Are Incomplete Denitrification Pathways a Common Trait in Thermus

McNair Poster Presentations
McNair Scholars Institute
2013
Are Incomplete Denitrification Pathways a
Common Trait in Thermus Species from
Geothermal Springs in China?
Julienne J. Paraiso
University of Nevada, Las Vegas
Brian P. Hedlund
University of Nevada, Las Vegas, brian.hedlund@unlv.edu
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Repository Citation
Paraiso, J. J., Hedlund, B. P. (2013). Are Incomplete Denitrification Pathways a Common Trait in Thermus Species from Geothermal
Springs in China?.
Available at: http://digitalscholarship.unlv.edu/mcnair_posters/35
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Are Incomplete Denitrification Pathways a Common Trait in
Thermus species from Geothermal Springs in China?
Julienne Jochel Paraiso, McNair Scholar, Pre-Professional Biology
Dr. Brian P. Hedlund, Faculty Mentor, School of Life Sciences
METHODS
ABSTRACT
I. Microbial Cultivation
12.0
A
10.0
Concentration of N (mM)
Temperature has strong impacts on ecosystem function and biogeochemical
cycles, particularly within extreme environments such as geothermal springs above
60 °C. The primary focus of this study was to investigate the denitrification pathways
of Thermus (Bacteria) isolates from geothermal springs from Tengchong, China. This
study tested the hypothesis that incomplete denitrification is a common
characteristic of the genus Thermus, regardless of geographic origin or species
affiliation, which would implicate them in the efflux of nitrous oxide (a strong greenhouse gas) to the atmosphere. In this study, we cultivated 25 isolates, including six
known Thermus species, and measured the stoichiometry of nitrogenous products of
nitrate respiration using gas chromatography and colorimetric assays. We also
designed custom primers for polymerase chain reaction (PCR) amplification of
denitrification genes including narG, nirS, nirK, and norB to screen for the genetic
capacity for each step in denitrification. Experimental results show that all Thermus
strains tested display incomplete denitrification pathways terminating at nitrite
(NO2-) or nitrous oxide (N2O), and possibly nitric oxide (NO).
RESULTS & DISCUSSION
Strain 442 (640x)
•
Isolates were grown anaerobically on 9 mM nitrate-amended Castenholz medium D (CMD).
o
Tryptone and yeast extract were utilized as carbon sources.
o
Isolates use nitrate as the terminal electron acceptor (in the absence of oxygen).
Isolates were also grown anaerobically on 1 mM nitrate-amended CMD medium with starch
and sodium pyruvate as alternative carbon sources.
•
II. Denitrification Measurements
A
A A
8.0
A
A
B
B
B B
B B
A A
1.) Group A produces nitrite (NO2-) as the only denitrification
product. This physiological phenotype may be due to a
missing or mutated nirK/nirS gene. This interpretation infers
that only narG gene is present.
A
A
B
B
A
AA
B
B
• Initial results from physiological experiments indicate 3 distinct
denitrification phenotypes:
C
6.0
Nitrous
oxide
2.) Group B produces nitrous oxide (N2O) as a denitrification
product, but has some unmeasured amount of nitrogen and
may be due to nitric oxide (NO). This physiological phenotype
may be due to a missing or mutated nosZ gene. This
interpretation infers that narG, nirK/S and norB are present.
Nitrite
4.0
2.0
B
B B
B
Nitrate
B
3.) Group C produces nitrite (NO2-), but has missing N. This
phenotype may be due nitric oxide (NO), which was not
measured. This interpretation infers that narG and nirK/S are
present.
0.0
Colorimetric Assays
N/A
T. tenchongensis
T. antranikianii T. brockianus
T. scotoductus
T. thermophilus
Strains
T. tenchongensis (Strain 410)
1.6
INTRODUCTION
Results from endpoint experiments show final products of nitrate reduction from 25 Thermus strains. Nitrate
has been corrected for interference from nitrite. The blue bar indicates a starting concentration of 9 mM
nitrate. Group A: Thermus strains reduce nitrate into nitrite, Group B: Thermus strains reduce nitrate into
nitrous oxide, and Group C: Thermus strains reduce nitrate into nitrite, but 3.0 mM of nitrogen was
unaccounted for in the expected total nitrogen output.
Nitrogen cycles in geothermal environments are poorly understood.
Many thermophilic microorganisms carry out denitrification.
o Denitrification is the process of reducing nitrate to nitrogenous gases through
anaerobic respiration.
Nitrate
NO3-
narG
Nitrate
reductase
•
•
•
•
Nitric
oxide
Nitrite
NO2-
nirS/K
Nitrite
reductase
NO
Nitrous
oxide
norB
Nitric oxide
reductase
N2 O
Nitrate, nitrite and ammonium ions were measured in each sample using colorimetric
assays.
Gas Chromatography
nosZ
Inert
Gas
Nitrous oxide
reductase
Isotopes
Capillary Column
•
Detector
http://www.etslabs.com/images/methods/10.gif
• This study demonstrates that incomplete denitrification pathways are
common phenotypes in Thermus and that Thermus may may have a
role in greenhouse gas production.
Table 1. Selected primers used for amplification for nitrate-reducing genes
(Murugapiran, 2013)
0.6
Nitrous
oxide
0.4
a
Primera
Primer sequence (5'-3')b
Reference
narG1960f
narG2669r
nirKF1
nirKF2
nirKR1
nirKR2
nirSF1
nirSF1
nirSR1
nirSR2
norBF1
cnorB6R
TAY GTS GGS CAR GAR AA
TTY TCR TAC CAB GTB GC
TGY GCN CCN GGN GGN CA
TGC GCS CCS GGS GGS CA
TTN GGR AAN WSR TTN CC
TTW GGG AAW WSG TTW CC
CAR CAN TGG CTN GTN GAY TA
CAG ACS TGG CTS GTSGAC TA
TAD TTY TGN GTR GGN TT
ATC AAG ACS CAC CCS AA
GCN CCN AGR TGG GCN GA
GAA NCC CCA NAC NCC NGC
Phillipot et al. 2002
Phillipot et al. 2002
This study
This study
This study
This study
This study
This study
This study
This study
This study
Braker & Tiedje, 2003
Primers are named according to corresponding nitrate-reducing gene; forward and reverse primers are
indicated by F and R.
b
N = A, C, G, or T; Y = C or T; R = A or G; D = G, A, or T; B = C, G, or T; S = C or G
50
100
150
200
250
300
Time (hours)
N/A (Strain 428)
1.4
stationary cells @ 24-96 hrs (>3x108 cells)
1.2
1
Nitrate
0.8
Nitrite
0.6
0.4
Nitrous
oxide
0.2
0
0
50
100
150
200
250
300
-0.2
Time (hours)
Selected strains were chosen to measure the stoichiometry of nitrogenous products of denitrification through a
time course experiment. Nitrate and nitrite assays along with nitrous oxide GC measurements show Thermus
isolates to reduce nitrate to nitrite and, in some cases, nitrous oxide. Endpoint and time course data show
denitrification activity varies within genus Thermus.
ACKNOWLEDGEMENTS
FUTURE WORK
IV. PCR Amplification
•
Nitrite
-0.2
DNA extractions were performed on cell pellets using the FastDNA SPIN Kit for Soil.
16S rRNA genes were amplified using bacterial primers to determine if sufficient DNA
was present.
Custom, degenerate primer sets were designed to screen for denitrification genes.
o
narG primers were designed from Philippot, 2002.
o
nirS, nirK, and norB primers were designed using alignments of Thermus
denitrification genes and other denitrification genes.
0.8
0
• This study showed highly variable denitrification phenotypes among
members of the genus Thermus, which suggests denitrification genes
are part of the dispensable genome in Thermus. Denitrification gene
“islands” are found on conjugative plasmids in some Thermus strains
(Ramirez-Arcos, 1998), which have been shown to evolve significantly
faster than the chromosome (Bruggemann, 2006).
III. DNA Extraction
•
Nitrate
1
0.2
• In contrast, the study from Hedlund (2011) investigated several isolates
of T. thermophilus and T. oshimai from the U.S. Great Basin, which
displayed incomplete denitrification pathways due to the absence of
the nitrous oxide reducatase (nosZ) gene (Murugapiran, 2013).
Headspace concentrations of nitrous oxide were determined using gas chromatograph
electron capture detector (GC-ECD).
•
1.2
0
Oven
N2
Truncated denitrification pathways are known to terminate at nitrous oxide (N2O)
(Hart, 1965).
Thermus thermophilus and T. oshimai from U.S. Great Basin have been shown to have
incomplete denitrification pathways terminating at N2O (Hedlund, 2011).
o Due to the absence of the N2O reductase (nosZ) gene (Murugapiran, 2013).
o T. scotoductus SA-01 also lacks the nosZ gene (Gounder, 2011).
A positive relationship has been observed between temperature and nitrous oxide flux
(Hedlund, 2011).
o High flux of nitrous oxide measured in Great Basin hot springs are consistent with
incomplete denitrification pathways of Thermus strains that were isolated from
that spring.
Ecological implications
o Hot spring environments serve as sources of nitrous oxide, a strong greenhouse
gas and atmospheric reactant.
o Incomplete denitrification pathways may promote N-cycling within hot spring
systems and slow the rate at which nitrogen is removed from the system.
Detection by
Election Capture
Sample
Gaseous
nitrogen
• One previous study indicates that some T. thermophilus isolates can
denitrify completely to dinitrogen (Cava, 2008).
Concentration (mM)
•
•
•
1.4
Concentration(mM)
http://parts.igem.org/wiki/images/5/58/ICL_salkowski_cuvettes.JPG
stationary cells @ 24-287 hrs (107 cells)
1.) Test and optimize PCR conditions
for denitrification genes
2.) Sequence PCR product to determine
diversity of denitrification genes
fromThermus species from different
geographic locations
3.) Repeat end point experiment with
1 mM nitrate-amended CMD
medium
4.) Determine amount of nitric oxide
through reliable assay
5.) Grow Thermus strains with various
denitrification intermediates as
sole terminal electron acceptor
I thank Gisele Goertz for assistance with DNA extraction, Namritha Manoharan and Jeremy Dodsworth for guidance on
PCR amplification work, and the UNLV McNair Scholars Summer Research Institute and the National Science Foundation
(OISE-0968421 and EPS-0814372) for funding. I thank Dr. Wen-Jun Li and Enmin Zhou for providing Thermus isolates
isolated from China for this study.
REFERENCES
1.)
2.)
3.)
4.)
5.)
6.)
7.)
8.)
9.)
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