff-Mangel wurde NtrB benötigt. Für TCS ist die Kommunikation mit der eigenen Sensorkinase oftmals von größerer Relevanz als der „cross-talk“ mit einer System-fremden Sensorkinase (Verhamme et al., 2002). Es ist daher zu vermuten, dass der „cross-talk“
mit NtrY der Feinabstimmung des NtrB/NtrC-Systems dient.
1.2
NtrY registriert den periplasmatischen Stickstoff-Status
Um zu klären, welche Umweltinformation NtrY in das NtrB/NtrC-System einbringt, muss
zunächst die Struktur der beiden Sensorkinasen betrachtet werden. Wie bereits erwähnt sind
die C-terminalen Kinasedomänen von NtrB und NtrY vergleichbar strukturiert. Die für die
Signalaufnahme zuständigen N-terminalen Domänen beider Sensorkinasen differieren hingegen stark. NtrB weist eine N-terminale PAS-Domäne auf, welche ein bei cytoplasmatischen
Sensorkinasen gängiges Sensormodul darstellt (Galperin et al., 2001; Galperin, 2004). Bei
NtrY hingegen liegt eine zusätzliche N-terminale Extension vor, und die Sensorkinase ist
dementsprechend mit einem Molekulargewicht von 82 kDa mehr als doppelt so groß wie
NtrB (38,3 kDa). N-terminal sind vier putative Transmembranregionen lokalisiert, wobei drei
Regionen durch einen Bereich von 174 Aminosäure-Resten von der vierten separiert sind
(Abb. 12). Diese stark hydrophobe Region ist über einen HAMP-Linker mit einer PASDomäne verbunden. Die für Transmembranrezeptoren typischen HAMP-Linker dienen der
Signalübertragung zwischen Input- und Output-Domänen und sind in der Cytoplasmamembran lokalisiert (Anantharaman et al., 2006; Appleman et al., 2003; Appelman & Steward, 2003; Hulko et al., 2006). Die Domänenstruktur von NtrY legt somit nahe, dass es sich
um ein Transmembranprotein handelt. Membran-assozierte Sensorkinasen setzen sich typischerweise aus einer extracytoplasmatischen Input und einer cytoplasmatischen Kinasedomä91
Diskussion
ne zusammen, wobei diese durch Transmembranhelices voneinander separiert werden (Mascher, 2006). Entsprechend der registrierten Stimuli kann zwischen zwei Typen von Membran-Sensorkinasen unterschieden werden (Mascher et al., 2006). Periplasmatische Reize werden typischerweise über einen ausgedehnten extracytoplasmatischen Loop aufgenommen.
Andere Sensorkinasen überwachen über Transmembranhelices hauptsächlich Membranassoziierte Faktoren wie Ionenstärke, Osmolarität und Turgor. NtrY zählt daher vermutlich
zum ersten Sensorkinase-Typ und ein extracytoplasmatischer Loop könnte die Registrierung
periplasmatischer Stimuli ermöglichen. Es ist allerdings nicht auszuschließen, dass die PASDomäne ein zusätzliches Sensormodul darstellt. So zeigt ResE aus B. subtilis eine zu NtrY
vergleichbare Domänenstruktur und registriert Reize sowohl über einen extrazellulären Loop
als auch über eine intrazelluläre PAS-Domäne (Baruah et al., 2004).
L
Periplasma
T T T T
H
Mem bran
Cytoplasm a
P
K
Abb. 12: Domänenstruktur der Sensorkinase NtrY
aus R. capsulatus. Die über Sequenzvergleiche identifizierten NtrY-Domänen sind schematisch dargestellt.
NtrY ist ein putatives Transmembranprotein mit vier Nterminalen hydrophoben Helices (T). Die Signalaufnahme erfolgt entweder über einen periplasmatischen Loop
(L) oder eine cytoplasmatische PAS-Domäne (P), beide
jeweils in rot dargestellt. Die Kinasedomäne (K) dient
dem Signaloutput. HAMP-Linker (H) fungieren als Signalübertragungsmodul zwischen Input- und Outputdomänen.
Da das ure-Operon sowohl im R. capsulatus Wildtyp als auch in einer ntrB-Mutante der
Stickstoff-Regulation unterlag, dient NtrY vermutlich wie NtrB der Registrierung des Stickstoff-Status (Drepper et al., 2006). Es ist jedoch fraglich, welches der beiden Sensormodule
dies leistet. Da die Kommunikation eines Responseregulators mit zwei Sensorkinasen der
Steigerung des Informationsinputs dient, ist anzunehmen, dass NtrY den periplasmatischen
Stickstoff-Status z. B. in Form von Ammonium überwacht. Davon ausgehend ließe sich zudem eine im Vergleich zur Phosphorylierungs-Aktivität gesteigerte DephosphorylierungsAktivität von NtrY physiologisch erklären. Bei niedriger periplasmatischer AmmoniumKonzentration und hoher cytoplasmatischer Stickstoff-Verfügbarkeit überwiegt die Dephosphorylierung von NtrC durch NtrB. Hierdurch wird zunächst der intrazellulär gebundene
Stickstoff genutzt, bevor energieaufwendige Reaktionen eingeleitet werden. Steigt die periplasmatische Ammonium-Konzentration hingegen bei einer niedrigen cytoplasmatischen
92
Diskussion
Stickstoff-Verfügbarkeit an, überwiegt die Dehosphorylierung von NtrC durch NtrY. Dies
unterbindet frühzeitig die Synthese von Enzymen zur Nutzung alternativer Stickstoff-Quellen.
Die bezüglich des „cross-talks“ zwischen NtrY und NtrC gewonnen Erkenntnisse sind in Abbildung 13 zusammengefasst.
+N
-N
NtrY
Periplasma
NtrY
Cytoplasma
P
NtrC
NtrC
NtrC
NtrC
P
P
NtrB
NtrB
-N
+N
P
GlnB
+
+
ure
nif
ure
nif
Abb. 13: Modell zur Rolle von NtrY bei der Stickstoff-abhängigen Genregulation in R. capsulatus. Der periplasmatische Stickstoff-Status (- N: Mangel; + N: Verfügbarkeit) wird über die putative
Transmembran-Sensorkinase NtrY registriert. Durch „cross-talk“ erfolgt die Signaltransduktion auf
NtrC. NtrY kann NtrB nicht vollständig als Phospho-Donor ersetzen (dünner Pfeil), scheint jedoch
eine ausgeprägtere Dephosphorylierungs-Aktivität (dicker Pfeil) aufzuweisen. NtrC-P aktiviert (+) die
Transkription von ure- und nif-Genen.
1.3
Der Responseregulator NtrX
Der Responseregulator NtrX ist in eine N-terminale Regulatordomäne, eine zentrale AAA+Typ ATPase-Domäne und eine C-terminale Fis-Typ DNA-Bindedomäne unterteilt und zählt
als Vertreter der NtrC-Familie zu den σ54-abhängigen Transkriptionsregulatoren (Fischer,
1994). Die Phosphorylierung von NtrX durch NtrY wurde bereits für das entsprechende TCS
aus Ehrlichia chaffeensis nachgewiesen (Kumagai et al., 2006). Daher unterliegt NtrX in
R. capsulatus vermutlich ebenfalls der Modifikation durch NtrY, wobei das in allen NtrX
konservierte Asp52 die putative Phosphorylierungsstelle ist (Ishida et al., 2002). Wie bereits
diskutiert ist hierbei vermutlich der periplasmatische Stickstoff-Status entscheidend. Davon
ausgehend steuert NtrX wahrscheinlich die Expression von Genen, welche direkt oder indi93
Diskussion
rekt am Stickstoff-Metabolismus beteiligt sind. So sind NtrX/NtrY in A. caulinodans an der
Transkriptionsaktivierung von nifA beteiligt (Pawlowski et al., 1991). In Azospirillum brasilense hingegen scheint NtrX Gene zu aktivieren, welche die Nutzung alternativer StickstoffQuellen wie Nitrat ermöglichen (Assumpcao et al., 2007; Ishida et al., 2002). Bei R. capsulatus jedoch ist NtrC absolut essentiell für die Nutzung von molekularem Stickstoff und Harnstoff als Stickstoff-Quellen und kann nicht durch NtrX ersetzt werden (Drepper et al., 2006).
Zudem wies eine ntrX-Mutante auf Vollmedium einen deutlichen Wuchsdefekt auf und die
durch NtrX regulierten Gene scheinen somit von globalerer Bedeutung zu sein. Interessanterweise zeigte weder eine ntrY-Mutante noch eine ntrB/ntrY-Doppelmutante einen vergleichbaren Phänotyp. Es lässt sich daher spekulieren, dass NtrX mit einer alternativen Sensorkinase
interagiert, die NtrY als Phospho-Donor ersetzen kann.
2.
Die Molybdat-abhängigen Regulatoren MopA und MopB
2.1
MopA und MopB regulieren den Molybdat-Metabolismus
In verschiedenen Prokaryoten erfolgt die Molybdat-abhängige Genregulation durch Vertreter
der ModE-Familie. Diese Regulatoren wurden bisher in E. coli und Azotobacter vinelandii,
welche jeweils über ein ModE-Protein verfügen, näher charakterisiert (Mouncey et al., 1995,
1996; Rosentel et al., 1995). Im Gegensatz dazu kodiert R. capsulatus für zwei ModEhomologe Proteine, MopA und MopB. Diese sind auf Sequenzebene zu 32 % (MopA) bzw.
33 % (MopB) mit E. coli ModE identisch und lassen sich entsprechend in eine N-terminale DNA-Bindedomäne und eine C-terminale Molybdat-bindende di-mop-Domäne unterteilen. MopA und MopB konnten sich als Molybdat-abhängiger Repressoren verschiedener Gene ersetzen (Kutsche et al., 1996; Wiethaus et al., 2006b). Diese kodieren für MopA selbst,
den Transkriptionsaktivator der alternativen Nitrogenase-Gene AnfA, den hoch affinen Molybdat-Transporter ModABC und den putativen ABC-Transporter MorABC. Daneben aktivierte spezifisch MopA in Anwesenheit von Molybdat die Expression des mop-Gens, welches
für ein putatives mono-mop-Molbindin kodiert. Somit stimmen MopA und MopB die Expression verschiedener Gene des Molybdat-Metabolismus von R. capsulatus auf die MolybdatVerfügbarkeit ab.
Eine physiologische intrazelluläre Metall-Konzentration wird vermutlich vornehmlich durch
die Expression der modABC-Gene unter Molybdat-Mangel und des mop-Gens unter Molyb94
Diskussion
dat-Verfügbarkeit erzielt. Hierdurch wird einerseits die Molybdat-Aufnahme durch den hoch
affinen ModABC-Transporter bei Bedarf induziert. Andererseits weist Mop Homologien zu
putativen Molybdat-Speicherproteinen aus Clostridium pasteurianum und Sporomusa ovata
auf (Harrison et al., 2001; Schüttelkopf et al., 2002; Wagner et al., 2000). Diese mono-mopMolbindine binden acht Molybdat pro Hexamer, wobei zwei Oxyanionen durch Typ1- und
sechs durch Typ2-Bindestellen koordiniert werden. Da diese Bindestellen unter Beteiligung
von drei (Typ1) bzw. zwei (Typ2) mop-Domänen gebildet werden, geht der MolybdatBindung jeweils die Oligomerisierung des Proteins voraus (Wagner et al., 2000). Sowohl die
Typ2-Bindestelle als auch das an der Typ1-Bindung beteiligte Valin sind in der mono-mopDomäne von R. capsulatus Mop konserviert (Abb. 14). Zudem erfolgte die Formierung des
Mop-Hexamers Molybdat-unabhängig. Das Hexamer wurde jedoch durch Zugabe von Molybdat stabilisiert, was auf die Bindung des Oxyanions hindeutet. Mop dient daher vermutlich
der Speicherung von Molybdat und die mop-Expression wird entsprechend durch MopA in
Überschusssituationen aktiviert.
Das mopA-modABCD-Operon sowie das anfA-Gen unterliegen neben der Molybdat-abhängigen Repression durch MopA bzw. MopB zusätzlich der Stickstoff-abhängigen Aktivierung
durch NtrC (Kutsche et al., 1996). Diese Verbindung von Stickstoff- und Molybdat-Regulation dient der Sicherung einer möglichst hohen Stickstoff-Fixierungsrate. Die Molybdän-Ni-
S R
35 Typ2
MRTSNRNTLRCTVTRVTLGAVNAEVELALTDGHSL
GRISACNRLTGIVAARTDGPVNTEIILDLGNCKSI
LRTSARNAWACKVWSVAADDVAAQVRMRLGEGQDL
ERLSVRNRLRGRVIERIDAPLSSEVTLDLGGGKTI
MKLSARNILAGKVTAVETGNVTTHVKIDIG-GTVV
XVX
Typ1
1
MopA1
MopA2
MopB1
MopB2
Mop
S A
S
T S
KA
69
TAVITERSATEMGLAPGVEVFALIKASFVMLAAG
TAVITHTSADALGLAPGVPATALFKASHVILAMP
TAVITARSAAEMRLAPGSEVLALVKSNFVLLAGA
TATITRDSAEMLDLHPGVETTALIKSSHVILALP
TASITNEAAADLALKVGDEACAIIKASDVIVGKN
36
MopA1
MopA2
MopB1
MopB2
Mop
Typ2
Abb. 14: Sequenzvergleich der mop-Domänen von R. capsulatus MopA, MopB und Mop. Die bei
Molbindinen an der Molybdat-Bindung beteiligten Aminosäure-Reste (Typ1, Typ2) sind angegeben
(X: nicht konserviert). Typ2-Bindestellen sind in den di-mop-Domänen von MopA (MopA1, MopA2)
und MopB (MopB1, MopB2) und in der mono-mop-Domäne von Mop konserviert (grün). Das Valin
der lediglich durch Molbindine gebildeten Typ1-Bindestelle ist in Mop enthalten (grün). Das SARNMotiv ist durch einen Kasten gekennzeichnet.
95
Diskussion
trogenase macht unter Stickstoff-Mangel große Mengen des löslichen Zellproteins aus (Masepohl et al., 1988). Der enorme Molybdat-Bedarf des Enzymsystems wird durch die NtrCabhängige Aktivierung der modABC-Gene gedeckt. Lediglich bei gleichzeitigem Stickstoffund Molybdat-Mangel erfolgt die Stickstoff-Fixierung über die alternative Nitrogenase, welche eine wesentlich geringere Aktivität als die Molybdän-Nitrogenase aufweist (Masepohl et
al., 2002b).
Die genaue Funktion des putativen MorABC-Transporters ist unklar. Auf Grund der Molybdat-abhängigen Repression der mor-Gene durch MopA bzw. MopB ist die direkte oder indirekte Beteiligung von MorABC am Molybdat-Metabolismus denkbar. Da eine Deletion der
morABC-Genregion die Aktivität der Molybdän-Nitrogenase nicht beeinflusste, kann eine
Funktion als Molybdat-Transporter jedoch ausgeschlossen werden (Wiethaus et al., 2006b).
Zudem weist MorC im Gegensatz zu ModC keine C-terminale mop-Domäne auf. Dennoch
deuten die vorhandenen Sequenzhomologien darauf hin, dass es sich bei MorABC um ein
funktionelles ABC-Transportsystem handelt. So verfügt MorA über eine extrazelluläre Metall-Bindedomäne, welche ebenfalls in Molybdat-, Sulfat- und Eisen(III)-bindenden periplasmatischen Proteinen verschiedenster ABC-Transporter vorliegt (Anderson et al., 2004).
2.2
MopA und MopB sind DNA-bindende Regulatoren
Wie bereits erwähnt, weisen MopA und MopB N-terminale DNA-Bindedomänen mit wHTHMotiv auf. Dementsprechend konnte die Bindung von MopA und MopB an negativ regulierte
Promotoren gezeigt werden (Wiethaus et al., 2006b). Der mop-Promotor hingegen wurde lediglich durch MopA, dem alleinigen Aktivator der mop-Expression, gebunden. Bei E. coli
ModE sind vermutlich neun Aminosäure-Reste innerhalb des wHTH-Motivs entscheidend für
die DNA-Sequenzerkennung und –Interaktion (Hall et al., 1999; McNicholas et al., 1998b).
Fünf dieser Aminosäure-Reste sind in der N-terminalen DNA-Bindedomäne ModE-ähnlicher
Proteine konserviert und dienen vermutlich der Anlagerung an gleichartige DNA-Sequenzen
(Abb. 15 A) (Studholme & Pau, 2003). Entsprechend sind in den Promotorbereichen MopAund MopB-regulierter Gene so genannte Mo-Boxen lokalisiert (Abb. 15 B) (Kutsche et al.,
1996). Diese palindromisch organisierten Sequenzen weisen Homologien zu charakterisierten
und putativen DNA-Bindestellen ModE-homologer Proteine auf (Rodionov et al., 2004). Die
Lage der einzelnen R. capsulatus Mo-Boxen relativ zum Transkriptionsstartpunkt ermöglicht
96
Diskussion
A
Ec
Av
Hi
Rc
Rc
B
Konsensus
anfA
mopA-mod
morA
morC
mop
ModE
ModE
ModE
MopA
MopB
** *
**** **
SQGAKDAGISYKSAWD
NRAAKVVPLSYKAAWD
NQAAKNAKVSYKSAWD
AGAAREVGLSYKTAWD
SAAAREVGLSYKAAWD
ATCGCTATATA-N6 -TATATAACGAT
GTCGTTATATG-N7 -TATATAACGGA
ATCGCTATTAA-N7 -TATATAACGAT
TTCGCTATAAG-N7 -TACATAGCGAC
GTCGCTATGTA-N7 -CTTATAGCGAA
GCCGATATGTG-N7 -CTCATATTGAA
Abb. 15: Konservierte Elemente Molybdat-abhängiger Regulatoren und ihrer DNA-Bindestellen. (A) Sequenzvergleich eines Abschnitts des wHTH-Motivs ModE-homologer Proteine. Die mit
einem Stern gekennzeichneten Aminosäure-Reste von E. coli (Ec) ModE sind vermutlich an der
DNA–Interaktion beteiligt. Diese sind zum Teil in homologen Proteinen aus A. vinelandii (Av), Haemophilus influenzae (Hi) und R. capsulatus (Rc) konserviert (grün). (B) Sequenzvergleich der R.
capsulatus Mo-Boxen. Mit dem palindromisch organisierten Konsensus (Studholme & Pau, 2003)
übereinstimmende Basen sind in grün dargestellt. Die an der jeweiligen Position lediglich in der mopMo-Box vorhandenen Basen sind unterstrichen, und, falls die Basen der übrigen Mo-Boxen hier
durchgängig konserviert sind, fett dargestellt.
die optimale Positionierung von MopA und MopB als Repressor oder Aktivator (Wiethaus et
al., 2006b). So sind die Transkriptionsstartpunkte reprimierter Gene innerhalb oder stromaufwärts der Mo-Box lokalisiert, wodurch die Regulator-Bindung mit der Transkriptionsinitiation oder –elongation interferiert. Die Mo-Box des durch MopA aktivierten mop-Gens hingegen ist an Position – 43 relativ zum Transkriptionsstart zentriert. Dies positioniert MopA in
räumliche Nähe zur RNA-Polymerase und könnte somit die Transkriptionsinitiation erleichtern. Analog differieren die Positionen von E. coli ModE-Bindestellen zwischen reprimierten
und aktivierten Genen. So überlappt die ModE-Bindestelle den Transkriptionsstart des negativ regulierten modABCD-Operons (Anderson et al., 1997; Grunden et al., 1999). Die MoBox des positiv regulierten moa-Operons hingegen ist an Position - 55 relativ zum Transkriptionsstart lokalisiert (Anderson et al., 2000).
Wie bereits erwähnt, bindet ausschließlich MopA als Transkriptionsaktivator an den mopPromotor. Entsprechend der Sonderfunktion als exklusive MopA-Bindestelle ist die Sequenz
der mop-Mo-Box nur zu 50 % mit der Konsensussequenz identisch. Die Mo-Boxen der durch
MopA und MopB reprimierten Gene hingegen stimmen weitestgehend mit dem Konsensus
überein. Vergleicht man die R. capsulatus Mo-Boxen untereinander, so weicht die mop-Mo97
Diskussion
Box lediglich in vier Basen von den übrigen Sequenzen ab, wobei zwei dieser Basen sonst
durchgängig konserviert sind. Diese könnten somit für die Spezifität entscheidend sein und
die mop-Mo-Box zur optimierten MopA-Bindestelle machen. Dementsprechend zeigte MopA
im Vergleich zu MopB eine verringerte Bindeaffinität an gemeinsam regulierte Promotoren
(Wiethaus et al., 2006b). Dies ließ sich insbesondere bei dem mopA-modABCD-Promotor
beobachten, dessen Mo-Box die größte Übereinstimmung mit dem Konsensus sowie die
stärkste Abweichung von der mop-Mo-Box aufweist. Durch Mutationsanalysen der MoBoxen und anschließende Gelshiftanalysen sollen die für die MopA- bzw. MopB-Bindung
spezifischen Basen in Zukunft identifiziert werden.
2.3
Molybdat erhöht die DNA-Affinität von MopA- und MopBDimeren
Metall-Regulatoren sind in der Regel in oligomerer Form funktionell, wobei es entweder zur
Dimer- oder zur Tetramer-Formierung kommt (Pennella & Giedroc, 2005). Ist die DNABindestelle wie im Fall der Mo-Boxen palindromisch organisiert, deutet dies auf einen dimeren Regulator hin. Entsprechend bildeten MopA und MopB stabile Homodimere aus (Wiethaus et al., eingereicht). Zudem werden die R. capsulatus Mo-Boxen anscheinend durch Dimere und nicht etwa sequentiell durch zwei Monomere besetzt, da in Gelshiftexperimenten
nach Protein-Zugabe lediglich eine zusätzliche Bande auftrat. Obwohl die Dimerisierung von
MopA und MopB Molybdat-unabhängig erfolgte, konnte die DNA-Bindeaffinität beider Regulatoren durch Molybdat signifikant gesteigert werden (Wiethaus et al., 2006b). Das Oxyanion wird vermutlich über die C-terminalen di-mop-Domänen beider Regulatoren gebunden,
was die Interaktion mit den Mo-Boxen positiv beeinflusst. So sind die Aminosäure-Reste der
Typ2-Molybdat-Bindestellen von Molbindinen bei MopA und MopB konserviert (Abb. 14).
Zwei dieser Aminosäure-Reste sind im so genannten SARN-Motiv lokalisiert, welches bei
E. coli ModE entscheidend für die Molybdat-Bindung sein könnte (McNicholas et al., 1996;
Grunden et al., 1996). Da sowohl die Typ2-Bindestelle, als auch das SARN-Motiv bei R. capsulatus MopA und MopB konserviert sind, binden beide Regulatoren höchstwahrscheinlich
Molybdat. Sowohl Molbindine als auch E. coli ModE bilden durch die Interaktion zweier dimop-Domänen zwei Typ2-Bindestellen aus (Wagner et al., 2000; Gourley et al., 2001). Da
MopA- und MopB-Dimere ebenfalls über zwei di-mop-Domänen verfügen, kommt es vermutlich zur Bindung von zwei Molekülen Molybdat pro Dimer. Auf Grund der vorhandenen
98
Diskussion
Sequenzhomologien kann hierbei von einem zu E. coli ModE vergleichbaren Mechanismus
ausgegangen werden. So führt die Bindung von zwei Molekülen Molybdat pro ModE-Dimer
zu massiven Konformationsänderungen innerhalb des Proteins, welche nicht nur die Molybdat-bindende sondern auch die DNA-bindende Domäne betreffen (Boxer et al., 2004; Schüttelkopf et al., 2003). Von besonderer Bedeutung ist hierbei die relative Neuorientierung der
beiden wHTH-Motive, welche anscheinend eine verbesserte DNA-Interaktion des aktivierten
Mo-ModE im Verhältnis zum Apo-ModE bedingen. MopA und MopB lagerten sich an einige
Promotoren auch ohne Zugabe von Molybdat an. Verschiedene E. coli Promotoren werden
ebenfalls durch Molybdat-freies ModE gebunden, wobei Molybdat-Zugabe die DNABindeaffinität jeweils signifikant steigert (McNicholas et al., 1997, 1998a; 2002; Self et al.,
1999). Hierbei interagiert anscheinend jede ModE-Untereinheit mit einer der beiden palindromischen Sequenzen (Hall et al., 1999).
Interessanterweise konnte die Formierung von MopA- und MopB-Heterodimeren gezeigt
werden, wobei deren Funktion in der Molybdat-abhängigen Genregulation von R. capsulatus
unklar ist (Wiethaus et al., in Vorbereitung). Da jedoch MopA- und MopB-Homodimere
offenbar als Molybdat-abhängige, DNA-bindende Regulatoren fungieren, gilt dies auf Grund
der signifikanten Sequenzhomologien beider Proteine vermutlich ebenso für MopA/MopBHeterodimere. Wie bereits diskutiert, differieren die DNA-Bindeaffinitäten von MopA und
MopB. So wurden negativ regulierte Promotoren effizienter durch MopB und der positiv regulierte mop-Promoter ausschließlich durch MopA gebunden. Die Kombination aus einem
MopA wHTH- und einem MopB wHTH-Motiv bedingt vermutlich ein intermediäres DNABindeverhalten des Heterodimers. Es kann zudem nicht ausgeschlossen werden, dass
MopA/MopB-Heterodimere nicht an Mo-Boxen binden können. Da mopA im Gegensatz zu
mopB sowohl der Reprimierung durch MopA bzw. MopB als auch der Aktivierung durch
NtrC unterliegt, wird das MopA/MopB-Verhältnis durch die intrazelluläre Molybdat- und
Stickstoff-Verfügbarkeit bestimmt. So lässt sich spekulieren, dass unter Molybdat- und Stickstoff-Mangel vornehmlich MopA/MopB- und MopA/MopA-Dimere ausgebildet werden.
Ausgehend von einem intermediären Bindeverhalten wäre hiervon insbesondere das mopAmodABCD-Operon betroffen, da der entsprechende Promotor mit besonders geringer Effizienz durch MopA gebunden wurde. Daher könnte die Heterodimer-Formierung die Repression des Operons bei höheren Molybdat-Konzentrationen bedingen. Somit wäre sichergestellt,
dass alle Molybdat-verwertenden Systeme wie die Molybdän-Nitrogenase und das putative
Speicherprotein Mop abgesättigt sind, bevor die Molybdat-Aufnahme gestoppt wird. Die
Formierung von Heterodimeren dient somit vermutlich der Feinabstimmung der Molybdat99
Diskussion
abhängigen Genregulation. Dies soll durch weiterführende Studien der mopA-modABCDExpressionsprofile im Wildtyp, in einer mopA- und einer mopB-Mutante unter steigenden
Molybdat-Konzentrationen geklärt werden. Die Repression des Operons in einer mopAMutante bei höheren und in einer mopB-Mutante bei geringeren Molybdat-Konzentrationen
im Vergleich zum Wildtyp würde die aufgestellte Hypothese unterstützen. Abbildung 16 zeigt
ein Model zur Molybdat-abhängigen Genregulation in R. capsulatus.
Mo
Mo
Mo
MopA
MopA
MopB
mopB
mopA
modABCD
MopA
Mo
Mo
MopB
MopA
Mo
MopB
Mo
Mo
MopA
MopB
Mo
MopA
?
Mo
Mo-aktiviert
Mo-aktiviert
Mo-reprimiert
Mo-reprimiert
?
+
mop
const.
anfA
morC
morAB
Abb. 16: Molybdat-abhängige Genregulation durch MopA und MopB in R. capsulatus. MopA
und MopB binden vermutlich zwei Moleküle Molybdat pro Dimer. Dies erhöht die Bindeaffinität an
konservierte Mo-Boxen (schwarze Kästen) im Promotorbereich von Molybdat-regulierten Genen.
Ferner sind vermutlich MopA/MopB-Heterodimere an der Molybdat-abhängigen Genregulation beteiligt. Die Positionen der Transkriptionsstartpunkte (Pfeile) relativ zu den Mo-Boxen differieren zwischen reprimierten (-) und aktivierten (+) Genen. const: konstitutive Expression.
2.4
MopA und MopB: mehr als nur Regulatoren
Die Funktion von MopA und MopB im Rahmen des Molybdat-Metabolismus von R. capsulatus scheint nicht auf die Molybdat-abhängige Genregulation beschränkt zu sein. So interagierten beide Proteine mit dem putativen Moco-Biosynthese Protein MogA in Yeast Two-HybridStudien (Wiethaus et al., in Vorbereitung). In E coli katalysieren MogA und MoeA die Incor100
Diskussion
poration von Molybdän in den Moco-Precursor (Nichols & Rajagopalan, 2002). Hierbei wird
die durch MoeA vermittelte Molybdän-Insertion durch MogA ATP-abhängig stimuliert (Nichols & Rajagopalan, 2005; Nichols et al., 2007). Auch in R. capsulatus ist MoeA an der Moco-Biosynthese beteiligt (Leimkühler et al., 1999). Im Gegensatz zu E. coli scheint MoeA
jedoch nicht essentiell für die Moco-Synthese zu sein. So kann in einer moeA-Mutante die
Wildtyp-Aktivität des Molybdoenzyms Xanthin-Dehydrogenase unter hohen extrazellulären
Molybdat-Konzentrationen wiederhergestellt werden. Die Funktion von R. capsulatus MogA
ist nicht bekannt. Die Interaktionen mit MopA und MopB deuten allerdings darauf hin, dass
Molybdat über MogA in die Moco-Biosynthese eingebracht wird (Abb. 17). Ob Molybdat
zuerst auf MoeA übertragen wird, oder ob Molybdän durch MogA direkt in den Cofaktor inseriert wird, ist unklar. Eine weitere Molybdat-Quelle für die Moco-Biosynthese scheint die
ATPase ModC zu sein, wobei diese ebenfalls mit MogA und nicht mit MoeA interagierte.
MoeA sowie MogA bildeten Homomere. In Analogie zu den entsprechenden E. coli Proteinen
ist die Formierung eines Dimers (MoeA) bzw. eines Trimers (MogA) denkbar (Liu et al.,
2000; Schrag et al., 2001; Xiang et al., 2001).
Es konnte nicht geklärt werden, wie Molybdat in die FeMoco-Synthese eingebracht wird.
NifQ weist ein typisches Metall-bindendes Cystein-Motiv auf und ist in verschiedenen Bakterien an der Synthese des FeMoco beteiligt (Allen et al., 1994; Moreno-Vivian et al., 1989;
Rodriguez-Quinones et al., 1993). Es wird vermutet, dass NifQ die Molybdän-Incorporation
in den Eisen-Schwefel-Precursor katalysiert. NifQ interagierte jedoch mit keinem der untersuchten Proteine des Molybdat-Metabolismus (Wiethaus et al., in Vorbereitung). Das Ausbleiben von Protein-Protein-Interaktionen im Yeast Two-Hybrid-System schließt die Interaktion in R. capsulatus allerdings nicht kategorisch aus. So kann sich u. a. die Fusion mit der
Aktivierungsdomäne des Yeast Two-Hybrid-Systems negativ auf die Protein-Protein-Interaktion auswirken. Zur Aufklärung der Molybdat-Einspeisung in die FeMoco-Biosynthese sollen
weitere Interaktionstudien mit NifQ in alternativen Testsystemen durchgeführt werden. Hier
sind u. a. Biacore-, Crosslinking- und Coreinigungsexperimente zu nennen.
Überraschenderweise konnte keine Interaktion von ModC mit ModC, MopA, MopB oder
Mop nachgewiesen werden (Wiethaus et al., in Vorbereitung). ModC-Proteine verfügen über
eine C-terminale mop-Domäne (Pau et al., 1997). Daher kann lediglich durch Interaktion mit
der mop-Domäne eines zweiten Proteins eine Molybdat-Bindestelle ausgebildet werden (Pau,
2003). Wie für ABC-Transporter typisch weist der putative Molybdat-Transporter aus Ar-
101
Diskussion
MopA/MopB
ModA
Periplasma
ModB
Membran
ModC
Cytoplasma
MopA
MopB
Mop
?
?
NifQ
FeSPrecursor
MogA
FeMoco
MPT
MoeA
Moco
Abb. 17: Modell zur Molybdat-Einspeisung in die Moco-Biosynthese von R. capsulatus. Molybdat
könnte direkt über ModC und indirekt über Mop, MopA und MopB an MogA weitergeleitet werden.
MogA bringt Molybdat in die Moco-Synthese ein. Die Inkorporation von Molybdän in das Molybdopterin-Molekül (MPT) könnte durch MogA selbst oder MoeA katalysiert werden. Wie die Versorgung
der FeMoco-Synthese mit Molybdat gewährleistet wird, ist unklar. Eine genaue Erläuterung des Modells erfolgt im Text.
chaeoglobus fulgidus eine dimere ATPase auf (Hollenstein et al., 2007; Tomii & Kanehisa,
1998). Folglich könnten zwei ModC-Moleküle eine Molybdat-Bindestelle ausbilden. Strukturell ist zudem eine Interaktion von ModC mit ModE-Proteinen wahrscheinlich (Pau, 2003).
So sind ModE-Proteine im Gegensatz zu Molbindinen nicht aus drei sondern aus zwei dimop-Domänen aufgebaut. Somit ist die Anlagerung einer dritten di-mop-Domäne denkbar.
Diese Domäne könnte durch ModC aber auch durch Molbindine gestellt werden (Pau, 2003).
Interessanterweise wurde die Interaktion von MopB und Mop aus R. capsulatus nachgewiesen. Es ist jedoch unklar, ob es tatsächlich zur Anordnung von insgesamt sechs heterogenen
mop-Domänen kommt und in welchem Verhältnis MopB und Mop im Heteromer vorliegen.
Die physiologische Bedeutung von MopB/Mop-Heteromeren erschließt sich wohlmöglich aus
der Interaktion von MopB mit dem Moco-Biosyntheseprotein MogA. So könnte Mop gespeichertes Molybdat über MopB der Cofaktor-Biosynthese zuführen.
Das Modell zur Molybdat-Einspeisung in die Cofaktor-Biosynthese muss durch weiterführende Experimente erhärtet bzw. erweitert werden. Vor allem Molybdat-Gehalt und –Bindekonstanten der beteiligten Proteine sollten über ICP-MS (inductively coupled plasma mass
102
Diskussion
spectrometry) und ITC (isothermal titration calorimetry) bestimmt werden. Hierdurch kann
geklärt werden, ob die postulierte Molybdat-Weitergabe von einem zum anderen Protein prinzipiell möglich ist.
3.
Der neuartige Kupfer-abhängige Regulator CutR
Eine gut regulierte Kupfer-Homöostase muss den physiologischen Kupfer-Bedarf sichern und
die intrazelluläre Akkumulierung toxischer Cu(I)-Ionen vermeiden. In Bakterien werden entsprechende Homöostase-Mechanismen auf genetischer Ebene durch Kupfer-abhängige Regulatoren an die Bioverfügbarkeit des Metalls angepasst. In R. capsulatus wurde CutR als Kupfer-abhängiger Regulator des orf635-cutOR-Operons identifiziert (Wiethaus et al., 2006a).
Die Expression des Operons wurde im Wildtyp durch Kupfer induziert. Bei konstitutiver
Transkription des cutR-Gens wurde die Expression auch in Gegenwart von Kupfer weitgehend reprimiert. Wurde die Transkription des cutR-Gens jedoch unterbunden, führte dies zu
einer hohen Expression des cut-Operons auch in Abwesenheit von Kupfer. Somit handelt es
sich bei CutR um einen Repressor des orf635-cutOR-Operons, wobei Kupfer-Verfügbarkeit
zur Derepression des Operons führt.
Das cutO-Gen kodiert für eine Multicopper-Oxidase, welche Kupfer-Toleranz vermittelt.
Dem orf635-Genprodukt konnte bislang keine Funktion zugeordnet werden. Auf Grund der
Kupfer-abhängigen Regulation ist allerdings eine Beteiligung an der Kupfer-Homöostase
denkbar. Zudem weist das putative Membran-assoziierte Protein ORF635 mehrere Cysteinund Histidin-Reste auf, welche die Bindung von Cu(I)-Ionen ermöglichen könnten (Adman,
1991; Abolmaali et al., 1998).
CutR zählt zur bislang uncharakterisierten DUF411-Proteinfamilie, deren Vertreter mit der
Vermittlung von Resistenzen gegenüber verschiedenen Kationen in Verbindung gebracht
werden. Als putatives Metall-Bindeprotein verfügt CutR über vier Cystein- und zwei HistidinReste, welche in CutR-homologen Proteinen konserviert sind (Abb. 18 A). CutR könnte folglich durch direkte Cu(I)-Bindung als Sensor des intrazellulären Kupfer-Status fungieren. Interessanterweise zählt CutR zu den CopG-ähnlichen Proteinen. CopG ist ein homodimerer Repressor der „ribbon-helix-helix“-Familie, welcher durch direkte DNA-Bindung die Kopienzahl von streptococcalen Plasmiden kontrolliert (Costa et al., 2001; Gomis-Rüth et al., 1998).
Dies deutet darauf hin, dass CutR die Expression seiner Zielgene durch Interaktion mit der
103
Diskussion
A
1
****
MTKHDLSRRTVLALVAGCLASAPLRAAAPVAITVVKDPDCGCCEAWIDILRA
*****
*****
DGFAVTTQVIDYDALQALKGQSGIPEPMRSCHTARVEGYVIEGHVPPADIRR
LLAERPAALGLAVPGMPLGAPGMGPEDQREAYDVHLITADGQTRIFAHYDAA
1 56
B
CCCGGTCAGCACCAGCGCGAGATCCGGGGCTGACCGTGATGCCGCGGCGTCATG
Abb. 18: Der Regulator CutR aus R. capsulatus und seine putative DNA-Bindestelle. (A) Drei
Cystein- bzw. Histidin-haltige Regionen sind in CutR-homologen Proteinen konserviert und mit einem
Stern gekennzeichnet. Die bei R. capsulatus CutR konservierten Cystein- und Histidin-Reste sind blau
dargestellt. (B) Stromaufwärts des orf635-Startcodons (ATG) ist eine palindromisch organisierte
DNA-Sequenz lokalisiert (blau).
Promotor-DNA beeinflusst. Unmittelbar stromaufwärts des putativen Translationsstarts des
orf635-Gens ist eine nahezu perfekte palindromische DNA-Sequenz von je 11 Basenpaaren
lokalisiert (Abb. 18 B). In Übereinstimmung mit der Repressor-Funktion von CutR würde die
Bindung eines Regulators an dieser Position die Transkription des orf635-cutOR-Operons
unterbinden. Die palindromische Organisation der putativen Bindestelle lässt vermuten, dass
CutR in dimerer Form bindet. Entsprechend könnte die Bindung von Cu(I)-Ionen die DNAAffinität des CutR-Dimers reduzieren (Abb. 19). Dies würde die Ablösung vom orf635cutOR-Promotor und somit die Derepression der regulierten Gene bewirken. Ein vergleichbarer Regulationsmechanismus ist bei CopY aus Enterococcus hirae und CsoR aus Mycobacterium tuberculosis realisiert (Liu et al., 2007; Strausak & Solioz, 1997). Es ist jedoch fraglich,
ob die Kupfer-Bindung lediglich die DNA-Affinität beeinflusst oder auch die Dimerisierung.
So ist nicht auszuschließen, dass ein CutR-Dimer durch Kupfer-Bindung in zwei Monomere
dissoziiert. Die genaue Funktionsweise von CutR als Transkriptionsregulator soll in weiterführenden Studien geklärt werden. Grundlegend hierbei sind Gelshiftanalysen mit dem
orf635-cutOR-Promotor. Ortspezifische Mutationen der palindromischen DNA-Sequenz sollen die Bindestelle weiter einschränken. Zudem sollte durch Gelfiltration die Oligomerisierung von CutR in Abhängigkeit von Kupfer geklärt werden.
CutR-homologe Proteine sind in Proteobakterien weit verbreitet, wobei cutR-Gene in P. putita, Ralstonia eutropha und Vibrio fischeri in räumlicher Nähe zu Kupfer-Resistenz-Genen
lokalisiert sind. So ist bei V. fischeri unmittelbar stromaufwärts des cutR-ähnlichen Gens eine
104
Diskussion
putative Kupfer-Efflux P-Typ ATPase kodiert (Ruby et al., 2005). In Enterobakterien wie
E. coli, Klebsiella pneumonia und Salmonella typhimurium hingegen sind cutR-ähnliche Gene
in Silber-Resistenz-Operons organisiert. Es lässt sich daher spekulieren, dass CutR-homologe
Proteine als Regulatoren verschiedener Resistenzmechanismen gegenüber Kationen fungieren. R. capsulatus CutR ist der erste näher charakterisierte Vertreter dieser Gruppe.
[Cu] niedrig
[Cu] niedrig
Cu
?
CutR
Cu
CutR
CutR
CutR
CutR
Cu
[Cu]hoch
hoch
[Cu]
orf635
cutO
cutR
Abb. 19: Model zur Kupfer-abhängigen Genregulation durch CutR in R. capsulatus. CutR reprimiert (-) die Expression des orf635-cutOR-Operons in Abhängigkeit von der Kupfer-Konzentration
([Cu]). Welchen Einfluss die vermutliche Bindung von Cu(I)-Ionen auf CutR hat ist unklar (?). Eine
genaue Erläuterung des Models erfolgt im Text.
4.
TauR, der chimäre Aktivator der Taurin-Assimilation
Sulfonate werden von verschiedenen Bakterien als alternative Schwefel-Quelle genutzt. So
assimiliert R. capsulatus unter phototrophen Bedingungen effektiv Taurin-Schwefel (Masepohl et al., 2001a). Im Rahmen dieser Arbeit wurde TauR als Taurin-abhängiger Aktivator
des tpa-tauR-xsc-Operons identifiziert (Schubert et al., in Vorbereitung). So ließ sich die Taurin-Induktion des tpa-Gens im Wildtyp, nicht aber in einer tauR-Mutante beobachten. Die
Gene tpa und xsc kodieren für die putativen Enzyme Taurin:Pyruvat-Aminotransferase (Tpa)
und Sulfoacetaldehyd-Acetyltransferase (Xsc), welche vermutlich die anaerobe Taurin-Assimilation in R. capsulatus einleiten (Masepohl et al., 2001a). TauR vermittelt die für katabolische Gene typische Substratinduktion durch Taurin.
105
Diskussion
TauR weist eine N-terminale DNA-Bindedomäne mit dem wHTH-Motiv der GntR-Familie
bakterieller Regulatoren auf (Abb. 20). Entsprechend wurde die Bindung von TauR an den
tpa-tauR-xsc-Promotor nachgewiesen (Schubert et al., in Vorbereitung). Die Bindestelle wurde durch Mutations- und Gelshift-Analysen auf eine Region mit zwei „direct repeats“ (DR)
von je 10 Basenpaaren eingegrenzt. Diese DNA-Region ist an Position - 61 relativ zum Transkriptionsstart zentriert, so dass eine Aktivierung des tpa-tauR-xsc-Operons durch direkte
Interaktion des Regulators mit der RNA-Polymerase wahrscheinlich ist. In Analogie zu anderen GntR-Regulatoren und entsprechend der DR im Promotorbereich des tpa-tauR-xscOperons ist die DNA-Bindung von TauR in dimerer Form denkbar (Gorelik et al., 2007; van
Aalten et al., 2000).
wHTH
Klasse I Aminotransferase
Abb. 20: Domänenstruktur von TauR aus R. capsulatus. Als Vertreter der MocR-Subfamilie bakterieller Transkriptionsregulatoren kann TauR in eine N-terminale DNA-Bindedomäne mit wHTHMotiv und eine C-terminale Aminotransferase-Domäne unterteilt werden. TauR zählt zu den Pyridoxal-5`-Phosphat-abhängigen Klasse I Aminotransferasen.
Interessanterweise wurde die DNA-Bindeaffinität von TauR durch Zugabe von Taurin nicht
gesteigert. Dies ist vermutlich in der C-terminalen Domäne des Regulators begründet. Regulatoren der GntR-Familie weisen zwar homologe N-terminale DNA-Bindedomänen auf, zeigen jedoch maximale Divergenz in der C-terminalen Domäne (Vindal et al., 2007). Die GntRFamilie wird daher weiter in die FadR-, HutC-, YtrA-, AraA-, PlmA- und MocR-Subfamilien
unterteilt (Franco et al., 2006; Lee et al., 2002; Rigali et al., 2002). TauR ist ein Vertreter der
MocR-Subfamilie, welche sich durch Homologien zu Klasse I Aminotransferasen auszeichnet. Da es sich um Pyridoxal-5`-Phosphat (PLP)-abhängige Aminotransferasen handelt, wird
für den MocR-Regulator des Rhizopin-Katabolismus aus Rhizobium meliloti eine PLPabhängige enzymatische Funktion postuliert (Rossbach et al., 1994). GabR, ein weiterer Vertreter der MocR-Subfamilie, reguliert die Nutzung von GABA als einzige Stickstoff-Quelle in
B. subtilis (Belitsky & Sonenshein, 2002). Die Aktivierung durch GabR-Dimere erfolgt
GABA- und PLP-abhängig. Somit ist eine durch GabR katalysierte Aminotransferase-Reaktion zwischen GABA und PLP vermutlich essentiell für die Transkriptionsaktivierung. Da
TauR ebenfalls zu den chimären Proteinen der MocR-Subfamilie zählt, könnte neben Taurin
auch PLP für die Transkriptionsaktivierung des tpa-tauR-xsc-Operons benötigt werden. TauR
106
Diskussion
katalysiert möglicherweise eine Aminotransferase-Reaktion zwischen Taurin und PLP. Somit
wäre Taurin lediglich für die Transkriptionsaktivierung, nicht aber für die DNA-Bindung essentiell (Abb. 21). Weitere Gelshift- und in vivo bzw. in vitro Transkriptionsanalysen unter
Zugabe von Taurin und/oder PLP könnten zur Aufklärung des genauen Mechanismus der
B
TauR
tpa
tauR
xsc
tpa
tauR
xsc
TauR
PLP
Taurin
?
TauR
A
TauR
Transkriptionsaktivierung durch TauR beitragen.
+
Abb. 21: Model der TauR-abhängigen Regulation des tpa-tauR-xsc-Operons in R. capsulatus. (A)
TauR bindet vermutlich als Dimer an DR des tpa-tauR-xsc-Promotors. (B) Möglicherweise wird neben Taurin auch PLP für die Transkriptionsaktivierung (+) benötigt. Ob die Aktivierung des Operons
eine durch TauR katalysierte enzymatische Reaktion einschließt ist unklar (?). Eine genaue Erläuterung des Models erfolgt im Text.
107
Zusammenfassung
H
Zusammenfassung
Die Adaptation an wechselnde Umweltbedingungen wird in Bakterien vorwiegend durch
Transkriptionsregulatoren gewährleistet. Im Rahmen dieser Arbeit wurden vier regulatorische
Systeme des phototrophen Purpurbakteriums Rhodobacter capsulatus näher untersucht. Diese
reagieren auf die Verfügbarkeit von Stickstoff (NtrY), Molybdän (MopA, MopB), Kupfer
(CutR) und Schwefel (TauR).
Die Stickstoff-abhängige Genregulation wird in R. capsulatus durch das zentrale Zwei-Komponenten-Regulationssystem NtrB/NtrC vermittelt. Die Sensorkinase NtrB autophosphoryliert
bei intrazellulärem Stickstoff-Mangel und überträgt ihre Phosphoryl-Gruppe auf den Responseregulator NtrC. Nachfolgend aktiviert NtrC-P die Expression von Genen des StickstoffMetabolismus. Im Rahmen dieser Arbeit wurde durch genetische Analysen gezeigt, dass die
Sensorkinase NtrY das NtrB-Protein als Phospho-Donor für NtrC ersetzen kann. Im Gegensatz zum cytoplasmatischen NtrB-Protein transduziert das Transmembranprotein NtrY anscheinend den periplasmatischen Stickstoff-Status durch „cross-talk“ auf NtrC.
Die Molybdat-abhängige Genregulation wird in R. capsulatus durch MopA und MopB vermittelt. Ziel dieser Arbeit war es, die genaue Funktionsweise beider Regulatoren zu klären.
MopA und MopB können sich als Molybdat-abhängige Repressoren von Genen des Molybdat-Metabolismus ersetzen, wie durch Mutations- und Transkriptionsanalysen gezeigt wurde.
Die Molybdat-abhängige Aktivierung des mop-Gens erfolgt hingegen ausschließlich durch
MopA. Mop ist ein hexameres, putatives Molybdat-Speicherprotein. Molybdat steigert die
DNA-Affinität von MopA und MopB, wobei konservierte palindromische Mo-Boxen als
DNA-Bindestellen dienen. Als typische Operatorbindestellen überlappen die Mo-Boxen reprimierter Gene die durch Primerextension bestimmten Transkriptionsstartpunkte. Entsprechend der Rolle als Aktivatorbindestelle liegt die Mo-Box des Molybdat-aktivierten mopGens stromaufwärts des Transkriptionsstarts. Yeast Two-Hybrid Studien, GlutaraldehydCrosslinking, Gelfiltrations-Chromatographie und Coreinigungen zeigten, dass MopA und
MopB Homodimere und Heterodimere bilden. MopA/MopB-Heterodimere dienen wahrscheinlich der Feinabstimmung der Molybdat-abhängigen Genregulation. Darüber hinaus interagieren MopA und MopB mit dem MogA-Protein, welches als Molybdat-Donor der Molybdopterin-Cofaktor-Biosynthese dient. Im Gegensatz zu MopA interagiert MopB mit Mop.
Kupfer-abhängige Regulatoren sichern zum einen bei niedrigen Kupfer-Konzentrationen die
zelluläre Kupfer-Versorgung und schützen zum anderen bei hohen Kupfer-Konzentrationen
vor toxischen Effekten. Mutations- und Expressionsanalysen zeigten, dass CutR in R. capsu108
Zusammenfassung
latus als Repressor des Kupfer-Toleranz-Operons orf635-cutO-cutR fungiert. Steigende Kupfer-Konzentrationen führen zur Derepression dieses Operons. Als erstem Vertreter der bislang
uncharakterisierten DUF411-Proteinfamilie konnte CutR somit im Rahmen dieser Arbeit eine
spezifische Funktion zugeordnet werden.
R. capsulatus kann seinen Schwefel-Bedarf durch Assimilation von Taurin decken. Die entsprechenden Enzyme sind im tpa-tauR-xsc-Operon kodiert. Genetische Analysen zeigten,
dass TauR ein Taurin-abhängiger Aktivator dieses Operons ist. Der Regulator bindet an die
Promotor-DNA, wobei Mutations- und Gelshift-Analysen als Bindestelle zwei „direct repeats“ nahe legen. TauR ist ein chimäres Protein mit einer DNA-Bindedomäne und einer
Aminotransferase-Domäne. Für die Transkriptionsaktivierung ist möglicherweise eine TauRkatalysierte Aminotransferase-Reaktion zwischen Taurin und Pyridoxal-5´-Phosphat essentiell. Der Charakterisierung von R. capsulatus TauR als erstem Vertreter dieser RegulatorenGruppe kommt besondere Bedeutung zu, da TauR-homologe Proteine in Proteobakterien weit
verbreitet sind.
109
Summary
I
Summary
In bacteria, adaptation to changing environments is mainly controlled by transciptional regulators. This work deals with four regulatory systems of the phototrophic purple bacterium
Rhodobacter capsulatus. These systems respond to availability of nitrogen (NtrY), molybdenum (MopA, MopB), copper (CutR), and sulfur (TauR).
Nitrogen-dependent gene regulation in R. capsulatus is mediated by the central twocomponent system NtrB/NtrC. Under intracellular nitrogen-deficient conditions the sensor
kinase NtrB autophosphorylates and transfers its phosphoryl-group to the response regulator
NtrC. In turn, NtrC-P activates expression of nitrogen-metabolism genes. Genetic analysis
demonstrated that the sensor kinase NtrY can substitute for NtrB as phosphor-donor towards
NtrC. In contrast to the cytoplasmic protein NtrB, the transmembrane protein NtrY apparently
transduces the periplasmic nitrogen-status to NtrC via cross-talk.
Molybdate-dependent gene regulation in R. capsulatus is mediated by MopA and MopB. In
this study specific functions of these two regulators were studied by mutational and transcriptional analyses. MopA and MopB can substitute for each other in molybdate-dependent repression of several target genes. In addition to its role as a repressor, MopA (but not MopB)
activates expression of the mop gene in the presence of molybdate. Mop is a hexameric, putative molybdate-storage protein. Molybdate enhances affinity of MopA and MopB to their target promotors, which contain conserved palindromic Mo-boxes serving as binding sites for
MopA and MopB. Mo-boxes serving as repressor binding site overlap the transcription start
sites (determined by primer extension analysis). As expected for an activator binding site, the
Mo-box controlling mop expression is located upstream of the transcription start site. Yeast
two-hybrid studies, glutaraldehyde crosslinking, gelfiltration chromatography, and copurification experiments show that MopA and MopB form homodimers and heterodimers.
MopA/MopB heterodimers most-likely are involved in fine-tuning of molybdate-dependent
gene regulation. In addition, MopA and MopB interacted with the molybdopterin cofactor
biosynthesis protein MogA. In contrast to MopA, MopB interacts with Mop.
Copper-dependent regulation ensures cellular copper requirement at low copper concentrations and protects cells against toxic effects at high copper concentrations. Mutational and
expression analyses suggest that CutR functions as a repressor of the R. capsulatus coppertolerance operon orf635-cutO-cutR at low copper concentrations. Increasing copper concentrations lead to derepression of this operon. R. capsulatus CutR is the first member of the yet
uncharacterised DUF411 protein family.
110
Summary
R. capsulatus can cover its sulfur requirement by assimilation of taurine, and the tpa-tauRxsc-Operon is essential for taurine utilisation. TauR is the activator of this operon as shown by
genetic analysis. TauR binds to two direct repeats within its target promoter as revealed by
mutational and gelshift analyses. TauR is a chimeric protein consisting of a DNA-binding and
an aminotransferase domain. A TauR-catalysed aminotransferase reaction between taurine
and pyridoxal-5`-phosphate may be essential for transcriptional activation. R. capsulatus
TauR is the first member of a large family of regulators widespread in proteobacteria.
111
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Structure 8(11):1127-36
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M., Bereswill S. (2005)
The Helicobacter pylori CrdRS twocomponent regulation system (HP1364HP1365) is required for copper-mediated induction of the copper resistance determinant
CrdA.
J. Bacteriol. 187(13):4683-8
Wang G., Angermüller S., Klipp W. (1993)
Characterization of Rhodobacter capsulatus
genes encoding a molybdenum transport system and putative molybdenum-pterin-binding
proteins.
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The bacterial enhancer-binding protein NTRC
is a molecular machine: ATP hydrolysis is
coupled to transcriptional activation.
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Zhao J., Tan H., Xu H., Jiang W., Li Y. (2007)
Comparative Analysis of Two-component
Signal Transduction System in Two Streptomycete Genomes.
Acta. Biochim. Biophys. Sin. 39(5):317-25
Williams C. E., White D. J., Delarbre L.,
Mitchenall L. A., Pau R. N., Lawson D. M.
(1999)
Crystallization and preliminary X-ray studies
on the molbindin ModG from Azotobacter
vinelandii.
55(7):1356-8
Xiang S., Nichols J., Rajagopalan K. V.,
Schindelin H. (2001)
The crystal structure of Escherichia coli MoeA
and its relationship to the multifunctional protein gephyrin.
Structure 9(4):299-310
Xu H. & Hoover T. R. (2001)
Transcriptional regulation at a distance in bacteria.
Curr. Opin. Microbiol. 4(2):138-44
Yamamoto K., Hirao K., Oshima T., Aiba H.,
Utsumi R., Ishihama A. (2005)
Functional characterization in vitro of all twocomponent signal transduction systems from
Escherichia coli.
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Yamamoto K. & Ishihama A. (2005)
Transcriptional response of Escherichia coli to
external copper.
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Toxic metal-responsive gene transcription.
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124
Publikationen
K
Publikationen
1.
Artikel
Drepper T., Wiethaus J., Giaourakis D., Gross S., Schubert B., Vogt M., Wiencek Y., McEwan A. G., Masepohl B. (2006)
Cross-talk towards the response regulator NtrC controlling nitrogen metabolism in Rhodobacter capsulatus.
Schubert B., Wiethaus J., Pfänder Y., Narberhaus F., Masepohl B. (in Vorbereitung)
The GntR-like regulator TauR activates expression of taurine utilization genes in Rhodobacter capsulatus.
Wiethaus J., Wildner G. F., Masepohl B. (2006a)
The multicopper oxidase CutO confers copper tolerance to Rhodobacter capsulatus.
FEMS Microbiol Lett. 256(1):67-74
Wiethaus J., Wirsing A., Narberhaus F., Masepohl B. (2006b)
Overlapping and specialized functions of the molybdenum-dependent regulators MopA and
MopB in Rhodobacter capsulatus.
J. Bacteriol. 188(24):8441-51
Wiethaus J., Narberhaus F., Masepohl B. (zur Publikation eingereicht)
Protein-protein interactions between MopA, MopB, and Mop from Rhodobacter capsulatus.
2.
Kongress-Beiträge
Masepohl B., Aktas M., Brusch M., Drepper T., Schubert T., Sicking C., Vermöhlen S.,
Wiethaus J., Schneider K. (2005)
Regulation of nitrogen fixation in Rhodobacter capsulatus.
14th International Congress on Nitogen Fixation (Beijing, China)
Wiethaus J. & Masepohl B. (2006)
Molybdenum regulation of nitrogen fixation in Rhodobacter capsulatus.
7th European Nitrogen Fixation Conference (Aarhus, Dänemark)
Wiethaus J. & Masepohl B. (2007)
Molybdenum regulation of nitrogen fixation and Mo-metabolism in Rhodobacter capsulatus.
Proceedings of the 15th International Conference on Nitrogen Fixation (Kapstadt, Südafrika)
125
Publikationen
Wiethaus J., Schubert B., Wirsing A., Masepohl B. (2006)
Regulation of nitrogen fixation and molybdenum transport in Rhodobacter capsulatus.
7th European Nitrogen Fixation Conference (Aarhus, Denmark)
Wiethaus J., Wildner G. F., Masepohl B. (2004)
The multicopper oxidase CutO confers copper tolerance in the phototrophic purple bacterium
Rhodobacter capsulatus under both aerobic and anaerobic conditions.
4th International Biometals Symposium (Garmisch-Partenkirchen, Deutschland)
126
Anhang
L
Anhang
1.
Erklärung
Hiermit erkläre ich, dass ich die Arbeit selbständig verfasst und bei keiner anderen Fakultät
eingereicht und dass ich keine anderen als die angegebenen Hilfsmittel verwendet habe. Es
handelt sich bei der heute von mir eingereichten Dissertation um fünf in Wort und Bild völlig
übereinstimmende Exemplare.
Weiterhin erkläre ich, dass digitale Abbildungen nur die originalen Daten enthalten und in
keinem Fall inhaltsverändernde Bildbearbeitung vorgenommen wurde.
Bochum, den 01. 07. 2007
127
Anhang
2.
Lebenslauf
zur Person
Name
Vorname
Anschrift
Geburtsdatum
Geburtsort
Staatsangehörigkeit
Wiethaus
Jessica
Wellinghoferstr. 148
44263 Dortmund
Deutschland
09.02.1980
Dortmund
deutsch
Schulausbildung
1986 - 1990
1990 - 1999
Brücherhof Grundschule, Dortmund
Goethe-Gymnasium, Dortmund
Abschluss Abitur
Hochschulausbildung
1999 - 2004
seit 2004
Diplomstudium der Biologie
Ruhr-Universität Bochum
Abschluss Diplom-Biologin
Anfertigung der Doktorarbeit
Beruflicher Werdegang
seit 2004
Wissenschaftliche Mitarbeiterin
128

Genregulation in Rhodobacter capsulatus durch Stickstoff

Transcription

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