APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2008, p. 5850–5853
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 18
Quantification of Desulfovibrio vulgaris Dissimilatory Sulfite Reductase
Gene Expression during Electron Donor- and Electron
Laura Villanueva,* Shelley A. Haveman, Zara M. Summers, and Derek R. Lovley
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts
Received 17 February 2008/Accepted 15 July 2008
Previous studies have suggested that levels of transcripts for dsrA, a gene encoding a subunit of the
dissimilatory sulfite reductase, are not directly related to the rates of sulfate reduction in sediments under all
conditions. This phenomenon was further investigated with chemostat-grown Desulfovibrio vulgaris. Under
sulfate-limiting conditions, dsrA transcript levels increased as the bulk rates of sulfate reduction in the
chemostat increased, but transcript levels were similar at all sulfate reduction rates under electron donor-
limiting conditions. When both electron donor- and electron acceptor-limiting conditions were considered,
there was a direct correspondence between dsrA transcript levels and the rates of sulfate reduction per cell.
These results suggest that dsrA transcript levels may provide important information on the metabolic state of
Quantifying levels of transcripts for key genes in anaerobic
sedimentary environments can provide insights into the meta-
bolic state of microorganisms carrying out important processes
in those environments (4, 5, 16, 17, 20, 24). Information can be
gained about the relative rates of microbial metabolism under
different conditions or the factors limiting the rates of growth
and metabolism. Such information is especially useful in the
design of bioremediation strategies because it indicates the
level to which the genes for the desired bioremediation en-
zymes are being expressed and can suggest modifications in
amendments to overcome nutrient limitations or other envi-
ronmental stresses (24).
Methods to evaluate the metabolic state of sulfate-reducing
bacteria are of interest because of the important role that
sulfate reducers play in the biogeochemistry of aquatic sedi-
ments (32) and corrosion (8, 14), as well as in the degradation
of organic contaminants (9). For example, some sulfate reduc-
ers can degrade aromatic hydrocarbons (13), including preva-
lent contaminants such as benzene (23) and polycyclic aro-
matic hydrocarbons (2, 3, 6, 12, 25), and stimulating the activity
of aromatic hydrocarbon-degrading sulfate reducers can be an
effective bioremediation strategy (1, 29).
Dissimilatory sulfite reductase catalyzes the final step in
sulfate reduction, and sequences for this enzyme are highly
conserved among sulfate reducers (18, 19, 31). Pure culture
studies provided a preliminary indication that transcript levels
of dissimilatory sulfite reductase genes might be related to
rates of sulfate reduction (15, 26). However, relating transcript
levels to rates of sulfate reduction in marine sediments was
more problematic (5). Transcripts for dsrA, which encodes the
alpha subunit of dissimilatory sulfite reductase, were quanti-
fied. There was a general trend that sediments with no sulfate
reduction had low dsrA transcript levels and that dsrA levels
were higher with active sulfate reduction. In sediments with
similar sulfate concentrations, there was a direct correlation
between the rates of sulfate reduction and dsrA transcript
levels. However, in laboratory sediment incubations in which
sulfate was continually being depleted over time, dsrA tran-
scripts increased over time even though sulfate reduction rates
did not increase (5). These results suggested that dsrA levels
can only be related to sulfate reduction rates in sediments
under very similar conditions, limiting the predictive value of
dsrA transcript measurements over a range of environmental
In order to better understand the factors controlling dsrA
transcript levels in sulfate reducers, Desulfovibrio vulgaris
Hildenborough (ATCC 29579) was grown under strict anaer-
obic conditions (N2:CO2, 80:20 [vol/vol]) at 30°C in a freshwa-
ter medium (2.5 g liter?1NaHCO3, 0.25 g liter?1NH4Cl, 0.6 g
liter?1or 0.06 g liter?1[for batch or continuous cultures,
respectively] NaH2PO4? H2O, 0.1 g liter?1KCl, and trace
mineral and vitamin solutions) (21) supplemented with a sel-
enite-tungstate solution (400 mg liter?1NaOH, 6 mg liter?1
Na2SO3? 5H2O, 8 mg liter?1Na2WO4? 2H2O) and reduced
with sodium sulfide (1 mM final concentration). Continuous cul-
ture conditions under electron donor (10 mM lactate, 10 mM
sulfate) and electron acceptor (10 mM lactate, 2.5 mM sulfate)
limitations were described previously (10). The methods for mea-
suring substrates and products were as follows: for sulfate, ion
chromatography (22); for fatty acids, high-pressure liquid chro-
matography (10); for sulfide, colorimetry (7); for protein, a
bicinchoninic acid method (30); and for cell numbers, epiflu-
orescence microscopy with acridine orange staining (27).
Total RNA was isolated with the Qiagen RNeasy mini kit
(Qiagen, Inc., Valencia, CA) after digestion of the cell pellet
with 50 mg ml?1lysozyme. Primers DSR1F (forward 5?-AAG
GAA CCC CGC ACC AAC-3?) and DSR1R (reverse 5?-TTA
TCT CAG GTG TCT CTT GCG GT-3?) (position 1 to 102,
* Corresponding author. Present address: Harvard FAS Center for
Systems Biology, Harvard University, Cambridge, MA 02138. Phone:
(617) 496-9519. Fax: (617) 495-2196. E-mail: email@example.com
?Published ahead of print on 25 July 2008.
dsrA gene) were evaluated and optimized for quantitative PCR
as previously described (4). Reverse transcription was per-
formed with an Enhanced Avian First Strand synthesis kit
(Sigma-Aldrich, St. Louis, MO). A dilution series of purified
PCR products (105to 1011molecules) was the calibration stan-
dard for the real-time PCR quantification as previously de-
scribed (16). The cDNA obtained from the reverse transcrip-
tion reaction was quantified in a GeneAmp 5700 sequence
detection system with a reaction mixture of primers (150 nM
each), 9.5 ?l of cDNA (20 to 250 ng ?l?1), 12.5 ?l of Power
Sybr green PCR master mix (Applied Biosystems, Foster City,
CA) to a final volume of 25 ?l. dsr transcript levels were
normalized to the total RNA levels to help account for different
biomass-specific sulfate reduction rates (28).
D. vulgaris was initially grown in chemostats in which elec-
tron-donor availability limited growth (Table 1). This was de-
signed to simulate the conditions in superficial marine sedi-
ments in which sulfate is abundant. Under such conditions, the
growth of sulfate reducers is primarily controlled by the rates
at which complex organic matter is broken down into simpler
substrates that sulfate reducers can utilize. In this system, bulk
sulfate reduction rates (moles of sulfate reduced per liter per
h) at steady state are analogous to the bulk sulfate reduction
rates measured in sediments. The levels of dsrA transcripts
were comparable over the range of sulfate reduction rates
evaluated (Fig. 1A), suggesting that, as previously found in
sediment studies (5), dsrA transcript levels are not always cor-
related with bulk rates of sulfate reduction. In order to inves-
tigate this further, a second set of chemostats (Table 1) were
run in which the electron acceptor, sulfate, was the limiting
nutrient (Fig. 1B). In contrast to the results from the electron
donor-limited chemostats, in the electron acceptor-limited
chemostats there was an increase in dsrA transcript levels that
paralleled increasing sulfate reduction rates at increasing dilu-
tion rates (Fig. 1B).
Differences in the results of the electron donor- versus elec-
tron acceptor-limited chemostats could be reconciled when the
levels of dsrA transcripts were compared with the rate of sul-
fate reduction per cell (Fig. 2). There was a strong correlation
between this cell-specific sulfate reduction rate and dsrA tran-
High levels of dsrA transcripts in the sulfate-limited chemo-
stats could not be attributed solely to low sulfate levels. For
example, when D. vulgaris grown in sulfate-free medium either
with pyruvate as a fermentative substrate or with lactate as the
electron donor in coculture with Methanospirillum hungatei
(DSM864), dsrA levels were low (1.06 ? 104? 2,000 and
8.6 ? 104? 8,000 transcripts per nanogram of RNA, respec-
A comparison of specific sulfate reduction rates and bulk
sulfate reduction rates in the electron donor-limited and elec-
tron acceptor-limited chemostats demonstrated that electron
acceptor-limited cells consistently have a higher respiration
rate per cell (Fig. 3). This is consistent with an apparently
higher maintenance energy requirement (calculated as previ-
ously described by Esteve-Nun ˜ez et al. ) under electron
acceptor-limited conditions (6.3 mmol electrons per gram dry
weight per h) than under electron donor-limited conditions
(2.7 mmol electrons per gram per h). Higher expression of
FIG. 1. Sulfate reduction rate (mmol sulfate consumed at steady
state per liter and h; black squares) and number of dsrA mRNA
transcripts (gray circles) expressed by D. vulgaris cells grown under
electron donor (A) or electron acceptor (B) limitation at different
growth rates. Each point is the average of the results for three samples
from triplicate continuous cultures at each growth rate. (A) Sulfate
reduction rate, R2? 0.997. (B) Sulfate reduction rate, R2? 0.998; dsrA
mRNA expression, R2? 0.967.
TABLE 1. Data at steady-state conditions during continuous
growth of Desulfovibrio vulgaris
Growth type and ratea
6.8 ? 0.2
7.4 ? 0.1
6.9 ? 0.1
7.5 ? 0.5
7.6 ? 0.2
7.9 ? 0.4
7.4 ? 0.03
7.7 ? 0.5
0 ? 0
0 ? 0
0 ? 0
0 ? 0
5.3 ? 0.2
6.4 ? 0.3
5 ? 0.4
6.5 ? 0.4
0.4 ? 0.2
0 ? 0
0.4 ? 0.3
0 ? 0
1.8 ? 0.9
1.3 ? 0.5
2.5 ? 0.7
2.5 ? 0.2
aSpecific growth rate (h?1) or dilution rate in the continuous culture. Steady-
state concentrations of acetate, sulfate, and lactate (average of the results from
triplicate continuous cultures at a certain growth rate ? 3 sampling events and
standard deviation). Initial conditions: electron donor-limited growth, 10 mM
lactate and 10 mM sulfate; electron acceptor-limited growth, 10 mM lactate and
2.5 mM sulfate.
bBiomass values are given in milligrams of dry weight per liter.
VOL. 74, 2008QUANTIFICATION OF dsrA EXPRESSION IN D. VULGARIS5851
respiratory genes under electron acceptor-limiting versus elec-
tron donor-limiting conditions was previously noted in chemo-
stat cultures of Geobacter sulfurreducens grown with either
fumarate or Fe(III) as the electron acceptor (4).
Implications. These results suggest that the level of dsrA
transcripts in an environmental sample cannot be expected to
be related to the bulk rates of sulfate reduction because the
relationship between dsrA transcript levels and the rates of
sulfate reduction can be influenced by the metabolic state of
the cell. This may explain why in a previous study (5) there was
a good correlation between dsrA transcript levels and sulfate
reduction rates at comparable nonlimiting sulfate concentra-
tions, but dsrA levels continued to increase as sulfate concen-
trations declined even though sulfate reduction rates de-
creased. The inability of dsrA transcript levels to serve as a
proxy for bulk sulfate reduction rates may not be a significant
limitation for the study of sulfate reduction in most environ-
ments because several other more straightforward methods,
such as monitoring the reduction of [35S]sulfate to [35S]sulfide
(11), are available for estimating bulk sulfate reduction rates.
However, the results from this pure culture study suggest
that dsrA transcript levels can provide insight into the meta-
bolic state of sulfate reducers because dsrA levels are related to
the rates of sulfate reduction per cell. This basic information
on the metabolic state of sulfate reducers cannot readily be
determined with any other method currently applicable to sed-
imentary environments. For studies with natural communities,
the normalization of dsrA transcript levels to total RNA levels,
as used here for pure cultures, might not be appropriate be-
cause many organisms will contribute to the RNA pool. One
solution that has proven successful in diagnosing the metabolic
status of subsurface Fe(III)-reducing communities is to nor-
malize to the transcript levels of a housekeeping gene se-
quence specific for the organisms under study (16, 17). Thus,
further studies are warranted to determine whether the rela-
tionship between dsrA expression and cell-specific rates of sul-
fate reduction observed here in pure culture holds for diverse
communities typically found in sedimentary environments.
This research was supported by Office of Naval Research award no.
N00014-03-1-0315 and Office of Science (BER), U.S. Department of
Energy, cooperative agreement no. DE-FC02-02ER63446.
1. Anderson, R. T., and D. R. Lovley. 2000. Anaerobic bioremediation of
benzene under sulfate-reducing conditions in a petroleum-contaminated
aquifer. Environ. Sci. Technol. 34:2261–2266.
2. Annweiler, E., A. Materna, M. Safinowski, A. Kappler, H. H. Richnow, W.
Michaelis, and R. U. Meckenstock. 2000. Anaerobic degradation of 2-meth-
ylnaphthalene by a sulfate-reducing enrichment culture. Appl. Environ. Mi-
3. Chang, B. V., L. C. Schiung, and S. Y. Yuan. 2002. Anaerobic biodegradation
of polycyclic aromatic hydrocarbons in soil. Chemosphere 48:717–724.
4. Chin, K. J., A. Esteve-Nu ´n ˜ez, C. Leang, and D. R. Lovley. 2004. Direct
correlation between rates of anaerobic respiration and levels of mRNA for
key respiratory genes in Geobacter sulfurreducens. Appl. Environ. Microbiol.
5. Chin, K. J., M. L. Sharma, L. A. Russell, K. R. O’Neill, and D. R. Lovley.
2008. Quantifying expression of a dissimilatory (bi)sulfite reductase gene in
petroleum-contaminated marine harbor sediments. Microb. Ecol. 55:489–
6. Coates, J. D., J. C. Woodward, J. Allen, P. Philp, and D. R. Lovley. 1997.
Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in
petroleum-contaminated marine harbor sediments. Appl. Environ. Micro-
7. Cord-Ruwisch, R. 1985. A quick method for determination of dissolved and
precipitated sulfides in cultures of sulfate-reducing bacteria. J. Microbiol.
8. Dinh, H. T., J. Kuever, M. Mussmann, A. W. Hassel, M. Stratmann, and F.
Widdel. 2004. Iron corrosion by novel anaerobic microorganisms. Nature
9. Ensley, B. D., and J. M. Suflita. 1995. Metabolism of environmental con-
taminants by mixed and pure cultures of sulfate-reducing bacteria, p. 293–
332. In L. L. Barton (ed.), Sulfate-reducing bacteria. Biotechnology hand-
books, vol. 8. Plenum Press, New York, NY.
10. Esteve-Nu ´n ˜ez, A., M. M. Rothermich, M. Sharma, and D. R. Lovley. 2005.
Growth of Geobacter sulfurreducens under nutrient-limiting conditions in
continuous culture. Environ. Microbiol. 7:641–648.
11. Fossing, H., and B. B. Jørgensen. 1989. Measurement of bacterial sulfate
reduction in sediments: evaluation of a single-step chromium reduction
method. Biogeochemistry 8:205–222.
12. Galushko, A., D. Minz, B. Schink, and F. Widdel. 1999. Anaerobic degra-
dation of naphthalene by a pure culture of a novel type of marine sulphate-
reducing bacterium. Environ. Microbiol. 1:415–420.
13. Gibson, J., and C. S. Harwood. 2002. Metabolic diversity in aromatic com-
pound utilization by anaerobic microorganisms. Annu. Rev. Microbiol. 56:
FIG. 2. dsr mRNA transcripts expressed by D. vulgaris cells grown
under electron donor (gray circles) and acceptor (black squares) lim-
itation at a specific sulfate reduction rate (femtomol sulfate consumed
per cell and day). Each point is the average of the results for three
samples from triplicate continuous cultures at each growth rate. R2?
FIG. 3. Relationship between the specific sulfate reduction rate
(femtomol sulfate consumed per cell and day) and the bulk sulfate
reduction rate (mmol sulfate consumed per liter and h) in D. vulgaris
continuous culture under electron donor (gray circles; R2? 0.9894)
and acceptor (black squares; R2? 0.983) limitation. Each point is the
average of the results for three samples from triplicate continuous
cultures at each growth rate.
5852VILLANUEVA ET AL.APPL. ENVIRON. MICROBIOL.
14. Hamilton, W. A. 1985. Sulphate-reducing bacteria and anaerobic corrosion.
Annu. Rev. Microbiol. 39:195–217.
15. Hayes, L., and D. R. Lovley. 2002. Quantifying expression of dissimilatory
sulfite reductase as an estimate of the rate of sulfate reduction, abstr. N-95.
Abstr. 102nd Gen. Meet. Am. Soc. Microbiol. American Society for Micro-
biology, Washington, DC.
16. Holmes, D. E., K. P. Nevin, and D. R. Lovley. 2004. In situ expression of nifD
in Geobacteraceae in subsurface sediments. Appl. Environ. Microbiol. 70:
17. Holmes, D. E., K. P. Nevin, R. A. O’Neil, J. E. Ward, L. A. Adams, T. L.
Woodard, H. A. Vrionis, and D. R. Lovley. 2005. Potential for quantifying
expression of the Geobacteraceae citrate synthase gene to assess the activity
of Geobacteraceae in the subsurface and on current-harvesting electrodes.
Appl. Environ. Microbiol. 71:6870–6877.
18. Karkhoff-Schweizer, R. R., D. P. Huber, and G. Voordouw. 1995. Conserva-
tion of the genes for dissimilatory sulfite reductase from Desulfovibrio vul-
garis and Archaeoglobus fulgidus allows their detection by PCR. Appl. Envi-
ron. Microbiol. 61:290–296.
19. Klein, M., M. Friedrich, A. J. Roger, P. Hugenholtz, S. Fishbain, H. Abicht,
L. L. Blackall, D. A. Stahl, and M. Wagner. 2001. Multiple lateral transfers
of dissimilatory sulfite reductase genes between major lineages of sulfate-
reducing prokaryotes. J. Bacteriol. 183:6028–6035.
20. Lee, P. K. H., D. R. Johnson, V. F. Holmes, J. He, and L. Alvarez-Cohen.
2006. Reductive dehalogenase gene expression as a biomarker for physio-
logical activity of Dehalococcoides spp. Appl. Environ. Microbiol. 72:6161–
21. Lovley, D. R., R. C. Greening, and J. G. Ferry. 1984. Rapidly growing rumen
methanogenic organism that synthesizes coenzyme M and has a high affinity
for formate. Appl. Environ. Microbiol. 48:81–87.
22. Lovley, D. R., and E. J. P. Phillips. 1994. Novel processes for anaerobic
sulfate production from elemental sulfur by sulfate-reducing bacteria. Appl.
Environ. Microbiol. 60:2394–2399.
23. Lovley, D. R., J. D. Coates, J. C. Woodward, and E. J. P. Phillips. 1995.
Benzene oxidation coupled to sulfate reduction. Appl. Environ. Microbiol.
24. Lovley, D. R. 2003. Cleaning up with genomics: applying molecular biology to
bioremediation. Nat. Rev. Microbiol. 1:35–44.
25. Meckenstock, R. U., E. Annweiler, W. Michaelis, H. H. Richnow, and B.
Schink. 2000. Anaerobic naphthalene degradation by a sulfate-reducing en-
richment culture. Appl. Environ. Microbiol. 66:2743–2747.
26. Neretin, L. N., A. Schippers, A. Pernthaler, K. Hamann, R. Amann, and B. B.
Jørgensen. 2003. Quantification of dissimilatory (bi)sulphite reductase gene
expression in Desulfobacterium autotrophicum using real-time RT-PCR. En-
viron. Microbiol. 5:660–671.
27. Rapposch, S., P. Zangerl, and W. Ginzinger. 2000. Influence of fluorescence
of bacteria stained with acridine orange on the enumeration of microorgan-
isms in raw milk. J. Dairy Sci. 83:2753–2758.
28. Ravenschlag, K., K. Sahm, C. Knoblauch, B. B. Jørgensen, and R. Amann.
2000. Community structure, cellular rRNA content, and activity of sulfate-
reducing bacteria in marine Arctic sediments. Appl. Environ. Microbiol.
29. Rothermich, M. M., L. A. Hayes, and D. R. Lovley. 2002. Anaerobic, sulfate-
dependent degradation of polycyclic aromatic hydrocarbons in petroleum-
contaminated harbor sediment. Environ. Sci. Technol. 36:4811–4817.
30. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner,
M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C.
Klenk. 1985. Measurement of protein using bicinchoninic acid. Anal. Bio-
31. Wagner, M., A. J. Roger, J. L. Flax, G. A. Brusseau, and D. A. Stahl. 1998.
Phylogeny of dissimilatory sulfite reductases supports an early origin of
sulfate respiration. J. Bacteriol. 180:2975–2982.
32. Widdel, F., and T. A. Hansen. 1999. The dissimilatory sulfate- and sulfur-
reducing bacteria. In M. Dworkin, K.-H. Schleifer, and E. Stackebrandt
(ed.), The prokaryotes: an evolving electronic database for the microbiolog-
ical community, 3rd ed., release 3.0. Springer-Verlag, New York, NY.
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