Cell density-dependent gene expression controls luminescence in

Cell density-dependent gene expression controls luminescence
in marine bacteria and virulence in several pathogens
E. Peter Greenberg
t has been said that every novel idea
in science passes through three
stages. First people say it isn’t true,
then they say it’s true but not important, and finally they say it’s true
and important, but not new.
Over the past several years, there has been an
increasing appreciation among microbiologists
that bacteria can perceive and respond to other
bacteria. This capability is often important in the
colonization of animal and plant hosts by symbiotic or pathogenic bacterial species. Although
different bacterial groups have different mechanisms for monitoring their own abundance in a
local environment, one mechanism that has
emerged as common in gram-negative bacteria is
that initially discovered by J. W. (Woody) Hastings and his collaborators in the luminescent bacterium Vibrio fischeri (see box p. 376).
The phenomenon originally termed autoinduction has become known as quorum sensing
and response, and has been extensively reviewed
recently (see Suggested Reading). The term quorum sensing first appeared in a Journal of Bdcteriology minireview written by Clay Fuqua,
Steve Winans, and myself in 1995. It originated
with Steve Winans’ brother-in-law, a lawyer
who was trying to understand what we were
studying as Steve explained it to him during a
family gathering at Christmas.
Stage 1, the 1970s: the Discovery and
the Ecological Explanation
Hastings was driven by his curiosity to explain
why luminous bacteria like VI fischeri contain
high levels of luciferase only when cultures
reach the late-logarithmic phase of growth. The
basic framework for quorum sensing was es-
tablished in the early 1970s by Hastings, Terry
Platt, Ken Nealson, and Anatol Eberhard. They
showed that V. fischeri and another luminous
species, Vibrio harveyi, produce diffusible compounds, termed autoinducers, that accumulate
in the medium during growth. These autoinducer signals can accumulate to sufficient concentrations only when there is a critical mass of
cells in a confined environment. The signals
from V. fischeri and VI harveyi do not crossreact, showing species specificity. When I was a
student in the MBL Summer Microbiology Program at Woods Hole, Mass., in 1973, Ken Nealson, then an instructor in the course, explained
to me that V. fischeri occurs at very high densities ( 1 Ol” to 1 O1 l cells per ml) as a specific symbiont in light organs of certain fish and is also
found free in seawater at much lower densities
(perhaps 5 cells per ml). Autoinduction allows
VI fischeri to sense its elevated density in the
light organ and express the luminescence system
there, where it is required for the symbiosis, but
not in seawater, where luminescence, which is
energetically expensive, would be frivolous.
The concept that bacteria produce pheromones and communicate with one another was
met with considerable skepticism by many and
disinterest by others at the time. My own curiosity was stimulated, however, and upon completion of my Ph.D. in 1977 I moved directly to
Woody Hastings’ laboratory to begin my postdoctoral research on autoinduction. In 1978
Hastings, Shimon Ulitzur, and I showed that although V fischeri did not make a signal that activates the luminescence genes in V. harveyi,
many other gram-negative marine bacteria do.
This was the first suggestion that cell-to-cell signaling within and perhaps between bacterial
E. Peter Greenberg
is professor of microbiology at the
University of Iowa,
Iowa City.
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F I G U R E
1 1
Quorum sensing in Vibrio fischeri. (A) The luminescence gene cluster. The /ux/? gene encodes an autoinducer-dependent .
transcriptional activator of the luxl-G operon. The luxl product is the autoinducer synthase, /uxC, 0, and E form a complex responsible
for generation of one of the substrates for the luciferase reaction, the long chain fatty aldehyde, /uxA and B encode the two subunits
of luciferase, and the function of /uxG remains unknown. (B) Cartoon of L: fischeri cells, each producing the diffusible autoinducer
signal. At low cell densities the luminescence operon is transcribed at a basal level. At high cell densities the autoinducer signal can
reach a sufficient concentration and bind to the cellular LuxR protein, which will then activate transcription of the luminescence operon.
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ASM News /Volume 63, Number 7
species might be a common phenomenon.
However, because we lacked an understanding
of the genes, proteins, and signal molecules involved in autoinduction, this idea was not further developed until the 1990s.
F I G U R E
2 1
’ - Regulator domain - I e Activator domain e !
I
I
I
I
Stage 2, the 1980s: Proof of the Model
The 1980s brought many important scientific
discoveries about autoinduction in V. fischeri,
first physiological, then chemical, and finally genetic. A careful chemostat study confirmed that
luminescence required high cell density. The
structure of the autoinducer signal, N-3-( oxohexanoyl) homoserine lactone, was solved. This
molecule was shown to move out of and into
cells by passive diffusion. The genes for luminescence were cloned from VI fischeri into E .
coli. Fortunately, the genes for autoinduction
are linked to the luminescence structural genes
(Fig. l), and E. coli cells containing this lux gene
cluster produce light in a cell density-dependent
fashion. Thus, quorum sensing could be analyzed with the tools of E. coli genetics.
It was found that the regulatory region that
enables autoinduction of luminescence consists
of two genes: luxR, which encodes an autoinducer-responsive transcriptional activator, and
luxl, which encodes a protein required for autoinducer synthesis. The region between luxR
and 1~x1 contains the regulated lux promoter elements. Because 1~x1 is positively autoregulated,
basal levels of 1uminescence operon transcription lead to low rates of autoinducer production,
and quite high densities of cells are necessary for
activation of the luminescence genes. Once activation has occurred, the rate of autoinducer
synthesis increases, and cell density must drop
considerably before the rate of transcription of
the luminescence operon returns to the basal
level.
Not surprisingly, autoinduction is just one of
the regulatory systems that come to bear on luminescence gene expression. 1uxR requires activation by cyclic AMP (CAMP) and the CAMP receptor protein. Iron can influence expression of
luminescence. FNR seems to exert an effect on
luxR, and there may be other cellular regulatory
elements that affect expression of the luminescence genes of K fischeri.
Since the late 198Os, more details of the mechanism of autoinduction have become known.
Helix-turn-helix
Multimerization
IL fischeri MJI /UX box
/UX box-like consensus sequence
ACC
RNS
CGTA
NXTR
Elements of autoinducible lux gene expression. (Top) Key regions of LuxR,
the activator of luminescence gene transcription. The polypeptide consists of
two domains. There is a C-terminal helix-turn-helix (H-T-H) containing
activator domain extending from about residue 160 to the C-terminal residue,
250. This domain interacts with the transcription initiation complex. The
region from residue 230 to 250 is thought to be required for transcriptional
activation but not for DNA binding. There is an N-terminal regulatory domain
extending to about residue 160. A region of this domain is involved with
autoinducer binding, residues 79-l 27, and a region is involved in multimer
formation, around 120 to 160. In the absence of autoinducer the regulatory
domain interferes with the activity of the activator domain. (Bottom) The lux
box from L: fischeri strain MJ I and a consensus sequence for lux-box like
elements found in promoter regions of acylhomoserine lactone-regulated
genes from other bacterial species. The 20-bp lux box is centered at about
-40 from the start of /ux/ transcription. Consensus sequence abbreviations:
N = A, T, C, or G: R = A or G; S = C or G; Y = T or C; X = N or a gap in the
sequence.
Since the VI fischeri autoinducer is free to diffuse
out of and into cells, the cellular and environmental concentrations of this signal are equivalent. For this reason, the transcriptional activator LuxR, which is located on the cytoplasmic
side of the cell membrane, can respond to the environmental concentration of the autoinducer,
which increases with V. fischeri cell density.
LuxR is a 250-amino acid polypeptide that
consists of two domains and functions as a homomultimer, likely a dimer, with o70-RNA polymerase to activate lux gene expression. It is a
member of the LuxR superfamily*of transcription factors, all of which contain somewhat similar H-T-H motifs in their DNA binding regions.
This superfamily includes LuxR homologs (see
below) and other regulatory proteins such as
MalT, GerE, NarL, and region 4 of the u subunit of bacterial RNA polymerase. The N-terminal 160 amino acids or so of LuxR constitute
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F I G U R E
0
I
20
I
3 1
I
40
I
I
60
I
Amino acid number
80
100 120 140
I
I
I
N
I
I
I
I
I
I
160
I
I
180193
I
ll
C
Active site
Acyl-ACP substrate specificity?
A scheme for autoinducer synthesis and key regions of the Luxl protein.
(Top) Luxl binds an acyl-ACP and S-adenosylmethionine (SAM). The acyl
group is transferred from the bound ACP forming amide bond with the
SAM. The acyl-SAM is converted to acylhomoserine lactone with
release of 5’-methylthioadenosine (MTA) and release of the
acylhomoserine lactone. (Bottom) Luxl is 193 amino acids in length.
There is a region extending from about residue 25 to residue 104 that
appears to represent the active site for amide bond formation. There is
limited evidence to suggest that a region from about residue 133 to
residue 164 is involved in selection of the appropriate acyl-ACP from the
cellular pools.
an autoinducer-binding, regulatory domain
which, in the absence of sufficient autoinducer,
interferes with the C-terminal domain (the last
90 or so amino acids), which binds to RNA
polymerase and the lux regulatory DNA to activate transcription of the luminescence operon
(Fig. 2).
Tom Baldwin and his coworkers at Texas
A&M University in College Station, Tex., identified a 20-bp inverted repeat at about -40 from
the start of transcription of the luminescence
operon (Fig. 2) which is required for autoinduction of luminescence. In vitro studies of
LuxR have been difficult and slow, but from
such studies we believe LuxR and ~70 RNA
polymerase are the only transcription factors required for activation of the 1~x1 promoter and
that these two factors bind synergistically to the
promoter region.
Many autoinducer analogs with alterations in
the acyl side chain can bind to LuxR. Some serve
weakly as autoinducers, but others inhibit the
activity of the natural autoinducer, presumably
by competition for the autoinducer binding site.
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We are beginning to understand the mechanism by which the 1~x1 gene directs the synthesis of the autoinducer signal. We now know that
LuxI is an autoinducer synthase that catalyzes
the formation of an amide bond between its two
substrates, a six-carbon fatty acyl-acyl carrier
protein (acyl-ACP) and S-adenosylmethionine
(Fig. 3). Although it has been suggested that the
acyl group forms a covalent bond with an active site cysteine in LuxI, recent studies of cysteine substitution mutants indicate that this is
not the case. Studies of LuxI mutants have revealed that the active site in which amide bond
formation is catalyzed is roughly in the region
of residues 2.5-l 10 of this 19%amino-acid protein. A region in the C terminus may be involved
in selection of the appropriate acyl-ACP from
those existing in the cellular pools (Fig. 3).
Stage 3, the 1990s: the Discovery of
LuxR-Luxl-Type Systems in Other Bacteria
In the early l99Os, several groups made key discoveries that led to our current view that quorum sensing is common to many gram-negative
bacterial species. First, LuxR homologs were
discovered in Pseudomonas aeruginosa and
Agrobacterium tumefaciens, and several bacte-
rial species were shown to produce N-3-(0x0hexanoyl) homoserine lactone. Shortly thereafter, it was found that the A. tumefaciens and
P. aeruginosa autoinducers are analogs of the VI
fischeri autoinducer. For A. tumefaciens the autoinducer is N-3-( oxooctanoyl) homoserine lactone. I? aeruginosa has at least two quorum
sensing systems, one that uses N-3-(oxododecanoyl)homoserine lactone and one that uses Nbutyrylhomoserine lactone. The genes responsible for autoinducer production were sequenced,
and their products were found to be homologous to LUXI.
There are now over a dozen LuxI homologs
and over a dozen LuxR homologs in the protein
sequence data bases. Furthermore, lux box-like
sequences can be found in the promoter regions
of many of the genes regulated by LuxR homologs in bacteria other than VI fischeri. The
LuxI homologs direct the synthesis of acylhomoserine lactones with saturated or unsaturated
acyl chains of 4 to 14 carbons with either a hydroxyl group, a carbonyl group, or hydrogens
on the third carbon from the amide bond. The
constant among these homologs is the homoserine lactone component, while the acyl
group provides species specificity. Different
LuxI homologs produce different autoinducers,
and the cognate LuxR homologs respond best
to the appropriate autoinducer. Table 1 lists
some bacteria that we now know to produce
acylhomoserine lactones.
What does autoinduction control in these
other bacteria? Quorum sensing in A. tumefaciens controls conjugal transfer genes, probably
to ensure that the catabolic Ti plasmid is present when cells are at a high density in a crown
gall tumor. l? aeruginosa, an opportunistic
human pathogen, uses quorum sensing to regulate expression of a battery of extracellular virulence genes, including enzymes and exotoxins.
Autoinduction of extracellular enzymes is a
common theme. Production of extracellular enzymes at low cell densities would be of no value;
the enzymes would diffuse away from the cell
and convert relatively little substrate to product,
and because the environmental concentration of
the product would not change appreciably, the
bacterial cells would not benefit. When the bacteria have achieved a high enough density, production of an extracellular enzyme could have
an impact on the environment.
Why might quorum sensing control exotoxin
production? Here we can use the analogy of an
invading army. The bacterial pathogen first
masses its troops, but it does not reveal its
weapons until they can be deployed in sufficient
quantity to overwhelm the opposition. By not
producing exotoxins at low cell densities and
waiting until the host defenses can be overwhelmed, I? aeruginosa deprives the host of the
chance to respond immunologically. In fact, R
aeruginosa quorum sensing mutants can colonize neonatal mouse lungs, but the progression
of the disease is impaired; in contrast to the wild
type, infection with the quorum sensing mutants
does not lead to death.
Another example of LuxR-LuxI-type quorum
sensing occurs in Erwinia carotovora, in which
not only extracellular enzymes, but also carbepenem antibiotic synthesis and in fact virulence of this plant pathogen are controlled by
this mechanism. The significance of some quorum sensing systems is more difficult to picture.
For example, the autoinduction of a set of Rhizobium leguminosarum genes that is expressed
just prior to root hair penetration and of genes
that lead to stationary phase. One general theme
that has emerged is that the bacteria that exhibit
this type of cell density-dependent gene regulation experience a plant or animal host association as part of their lifestyle. Thus, we were quite
surprised to discover in collaboration with
Agnes Puskus and Sam Kaplan of the University
of Texas Medical School, Houston, Tex., that the
free-living photosynthetic bacterium Rhodobacter sphaeroides has a quorum sensing system.
Although the divergently transcribed 1~x1 and
1uxR genes in VI fischeri are linked to each other
and to the genes they regulate (Fig. l), this is not
always the case. In fact, every sort of arrangement imaginable has been reported. The 1~x1
and luxR genes can regulate unlinked genes, and
in some cases are not even linked to each other.
There can even be multiple luxR and 1~x1 homologs in a single bacterium. There is now some
evidence that Burkholderia cepacia, an opportunistic pathogen that can colonize lungs of cystic fibrosis patients, may sense and respond to
the density of another bacterial species infecting
the cystic fibrosis lung, I? aeruginosa. It appears
that the elements of the LuxR-LuxI system
evolved in gram-negative bacteria early or have
moved from species to species by gene transfer
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and that each species has adapted these elements
to its own needs.
A Case of Convergent Evolution. Understanding autoinduction of luminescence in VI harveyi
has come more slowly than our understanding
of autoinduction in VI fischeri, in large part because the K harveyi system is more complicated.
For instance, there are two signaling systems
that can function independently of each other.
One of the systems involves an acyl- homoserine lactone, N-( 3-hydroxybutyryl)homoserine
lactone; the structure of the signal for the other
system remains unknown and it may not be an
acylhomoserine lactone. LuxR homologs have
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not been identified in VI harveyi. Rather, the signal sensors are complex proteins with sequence
similarities to both components of two-component regulatory proteins. Two genes, 1uxL and
luxM, are required for synthesis of N-(3-hydroxybutyryl) homoserine lactone. Neither of
these genes encode a proteins with similarity to
the VI fischeri LuxI protein or any of its homologs. Paul Dunlap and coworkers, then at the
Woods Hole Oceanographic Institute in Woods
Hole, Mass., discovered that VI fischeri 1~x1 mutants produce octanoylhomoserine lactone,
which serves as a very poor substitute for the
LuxI-produced 3-oxohexanoylhomoserine lac-
tone in luminescence gene activation. The gene
required for octanoylhomoserine lactone synthesis was cloned and sequenced, and although
its product is not a LuxI homolog, there is a
38 % sequence identity between its amino-terminal region and the V. harveyi LuxM protein.
This suggests that there is a second family of
acylhomoserine lactone-synthesizing enzymes.
The mechanism of acylhomoserine lactone
synthesis by this family has not yet been
investigated.
It’s True and Important but Not New. The phenomenon of autoinduction noticed by Woody
Hastings 30 years ago is well established, and has
been found to be common among a diverse group
of gram-negative bacteria. It plays a role not only
in the curious light organ relationship between V
fischeri and its marine animal hosts but also in
the virulence of certain human and plant
pathogens. Although quorum sensing is no longer
considered “new,” the field of research is young
and there are many important areas to investigate. It is clear that LuxI and LuxR homologs in
pathogens are targets for development of novel
antimicrobial factors, but we need more knowledge about how to inhibit them. Staffan Kjelleberg and his collaborators at the University of
New South Wales in Sydney, Australia, have reported recently that at least one marine alga produces furanone compounds that can inhibit autoinduction. This may provide an explanation as
to why luminescent marine bacteria, which can
be isolated from a variety of marine habitats, are
not found on the surface of algae.
We know little about quorum sensing in natural environments and in the biofilms in which
bacteria often grow. Is communication between
bacterial species in complex natural environments common or important? Why do bacteria
have multiple quorum sensing systems, and how
many can be found in an individual strain?
What is the significance of the “second family”
of autoinducer synthesis enzymes? Finally, one
might expect that with the intimate associations
known to exist between mutualistic and pathogenic quorum sensing bacteria and their plant
and animal hosts, the hosts may have evolved
systems that can sense and respond to acylhomoserine lactone signals. It has been reported
by Alice Prince and collaborators at Columbia
University, New York City, N.Y., that one of the
I? aeruginosa autoinducers stimulates epithelial
cell production of interleukin-8, but in general
host detection and response to autoinducers remains an untapped avenue of investigation.
ACKNOWLEDGMENTS
I thank the Office of Naval Research for continued support of my research on quorum sensing from the early years of the
field up to the present.
The originator of the opening lines of this article (about the three stages through which scientific ideas pass) is unknown,
but the statement appeared in print in F. M. Harold’s work cited below.
SUGGESTED READING
Bassler, B. L., and M. R. Silverman. 1995. Intercellular communication in marine Vibrio species: density-dependent regulation of the expression of bioluminescence, p. 431-445.112 J. A. Hoch and T. J. Silhavy (ed.), Two-component signal transduction. ASM Press, Washington, D.C.
Fuqua, W. C., S. C. Winans, and E. I? Greenberg. 1996. Census and consensus in bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcriptional regulators. Annu. Rev. Microbial. 50:727-7.51.
Fuqua, W. C., S. C. Winans, and E. I! Greenberg. 1994. Quorum sensing in bacteria: the LuxR-LuxI family of cell densityresponsive transcriptional regulators. J. Bacterial. 176:269-275.
Harold, F. M. 1986. The vital force: a study of bioenergetics. W. H. Freeman and Co., New York.
Kaiser, D., and R. Losick. 1997. How and why bacteria talk to each other. Sci. Am. 276:68-73.
Salmond, G. I? C., B. W. Bycroft, G. S. A. B. Stewart, and P. Williams. 1995. The bacterial ‘enigma’: cracking the code of
cell-cell communication. Mol. Microbial. 16:615-624.
Sitnikov, D., J. B. Schineller, and T. 0. Baldwin. 1995. Transcriptional regulation of bioluminescence genes from Vibrio fischeri. Mol. Microbial. 17:801-812.
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