JOURNAL OF BACTERIOLOGY, Apr. 2006, p. 2792–2800
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 188, No. 8
DNA Microarray and Proteomic Analyses of the RpoS Regulon in
Cinthia Nu ´n ˜ez,1,2* Abraham Esteve-Nu ´n ˜ez,1Carol Giometti,3Sandra Tollaksen,3Tripti Khare,3
Winston Lin,1,4Derek R. Lovley,1and Barbara A. Methe ´5
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 010031; Departamento de Microbiologı ´a Molecular,
Instituto de Biotecnologı ´a, UNAM, Cuernavaca, Morelos 62210, Mexico2; Biosciences Division, Argonne National Laboratory,
Argonne, Illinois3; IIT Research Institute, Chicago, Illinois4; and The Institute for
Genomic Research, Rockville, Maryland 208505
Received 5 October 2005/Accepted 25 January 2006
The regulon of the sigma factor RpoS was defined in Geobacter sulfurreducens by using a combination of DNA
microarray expression profiles and proteomics. An rpoS mutant was examined under steady-state conditions
with acetate as an electron donor and fumarate as an electron acceptor and with additional transcriptional
profiling using Fe(III) as an electron acceptor. Expression analysis revealed that RpoS acts as both a positive
and negative regulator. Many of the RpoS-dependent genes determined play roles in energy metabolism,
including the tricarboxylic acid cycle, signal transduction, transport, protein synthesis and degradation, and
amino acid metabolism and transport. As expected, RpoS activated genes involved in oxidative stress resistance
and adaptation to nutrient limitation. Transcription of the cytochrome c oxidase operon, necessary for G.
sulfurreducens growth using oxygen as an electron acceptor, and expression of at least 13 c-type cytochromes,
including one previously shown to participate in Fe(III) reduction (MacA), were RpoS dependent. Analysis of
a subset of the rpoS mutant proteome indicated that 15 major protein species showed reproducible differences
in abundance relative to those of the wild-type strain. Protein identification using mass spectrometry indicated
that the expression of seven of these proteins correlated with the microarray data. Collectively, these results
indicate that RpoS exerts global effects on G. sulfurreducens physiology and that RpoS is vital to G. sulfurre-
ducens survival under conditions typically encountered in its native subsurface environments.
The regulatory networks that govern the physiology of
Geobacter species, which belong to the ? subclass of Proteobac-
teria, are not well known. This is despite the fact that these
organisms are of intense interest, in part because molecular
analyses have demonstrated that they are the predominant
dissimilatory metal-reducing microorganisms in subsurface en-
vironments in which organic contaminants are being degraded
with the reduction of Fe(III) (51, 52, 55) or in which in situ
bioremediation of uranium and vanadium has been stimulated
(36, 47). In addition to conserving energy from electron trans-
fer to metals, Geobacteraceae can generate electrical current
via the transfer of electrons to electrodes as a terminal electron
acceptor (5, 26), which may have applications for harvesting
electricity from a variety of organic matter sources.
The ability to adapt to changing environmental conditions is
crucial for growth and survival of bacteria in their natural
habitats. One important adaptive strategy is the modification
of the transcriptional apparatus in order to transcribe, or “turn
on,” genes necessary to cope with a new condition and to
repress, or “turn off,” genes that are no longer needed (28).
The sigma factor is a subunit of RNA polymerase that in
bacteria confers the property of promoter specificity on RNA
polymerase in the initiation of transcription (62). Thus, the
pool of sigma factors within the cell is a critical contributing
factor in the determination of the genes to be transcribed at a
particular time point and cell state (21, 28). The rpoS gene
encodes a sigma factor, ?Sor RpoS, which is the master reg-
ulator of the general stress response. First described for Esch-
erichia coli, homologs of RpoS in the ?, ?, and ? subclasses of
Proteobacteria have since been characterized (23, 24, 35, 44).
In previously studied bacterial systems, activities of RpoS-
dependent genes vary, with most related to mechanisms of
resistance to various stress conditions, such as high tempera-
ture (25, 50), oxidizing agents, UV irradiation (43), pH, and
osmotic pressure (25), and to pathogenesis (18, 29, 46). Hence,
expression of the RpoS regulon provides a mechanism for cell
adaptability to changing environments. E. coli contains one of
the best-studied examples of the RpoS regulon. Approximately
200 genes have been estimated to be members of its regulon
based on results from transcriptional fusion (61) and microar-
ray expression (30, 48) studies. Such studies not only have
confirmed previously described functions for the RpoS regulon
but also have revealed that RpoS regulates the uptake and
metabolism of amino acids, sugars, and iron; carbon compound
catabolism; and central intermediary metabolism (30). This
further supports the role of RpoS as a global regulator. Exten-
sive negative regulation of gene transcription by RpoS was also
uncovered, including the repression of almost all genes re-
quired for flagellum biosynthesis and genes encoding enzymes
of the tricarboxylic acid (TCA) cycle (48).
Previously we described an RpoS homolog in Geobacter
sulfurreducens (44). It was found that RpoS is necessary for
* Corresponding author. Mailing address: Departamento de Micro-
biologı ´a Molecular, Instituto de Biotecnologı ´a, Universidad Nacional
Auto ´noma de Me ´xico, Av. Universidad 2001, Col. Chamilpa Cuerna-
vaca, Morelos 62210, Mexico. Phone: (52) 777 329-16-29. Fax: (52) 777
317-23-88. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jb
survival in stationary phase and upon oxygen exposure as well
as for effective reduction of Fe(III) oxides, the primary elec-
tron acceptor for the Geobacteraceae in most sedimentary en-
vironments. Here we report an initial characterization of the
RpoS regulon in G. sulfurreducens in order to better under-
stand its mechanisms of stress response. This was achieved by
comparison of gene expression and protein profiles, using
DNA microarray and proteomics analyses, of the G. sulfur-
reducens wild-type strain and an rpoS mutant derivative grown
in continuous culture with acetate as the electron donor and
fumarate as the electron acceptor. Additional transcriptional
profiling was also conducted using Fe(III) as an electron ac-
ceptor. The results indicate that in G. sulfurreducens RpoS has
both negative and positive effects on gene transcription and
that the corresponding regulon comprises genes with very di-
MATERIALS AND METHODS
Bacterial strains and culture conditions. Wild-type G. sulfurreducens strain
DL1 (11, 12) and mutant DLCN16 (?rpoS::Km) (44) were grown under anaer-
obic conditions at 30°C in continuous culture with a 200-ml working volume as
previously described (16). Cells were cultured at a growth rate of 0.05 h?1.
Steady-state cell growth was obtained after five volume refills and was confirmed
by a constant cell density and constant concentrations of fumarate and succinate
or Fe(II). Acetate (5.5 mM) was the electron donor and the limiting substrate.
The electron acceptor was fumarate (30 mM) or Fe(III)-citrate (60 mM). Cells
derived from these cultures were used to carry out proteomics as well as DNA
microarray analyses as described below. Oxygen respiration assay was carried out
as previously described (33) using strain DLI, an rpoS mutant.
RNA isolation. Total RNA was extracted as previously described by first
mechanically disrupting cells using a FastPrep instrument (Qbiogene, Carlsbad,
CA) with Lysing Matrix B (Qbiogene, Carlsbad, CA), followed by nucleic acid
extraction with TRIzol reagent (Invitrogen, Carlsbad, CA) (a monophasic solu-
tion of phenol and guanidine isothiocyanate) (41). Any residual DNA was re-
moved using RNase-free DNase according to the manufacturer’s instructions
(Ambion, Austin, TX), and treated RNA was subsequently cleaned and concen-
trated with RNeasy minicolumns (QIAGEN Inc., Valencia, CA). The quality of
total RNA was assessed by agarose-formaldehyde gel electrophoresis and the
concentration determined using a NanoDrop ND-1000 spectrophotometer
(NanoDrop Technologies, Wilmington, DE).
DNA microarray transcriptional profiling. A DNA microarray, as previously
described (41), was used for transcriptional profiling. Briefly, the microarray
consisted of 3,417 unique PCR products representing predicted coding se-
quences of the G. sulfurreducens genome, and an additional 51 coding sequences
were represented in duplicate. Several intergenic regions of the genome were
also represented. Amplicons (reporters) were resuspended to a concentration of
100 to 200 nM in 50% dimethyl sulfoxide prior to printing onto UltraGaps
aminosilane-coated slides (Corning Life Sciences, Acton, MA) using a Brooks
Automation Systems (Cambridge, MA) array spotter. All reporters were printed
a total of six times per slide. After printing, all slides were cross-linked using a
Stratalinker UV cross-linker (Stratagene, La Jolla, CA) and stored under vac-
uum until use.
Total RNA was used for indirect labeling of targets with either Cyanine 3
(Cy3) or Cy5 fluorescent dye as previous described (41). Targets used for hy-
bridizations in both experiments consisted of approximately 4 to 5 ?g of cDNA,
with greater than 200 pmol of dye molecule incorporated per microgram of
cDNA synthesized. Triplicate control and treatment chemostat cultures were
extracted for the experiment conducted using fumarate as an electron acceptor
and in duplicate for the experiment using Fe(III) citrate as an electron acceptor.
Extracted RNA was subsequently paired to produce three biological replicates
and two biological replicates, respectively, for the fumarate and Fe(III) RpoS
experiments from which hybridizations could be repeated (technical replicates).
Prehybridization of slides consisted of their immersion in a solution of 5? SSC
(1? SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% sodium dodecyl
sulfate (SDS), and 1% bovine serum albumin for 45 min at 42°C, after which
slides were washed and dried. Labeled cDNA was resuspended in a solution of
50% formamide, 5? SSC, 0.1% SDS and allowed to hybridize for 18 to 20 h at
42°C under glass coverslips. After hybridization, slides were washed two times for
4 minutes each in the following series of solutions: 1? SSC, 0.2% SDS; 0.1?
SSC, 0.1% SDS; and 0.1? SSC. A final wash for 30 seconds in 0.05? SSC was
completed prior to drying by centrifugation. Slides were promptly scanned at a
10-?m resolution using an Axon 4000B scanner with GenePix 4.0 software
(Molecular Devices Corp., Sunnyvale, CA).
Microarray significance analysis. Processing of 16-bit TIFF images from hy-
bridized arrays was done using the TIGR TM4 package (www.tigr.org/software).
Intensity values for Cy3 and Cy5 channels were obtained using TIGR Spotfinder
software. Normalization was performed using the LOWESS algorithm available
in TIGR MIDAS, using block mode and a smooth parameter of 0.33. All
intensity values less than two times greater than background were removed from
subsequent analysis, and replicate reporter intensities on one slide (one technical
replicate) were reduced to a single value by computing the geometric mean. Six
hybridizations were performed from each of three biological replicate chemostat
pairs (control and treatment) for the fumarate condition. Four technical repli-
cates were performed from each of the two biological replicates for the Fe(III).
As part of overall quality assurance, half of the technical replicate dye labelings
in each experiment were performed as dye swaps (flip dyes).
To determine genes whose expression was significantly different from zero,
Significance Analysis of Microarrays (SAM) software (60) was employed, using
the one-class response with 1,000 permutations. The biological replicates for
each condition were analyzed individually. Significant genes were determined by
setting the number of falsely called genes to less than one and choosing similar
false discovery percentage medians for each biological replicate resulting in
similar total numbers of significant genes for output with greater than a 1.5-fold
change in expression. At these levels, the q values (a measure of significance in
terms of the false discovery rate) (57) for the three biological replicates for the
fumarate condition and two biological replicates for the Fe(III) conditions were
all less than 1 percent. The intersections from the significant gene sets from each
biological replicate were further analyzed. Significant reporters present in each
of the two biological replicates based on SAM analysis for the Fe(III) condition
totaled 162. In the fumarate condition, reporters which had significant changes in
expression based on SAM analysis from all three biological replicates totaled
152, and this number increased to a total of 294 when the criterion of significant
expression in any two of three biological replicates was considered.
EASE analysis. Expression analysis systematic explorer (EASE) analysis was
performed on the subset of genes determined to have significant changes in
expression in at least two biological replicates from the fumarate condition and
in the two biological replicates from the Fe(III) condition as identified by the
SAM analysis as previously described (41). EASE uses a modified Fisher exact
test (EASE score) to estimate the significance of classes of biological function
present in a subset of significant genes relative to the total as represented on the
array (27). TIGR role categories (www.tigr.org) and gene ontology (GO) terms
(3) determined as part of the whole-genome annotation of G. sulfurreducens (40)
were used as the biological classes examined for overrepresentation in the lists of
significant genes. Only biological classes with EASE scores of ?10?3are re-
Microarray data. All microarray data presented here are in accordance with
the Microarray Gene Expression Data Society’s recommendations for minimum
information about a microarray experiment (7).
Two-dimensional electrophoresis (2DE) of G. sulfurreducens. Aliquots of the
wild-type and rpoS mutant cytosol fractions from triplicate experiments with cells
grown with acetate as the limiting substrate and fumarate as electron donor were
mixed with 2 volumes of a solution containing 9 M urea, 2% 2-mercaptoethanol,
2% ampholytes (pH 8 to 10; Bio-Rad, Hercules CA), and 2% Nonidet P-40
(Roche Diagnostics, Indianapolis, IN). The soluble, denatured proteins were
recovered in supernatants by centrifugation at 435,000 ? g for 10 min using a
Beckman TL100 tabletop ultracentrifuge. Protein concentrations were deter-
mined using a modification of the Bradford protein assay (49).
Aliquots of sample containing 40 ?g of protein were separated in the first
dimension by isoelectric focusing using polyacrylamide gels containing 50% pH
5 to 7 with 50% pH 3 to 10 carrier ampholytes (1). After 14,000 V-h, the
first-dimension gels were equilibrated with SDS and the proteins were separated
by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described by
O’Farrell (45), using a linear gradient of 10 to 17% acrylamide (2). Proteins were
then detected by staining with silver nitrate (19).
Image acquisition and analysis of 2DE gels. The 2DE images were digitized
using an Eikonix1412 scanner interfaced with a VAX 4000-90 workstation. The
images were then transferred to a personal computer, converted to TIFF format,
and then processed for spot detection and pattern matching using the Progenesis
software (Nonlinear USA, Research Triangle Park, NC). One 2DE image of
cytosol proteins from wild-type cells was used as a reference pattern for the
experiment. All patterns in the experiment (six wild-type and five mutant pat-
VOL. 188, 2006RpoS REGULON IN GEOBACTER SULFURREDUCENS2793
terns representing duplicate or triplicate gels for duplicate biological replicates)
were matched to the reference pattern so that the protein spots were given
identification numbers. Statistical analysis of the relative abundance of each
matched protein spot across the data set was done using a two-tailed Student t
test as previously described (20).
Protein identification by mass spectrometry. Proteins to be identified were cut
from two or three replicate gels stained with Coomassie blue R250 (approxi-
mately 200 mg of protein was loaded on each gel), and the proteins were digested
in the gel with trypsin (sequence-grade trypsin; 12.5 ng/mg; Promega, Madison,
WI). The resulting peptides were eluted from the gel pieces by extracting three
times, first with equal parts of 25 mM ammonium bicarbonate and acetonitrile
and then twice with equal parts of 5% (vol/vol) formic acid and acetonitrile. The
eluted tryptic peptides were desalted and concentrated with a commercial ZipTip
C18pipette tip (Millipore, Bedford, MA). Peptide samples were then loaded
onto a 365- by 100-?m fused silica capillary column packed with 10-?m POROS
10 R2 packing material (PE Biosystem, Long Beach, CA) at a length of 10 to 15
cm. Peptides were separated with a 30-minute linear gradient of 0 to 60% solvent
containing 80% acetonitrile and 0.5% acetic acid and then entered into an LCQ
ion trap mass spectrometer (Finnigan MAT, Milford, MA). Tandem mass spec-
tra (MS/MS) were automatically collected under computer control during the
30-minute liquid chromatography/mass spectroscopy runs. MS/MS spectra were
then directly subjected to SEQUEST database searches (15, 53) by correlating
experimental MS/MS spectra to predicted protein sequences in the G. sulfur-
reducens open reading frame database. Protein identifications were accepted as
correct when, for multiple peptides associated with the same 2DE gel spot, the
SEQUEST search yielded a minimum DeltaCN of 0.08 and a minimum cross
correlation (Xcorr) score of 1.8 for all peptides of 1? charge state for fully tryptic
peptides, a minimum Xcorr of 2.5 for peptides of 2? charge state for a full or
partial tryptic peptide, and a minimum Xcorr of 3.5 for peptides of 3? charge
state for a full or partial tryptic peptide.
Microarray accession numbers. Descriptions of the microarray experiments,
quantitation data, and array design have been deposited into ArrayExpress
(www.ebi.ac.uk/arrayexpress) and have been assigned accession numbers
E-TIGR-123, E-TIGR-128, and A-TIGR-20.
RESULTS AND DISCUSSION
In order to analyze the RpoS regulon in G. sulfurreducens,
wild-type and DLCN16 (?rpoS::Km) strains were grown in
continuous culture to obtain stable cultures, ensuring that tran-
scriptome and protein expression patterns were consistent and
reproducible for comparison. Furthermore, there is evidence
indicating that a continuous culture is a better representation
of life in subsurface environments than batch growth (16).
Fumarate-respiring cells were used for parallel DNA microar-
ray hybridization and proteomic analysis to investigate the
RpoS-dependent gene transcription and translation correla-
tion. The transcriptional profile of the RpoS regulon was also
examined using soluble Fe(III) as an electron acceptor.
Identification of RpoS-regulated proteins by proteomic
analysis. Compared with the proteome of the wild-type G.
sulfurreducens strain, rpoS mutant cells contained a total of 12
polypeptide spots (spots 293, 339, 386, 498, 647, 714, 728, 737,
768, 769, 1159, and 1200) whose abundance was severely di-
minished, suggesting that they represent RpoS-regulated genes
(Fig. 1). Furthermore, RpoS appeared to have a negative effect
on the regulation of some G. sulfurreducens genes, as the in-
tensities of four polypeptides (spots 134, 404, 719, and 1167)
were significantly increased in the mutant cells relative to the
wild type. The proteins differentially expressed in the wild-type
strains versus the rpoS mutant (numbered proteins in Fig.1A
and B) were identified on the basis of their tryptic peptide
masses (Table 1). The extent of the differential expression
varies from proteins virtually undetectable in the rpoS mutant,
such as the Trp repressor-binding protein (WrbA), to proteins
such as ClpB, whose abundance increased twofold upon rpoS
Transcriptional profile of the RpoS regulon in G. sulfur-
reducens with fumarate as an electron acceptor. Transcripts
from the wild-type strain DL1 were compared to those from its
isogenic rpoS mutant by using a DNA microarray for expres-
sion analysis. In fumarate-grown cells 196 genes were down-
regulated (genes positively regulated or activated by RpoS)
and 98 were up-regulated (genes repressed by RpoS) as a
consequence of the lack of RpoS (see Tables S1 and S2 in the
FIG. 1. 2DE patterns of soluble-fraction proteins isolated from the
G. sulfurreducens DL1 wild-type strain (A) and its rpoS mutant deriv-
ative DLCN16 (B) grown with fumarate as electron acceptor. Protein
spots showing significant quantitative differences between the two strains
are indicated by arrows and numbers. The gel images are oriented with
the isoelectric focusing dimension horizontal and the SDS-PAGE dimen-
sion vertical. The approximate positions of the SDS-PAGE molecular
mass (MW) standards are presented along the vertical axis.
2794 NU ´N ˜EZ ET AL.J. BACTERIOL.
Genes activated by RpoS fall into 14 of 20 functional main-
role categories (40) (Fig. 2). Except for the genes categorized
as hypothetical (37 genes), conserved hypothetical (27 genes),
or of unknown function (19 genes), the majority of RpoS-
activated genes are involved in energy metabolism (51 genes).
RpoS also activated genes involved in transport and binding
proteins (17 genes), regulatory functions and signal transduc-
tion (16 genes), the cell envelope (9 genes), protein fate (7
FIG. 2. Global view of RpoS-regulated genes from fumarate-respiring cells. The genes were identified as either activated or repressed by RpoS
with the use of the comparison of gene expression profiles in DL1 versus DLCN16 (?rpoS::Km) grown as described in Materials and Methods.
TABLE 1. Identities of proteins in 2DE spots showing statistically significant differences in expression levels in lysates
from rpoS mutant and wild-type cells
293 0.296 GSU243544,3274.9 Dehydrogenase complex E2-component,
Rubredoxin-oxygen oxidoreductase, putative
Ribosomal protein S2 (rpsB)
Conserved hypothetical protein
Nitroreductase family proteins
Trp repressor-binding protein WrbA
Conserved hypothetical protein
Conserved hypothetical protein
Laccase family protein
Superoxide dismutase (sodA)
ATP-dependent Clp protease, ATP-binding
Aminopeptidase A/I (pepA)
carboxamide ribotide isomerase
Pyruvate phosphate dikinase (ppdK)
Amino acid biosynthesis2
1167 2.154 GSU0580 97,1115.6 Energy metabolism
aRatio of the average integrated densities (spot volumes) of protein spots displaying quantitative differences in integrated density with a P value of at least 0.05 after
statistical analysis using a two-tailed Student t test.
bND, not detected
cNumber of tryptic peptides with masses that matched, within the acceptance criteria (DeltaCN and Xcorr) specified in Materials and Methods, the open reading
frame (locus identification).
dPredicted protein molecular weight based on the corresponding open reading frame sequence.
ePredicted isoelectric point based on the corresponding open reading frame sequence.
fAnnotation from http://www.ncbi.nlm.nih.gov/.
VOL. 188, 2006RpoS REGULON IN GEOBACTER SULFURREDUCENS 2795
genes), and cellular processes (5 genes). Genes apparently
repressed by RpoS fall into 17 functional categories and in-
clude genes involved in protein synthesis (17 genes), hypothet-
ical genes (14 genes), genes involved in the cell envelope (11
genes), genes involved in protein fate (8 genes), genes of un-
known functions (9 genes), and conserved hypothetical genes
(5 genes) (Fig. 2).
Results of the EASE analysis indicate that GO terms rep-
resenting biological processes and molecular functions related
to electron transport, TCA, and transporter activity were sig-
nificantly overrepresented in the set of genes activated by
RpoS (see Table S3 in the supplemental material), while for
genes repressed by RpoS, EASE analysis indicates an overrep-
resentation of biological processes and functions related to
protein synthesis and metabolism as well as ribosome biogen-
esis and assembly (see Table S4 in the supplemental material).
These data indicate that the sigma factor RpoS coordinates the
global expression of many genes that are important for a va-
riety of functions in actively dividing cells of G. sulfurreducens.
G. sulfurreducens RpoS regulates growth on oxygen and the
expression of genes with antioxidant functions. Although G.
sulfurreducens was originally described as a strict anaerobe,
analysis of the G. sulfurreducens genome (40) and recent phys-
iological studies have shown that it can tolerate exposure to
oxygen and even grow with low levels of oxygen (5%) as the
sole electron acceptor (33). Transcriptome analysis of G. sul-
furreducens indicated that RpoS positively regulates the ex-
pression of the cytochrome c oxidase (aa3), as transcript levels
of two genes (GSU0219 and GSU0222) within the putative
cytochrome c oxidase operon were significantly reduced, by
approximately twofold, in the rpoS mutant (see Table S1 in the
supplemental material). The presence of a functional cyto-
chrome c oxidase is required for G. sulfurreducens to grow with
oxygen as a terminal electron acceptor (W. Lin, unpublished
data). Therefore, it is of interest that the rpoS mutant was
unable to grow with oxygen as the sole electron acceptor under
conditions which support the growth of the wild type (Fig. 3).
The expression of the two genes (GSU1640 and GSU1641)
encoding subunit I and subunit II of the cytochrome d ubiqui-
nol oxidase (a high-oxygen-affinity terminal oxidase) was also
significantly reduced in the rpoS mutant. Further work is
needed to assess the role of this oxidase in oxygen respiration
in G. sulfurreducens. The control of terminal oxidases by RpoS
is not unprecedented; in E. coli the expression of the cbdAB
genes, encoding the cytochrome bd-II oxidase, is dependent on
RpoS in either the logarithmic or stationary phase of growth
(4, 48, 61).
Previously, it was demonstrated that G. sulfurreducens re-
quires RpoS for optimal tolerance to oxygen exposure during
growth on fumarate (44). Consistent with the role of RpoS in
oxidative stress resistance was the positive transcriptional reg-
ulation of genes encoding a ferrodoxin (GSU3187), a rubredoxin
(GSU3188), and a desulfoferredoxin ferrous iron-binding pro-
tein (GSU0720). Furthermore, the proteomic analysis revealed
that protein levels of a superoxide dismutase (GSU1158) and
those of a putative rubredoxin-oxygen oxidoreductase (GSU
3294) were reduced approximately seven- and fourfold, respec-
tively, in the rpoS mutant, (Table 1). One gene strongly af-
fected by RpoS encoded a prismane protein (GSU0674). The
microarray and proteomic results suggest that both transcrip-
tion and translation appear to be largely dependent on RpoS.
Two polypeptide spots (spots 339 and 386 in Table 1) corre-
spond to the same prismane protein, suggesting the existence
of a modified version of this protein. Although it has been
implicated in the oxidative stress response, its exact function is
not clearly understood (9).
These effects on the G. sulfurreducens transcriptome and
proteome could, in part, account for the reduced survival of
the rpoS mutant in the presence of oxygen and are consistent
with the concept that RpoS contributes not only to the survival
but also to the prevalence of G. sulfurreducens under sediments
exposed to oxygen. Conversely it is important to point out the
existence of additional genes in the G. sulfurreducens genome
whose predicted functions are to cope with oxidative damage
but that apparently do not belong to the RpoS regulon, based
on their lack of differential expression in this study. These
include genes encoding several thiol peroxidases (GSU0352,
GSU0893, and GSU3246) which putatively participate in the
elimination of H2O2and a gene encoding a putative ru-
c-type cytochromes in the G. sulfurreducens RpoS regulon.
Transcript levels for 16 c-type cytochromes were diminished,
while those for 5 were up-regulated, as a result of the rpoS
mutation (see Tables S1 and S2 in the supplemental material).
Among the genes whose expression was significantly reduced
in the rpoS mutant were those encoding the periplasmic c-type
cytochrome MacA (GSU0466) (?5-fold reduction), which is
proposed to be an intermediary in the electron transfer to
Fe(III), and OmcC (GSU2731) (6-fold reduction on fuma-
rate), a homolog of the OmcB (GSU2737) cytochrome that
participates in the electron transfer to insoluble and soluble
Despite the high degree of identity (78%) between the
OmcB and OmcC proteins, only OmcB is necessary for Fe(III)
reduction under the conditions tested; thus, the function of
OmcC remains elusive. The omcB and omcC genes are part of
a tandem chromosomal duplication consisting of two repeated
FIG. 3. Growth of G. sulfurreducens on oxygen. Growth of strain
DL1 (circles) and its mutant derivative rpoS (squares) following the
introduction of 5% oxygen into the headspaces of cultures pregrown
on 5 mM fumarate is shown. Arrows indicate the times at which 5%
oxygen was added. Data shown are the means from two replicate
2796NU ´N ˜EZ ET AL.J. BACTERIOL.
clusters of four genes (31, 40). Transcriptional regulation of
these clusters is complex. Leang and Lovely (32) reported that
omcC is contained in two transcriptional units, the first one
monocistronic and the second one polycistronic (orf1-orf2-
omcC), both controlled by RpoS-dependent promoters. Thus,
in the rpoS mutant strain DLCN16, no transcripts of omcC
were detected by either Northern blot or primer extension
analysis, validating the DNA microarray result. Further, the
transcript levels of orf2 (GSU2732), which is cotranscribed
with omcC, were also diminished in the rpoS mutant. Microar-
ray expression data from the current study show that omcB was
down-regulated in the rpoS mutant approximately 2.4-fold
when cells were grown on fumarate. However, Leang and
Lovely (32) found that under fumarate-respiring conditions
this gene was fivefold upregulated as a result of the rpoS
mutation. This discrepancy may be due to the fact that these
later experiments were carried out with batch culture cells,
whereas the microarray expression experiment was done using
cells derived from continuous cultures. The potential role of
the additional c-type cytochromes in the Fe(III) reduction
capabilities of G. sulfurreducens remains to be investigated,
especially for those cytochromes whose transcription was
strongly affected by RpoS, such as one encoded by the
GSU0357 gene whose expression was greatly diminished (?6-
fold) in the rpoS mutant.
Regulation of citric acid cycle enzymes. The expression of
several genes encoding enzymes of the TCA cycle, such as
tate hydratase 2 (AcnB) (GSU1660), and NADP-dependent
isocitrate dehydrogenase (GSU1465), was significantly RpoS
dependent. This positive regulation indicates a role of RpoS in
regulating and increasing the efficiency of the TCA cycle under
the conditions tested. This is further supported by the signifi-
cant overrepresentation of the GO biological process term
related to the TCA cycle in the set of genes activated by RpoS
as determined from the EASE analysis (see Table S3 in the
These results, to the best of our knowledge, are the first
instance in which RpoS has been experimentally determined to
be involved in the positive regulation of enzymes of the TCA
cycle. In contrast, previous reports on E. coli have suggested
that RpoS represses the expression of several enzymes of the
TCA cycle during stationary phase, including AcnB and SdhA
(a subunit of the succinate dehydrogenase) (14, 48, 63). This
difference could be due to the fact that cells from continuous
culture are actively growing, in contrast to what is observed in
the E. coli stationary-phase batch culture.
RpoS-mediated control of the respiratory hydrogenases Hyb
and Hya. G. sulfurreducens can grow by coupling the oxidation
of hydrogen to the reduction of a variety of electron acceptors,
including Fe(III) and fumarate (11). The existence of two
hydrogen uptake respiratory dehydrogenases, encoded by the
hyaSLBP and hybSABLP operons, was previously demon-
strated in G. sulfurreducens (13, 40). Only Hyb was shown to be
essential for hydrogen-dependent growth. Transcriptional pro-
filing in the present study showed that RpoS regulates the
expression of these two operons, since the relative transcript
abundances of three (GSU0121, GSU0122, and GSU0123) and
four (GSU0782, GSU0783, GSU0784, and GSU0785) genes in
the hya and hyb operons, respectively, was significantly altered
in the rpoS mutant. Interestingly, the expression of the hya
operon was activated, while that of the hyb operon was re-
pressed, by RpoS (see Tables S1 and S2 in the supplemental
material). Differential expression of the operons involved in
hydrogen uptake was also reported in a previous microarray
study when G. sulfurreducens transcription was examined in
cells with Fe(III) as a terminal electron acceptor versus those
respiring with fumarate (41). The significance of this contrast-
ing regulation in the present study is not known, but it might be
related to a possible compensatory role of these two hydroge-
nases under specific conditions. In E. coli, Hya is induced
under carbon and phosphate starvation and during stationary
phase, and this induction is also RpoS dependent (4).
Genes for adaptation to adverse conditions. As in many
gram-negative bacteria, G. sulfurreducens RpoS contributes to
long-term survival (44). Expression data revealed that RpoS
positively regulates genes encoding a universal stress protein
(GSU1118); spore coat protein A, whose functions remains un-
clear (GSU2657); and a mechanosensitive channel (GSU2794),
which is proposed to protect cell integrity when cells are exposed
to hypo-osmotic shock. In E. coli, the expression of mechanosen-
sitive channels MscA and McsL are RpoS dependent upon entry
into stationary phase or hyperosmotic shock (56).
The gene greA (GSU1277), encoding a transcription elonga-
tion factor, was among those genes whose expression was sig-
nificantly increased in the rpoS mutant. During transcription,
the RNA polymerase forms an elongation complex with its
template DNA and the nascent RNA product. The rate of
elongation responds to intrinsic signals such as the lack of
nucleotides, which can lead to a transient pause or arrest of the
complex (59). The function of GreA is to reactivate RNA
polymerase once such a halt has occurred. In G. sulfurreducens
the absence of RpoS might lead to an arrest of RNA polymer-
ase-DNA-mRNA complexes, requiring the activation of the
greA gene to overcome this condition.
Transcriptional profile of the RpoS regulon in G. sulfur-
reducens with soluble Fe(III) as an electron acceptor. During
Fe(III) reduction RpoS positively regulates the expression of
137 genes (see Table S5 in the supplemental material), 33 of
which are associated with energy metabolism, constituting the
largest role category of genes. Other role categories whose
expression was significantly affected in the rpoS mutant include
genes encoding hypothetical proteins (25 genes), proteins of
unknown functions (22 genes), conserved hypothetical proteins
(19 genes), proteins for signal transduction and regulatory
functions (13 genes), transport and binding proteins (10
genes), and the cell envelope (7 genes). Only 44 out of the 137
RpoS-activated genes were specifically down-regulated under
Fe(III) respiring conditions; the remainder were also down-
regulated in the rpoS mutant grown with fumarate as the elec-
tron acceptor. The transcription of at least 25 genes was re-
pressed by RpoS under Fe(III)-respiring conditions (see Table
S6 in the supplemental material), including genes encoding
hypothetical proteins (six genes) or proteins of unknown func-
tion (five genes). Of these 25 genes, 15 were apparently neg-
atively regulated by RpoS exclusively under Fe(III)-respiring
conditions, while expression of the remaining 10 reporters was
also inhibited (as indicated by increased expression in the rpoS
mutant) during growth with fumarate as electron acceptor.
An EASE analysis of the set of genes regulated by RpoS
VOL. 188, 2006 RpoS REGULON IN GEOBACTER SULFURREDUCENS 2797
under Fe(III) respiration conditions indicates that the GO
term of molecular function relating to two-component re-
sponse regulator activity and the biological processes of elec-
tron transport and the TCA cycle were significantly overrep-
resented for genes activated by RpoS (see Table S7 in the
supplemental material). GO terms representing biological pro-
cesses and molecular functions related to transport of amino
acids and organic acids were significantly overrepresented
among RpoS-repressed genes (see Table S8 in the supplemen-
tal material). The apparent regulation of a number of pro-
teases and amino acid transporters by RpoS under either fu-
marate or Fe(III) reduction implies that this sigma factor
facilitates the turnover of proteins and the balance of amino
acid pools within the cell. Although this function has been
previously described for E. coli (30, 61), it is remarkably im-
portant for the survival of G. sulfurreducens under subsurface
environments, given the limitation of electron donor that
seems to predominate under this condition and in which the
synthesis de novo of amino acids would be energetically ex-
Transcription of 13 c-type cytochromes appeared to be ac-
tivated by RpoS during growth on Fe(III). Under fumarate-
respiring conditions, these cytochromes exhibited similar fold
changes in expression, indicating that their transcription is not
related exclusively to the presence of Fe(III). Although the
involvement of several c-type cytochromes in the reduction of
Fe(III) has been well established (10, 31, 34), the functions of
the majority of them remain to be elucidated. The c-type cy-
tochrome MacA is involved in the reduction of Fe(III) oxide
(10), suggesting that the decreased reduction of insoluble
Fe(III) by the rpoS mutant (44) might be due to the lack of
MacA activity or to the simultaneous low transcript levels of all
13 c-type cytochromes (or some combination thereof) in the
Expression of the cytochrome c oxidase operon was appar-
ently activated by RpoS under Fe(III)-respiring conditions,
since transcription of the first two genes (GSU0222 and
GSU0219) was inhibited in the rpoS mutant strain. Likewise,
expression of the prismane protein gene (GSU0674) and that
of a putative cytochrome c551 peroxidase gene (GSU2813)
which potentially participate in the G. sulfurreducens oxidative
stress response were positively regulated by RpoS in the pres-
ence of Fe(III). The significance of the RpoS-mediated acti-
vation of these genes remains unclear and requires further
investigation, because electron transfer to oxygen is unlikely
under the extreme anaerobic condition of Fe(III) reduction.
Concluding remarks. In this study a total of 294 and 162
genes under conditions of fumarate and Fe(III) respiration,
respectively, were identified as differentially expressed in a G.
sulfurreducens rpoS mutant evaluated by DNA microarray ex-
pression profiling. Approximately 30% of the genes in the
regulon were categorized as hypothetical genes, conserved hy-
pothetical genes, or genes with unknown function, suggesting
that investigation into their roles could provide further insights
into RpoS function in G. sulfurreducens. Using proteomic anal-
ysis, we successfully identified 16 polypeptides whose expres-
sion was regulated by RpoS under fumarate-respiring condi-
tions. The expression of seven proteins correlated with the low
levels of the corresponding mRNA in the rpoS mutant accord-
ing to the microarray expression data. However, there are eight
proteins (GSU2435, GSU3294, GSU1921, GSU1158, GSU658,
GSU0332, GSU3096, and GSU0580) whose abundance was
significantly affected by RpoS but for which the transcription of
the corresponding genes was not significantly changed based
on the DNA microarray expression profile. This result is not
unexpected, since there are reports indicating differences be-
tween parallel profiles of transcripts and proteins in a cell (22).
Further, a perfect correlation between the DNA microarray
and proteomic analyses was not anticipated due to the intrinsic
technical differences between the two experimental methods.
However, these results may also indicate important biolog-
ical consequences in G. sulfurreducens. Although the sigma
factor RpoS is a component of the transcription machinery, it
is likely to have an effect on the translation of some genes, as
has been reported for other gram-negative bacteria. For exam-
ple, in Erwinia carotovora, RpoS negatively affects the produc-
tion of extracellular enzymes by up-regulating the transcription
of the rsmA gene, encoding an RNA-binding protein that re-
presses translation of target genes (42).
Among the many genes in the RpoS regulon, some might be
directly regulated by RpoS and others might be indirectly reg-
ulated. Consistent with this idea are the findings in the current
study of RpoS-dependent expression of a variety of genes
which participate in two-component response regulatory sys-
tems and transcriptional regulators, which in turn regulate the
expression of other genes. The transcription of approximately
30% of the RpoS regulon was repressed. To the best of our
knowledge, the only sigma factor that is able to act directly as
a repressor is RpoN, by virtue of its intrinsic ability to bind
DNA in the absence of the RNA polymerase holoenzyme (6).
Thus, as has been proposed for other bacteria, the negative
regulation exerted by RpoS might be indirect and involve
sigma factor competition (17, 54). A decrease in one sigma
factor normally present under certain conditions allows other
sigma factors, for example, RpoD or ?70, to compete for a
limiting amount of core RNA polymerase more successfully.
Thus, promoters regulated by sigma factors other than RpoS
are expected to show elevated activity in an rpoS mutant in
which the RpoS protein is absent. In addition, the transcription
of some RpoS-repressed genes could be mediated by a nega-
tive regulator, the expression of which is activated by RpoS.
Genes directly regulated by RpoS are likely to contain an
RpoS-binding consensus sequence in their promoter region.
Work is currently under way to predict a consensus sequence
recognized by RpoS and the presence in such promoters of
binding sites recognized by any other transcriptional regulator
that are known to coregulate the expression of RpoS-depen-
In summary, the G. sulfurreducens RpoS regulon in actively
dividing cells derived from continuous culture was investigated; to
this point, RpoS has been characterized largely as a regulator of
cell responses to stress and nutrient limitation, a finding which
was confirmed in the present work. More importantly, how-
ever, this study demonstrates that the RpoS regulon is com-
posed of genes with very diverse functions, with those involved
in energy metabolism constituting the largest group, and in-
clude c-type cytochromes, TCA cycle enzymes, and cyto-
chrome oxidases, among others. These results reveal that in
addition to nutrient and stress responses, RpoS also plays a
fundamental and heretofore less appreciated role in regulating
2798NU ´N ˜EZ ET AL. J. BACTERIOL.
the normal physiology of the cell. The finding that RpoS reg-
ulates important normal physiological responses along with
genes involved in adaptation to adverse conditions and nutri-
ent limitation is consistent with the idea that RpoS has an
important role during growth and survival of G. sulfurreducens
in subsurface environments.
This research was funded by the Genomics: GTL program, U.S.
Department of Energy (DE-FC02-02ER63446). C.N. was the recipient
of a DGAPA/UNAM postdoctoral fellowship. A.E.N. was the recipi-
ent of a postdoctoral fellowship from the Secretarı ´a de Estado de
Educacio ´n y Universidades (Spain), cofunded by the European Social
Portions of the submitted manuscript were created by the University
of Chicago as operator of Argonne National Laboratory under con-
tract no. W-31-109-ENG-38 with the U.S. Department of Energy. The
U.S. Government retains for itself, and others acting on its behalf, a
paid-up, nonexclusive, irrevocable worldwide license in this article to
reproduce, prepare derivative works, distribute copies to the public,
and perform publicly and display publicly, by or on behalf of the
1. Anderson, N. G., and N. L. Anderson. 1978. Analytical techniques for cell
fractions. Two-dimensional analysis of serum and tissue proteins: multiple
isoelectric focusing. Anal. Biochem. 85:331–340.
2. Anderson, N. L., and N. G. Anderson. 1978. Analytical techniques for cell
fractions. XXI. Two-dimensional analysis of serum and tissue proteins: mul-
tiple gradient slab-gel electrophoresis. Anal. Biochem. 85:341–354.
3. Ashburner, M., C. A. Ball, J. A. Blake, H. Botstein, H. Butler, J. M. Cherry,
A. P. Davis, K. Dolinsky, S. S. Dwight, J. T. Eppig, M. A. Harris, D. P. Hill,
L. Issel-Tarver, A. Kasarskis, S. Lewis, J. C. Matese, J. E. Richardson, M.
Ringwald, G. M. Rubin, and G. Sherlock. 2000. Gene ontology, tool for the
unification of biology. Nat. Genet. 25:25–29.
4. Atlung, T., K. Knudsen, L. Heerfordt, and L. Brondsted. 1997. Effects of
sigma S and the transcriptional activator AppY on induction of the Esche-
richia coli hya and cbdAB-appA operons in response to carbon and phosphate
starvation. J. Bacteriol. 179:2141–2146.
5. Bond, D. R., D. E. Holmes, L. M. Tender, and D. R. Lovley. 2002. Electrode-
reducing microorganisms that harvest energy from marine sediments. Sci-
6. Boucher, J. C., M. J. Schurr, and V. Deretic. 2000. Dual regulation of
mucoidy in Pseudomonas aeruginosa and sigma factor antagonism. Mol.
7. Brazma, A., P. Hingamp, J. Quackenbush, G. Sherlock, P. Spellman, C.
Stoeckert, J. Aach, W. Ansorge, C. A. Ball, H. C. Causton, T. Gaasterland,
P. Glenisson, F. C. P. Holstege, I. F. Kim, V. Markowitz, J. C. Matese, H.
Parkinson, A. Robinson, U. Sarkans, S. Schulze-Kremer, J. Stewart, R.
Taylor, J. Vilo, and M. Vingron. 2001. Minimum information about a mi-
croarray experiment (MIAME)—towards standards for microarray data.
Nat. Genet. 29:365–371.
8. Reference deleted.
9. Briolat, V., and G. Reysset. 2002. Identification of the Clostridium perfringens
genes involved in the adaptive response to oxidative stress. J. Bacteriol.
10. Butler, J. E., F. Kaufmann, M. V. Coppi, C. Nunez, and D. R. Lovley. 2004.
MacA, a diheme c-type cytochrome involved in Fe(III) reduction by
Geobacter sulfurreducens. J. Bacteriol. 186:4042–4045.
11. Caccavo, F., Jr., D. J. Lonergan, D. R. Lovley, M. Davis, J. F. Stolz, and M. J.
McInerney. 1994. Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-
oxidizing dissimilatory metal-reducing microorganism. Appl. Environ Micro-
12. Coppi, M. V., C. Leang, S. J. Sandler, and D. R. Lovley. 2001. Development
of a genetic system for Geobacter sulfurreducens. Appl. Environ Microbiol.
13. Coppi, M. V., R. A. O’Neil, and D. R. Lovley. 2004. Identification of an
uptake hydrogenase required for hydrogen-dependent reduction of Fe(III)
and other electron acceptors by Geobacter sulfurreducens. J. Bacteriol. 186:
14. Cunningham, L., M. J. Gruer, and J. R. Guest. 1997. Transcriptional regu-
lation of the aconitase genes (acnA and acnB) of Escherichia coli. Microbi-
15. Eng, J. K., A. L. McCormack, and J. R. Yates III. 1994. An approach to
correlate mass spectral data of peptides with amino acid sequences in a
protein database. J. Am. Soc. Mass Spectrom. 5:976–989.
16. Esteve-Nun ˜ez, A., M. Rothermich, M. Sharma, and D. Lovley. 2005. Growth
of Geobacter sulfurreducens under nutrient-limiting conditions in continuous
culture. Environ. Microbiol. 7:641–648.
17. Farewell, A., K. Kvint, and T. Nystrom. 1998. Negative regulation by RpoS:
a case of sigma factor competition. Mol. Microbiol. 29:1039–1051.
18. Giddens, S. R., A. Tormo, and K. Mahanty. 2000. Expression of antifeeding
gene anfA1 in Serratia antomophila requires RpoS. Appl. Environ. Microbiol.
19. Giometti, C. S., M. A. Gemmell, S. L. Tollaksen, and J. Taylor. 1991.
Quantitation of human leukocyte proteins after silver staining: a study with
two-dimensional electrophoresis. Electrophoresis 12:536–543.
20. Giometti, C. S., and J. Taylor. 1991. The application of two-dimensional
electrophoresis to mutation studies. Walter de Gruyter and Co., New York,
21. Gruber, T. M., and C. A. Gross. 2003. Multiple sigma subunits and the
partitioning of bacterial transcription space. Annu. Rev. Microbiol. 57:441–
22. Hegde, P. S., I. R. White, and C. Debouck. 2003. Interplay of transcriptomics
and proteomics. Curr. Opin. Biotechnol. 14:647–651.
23. Hengge-Aronis, R. 1999. Interplay of global regulators and cell physiology in
the general stress response of Escherichia coli. Curr. Opin. Microbiol. 2:148–
24. Hengge-Aronis, R. 2002. Recent insights into the general stress response
regulatory network in Escherichia coli. J. Mol. Microbiol. Biotechnol. 4:341–
25. Hengge-Aronis, R., W. Klein, R. Lange, M. Rimmele, and W. Boos. 1991.
Trehalose synthesis genes are controlled by the putative sigma factor en-
coded by rpoS and are involved in stationary-phase thermotolerance in
Escherichia coli. J. Bacteriol. 173:7918–7924.
26. Holmes, D. E., J. S. Nicoll, D. R. Bond, and D. R. Lovley. 2004. Potential role
of a novel psychrotolerant member of the family Geobacteraceae, Geopsy-
chrobacter electrodiphilus gen. nov., sp. nov., in electricity production by a
marine sediment fuel cell. Appl. Environ. Microbiol. 70:6023–6030.
27. Hosack, D., G. Dennis, Jr., B. Sherman, H. Lane, and R. Lempicki. 2003.
Identifying biological themes within lists of genes with EASE. Genome Biol.
28. Ishihama, A. 2000. Functional modulation of Escherichia coli RNA polymer-
ase. Annu. Rev. Microbiol. 54:499–518.
29. Kowarz, L., C. Coynault, V. Robbe-Saule, and F. Norel. 1994. The Salmonella
typhimurium katF (rpoS) gene: cloning, nucleotide sequence, and regulation
of spvR and spvABCD virulence plasmid genes. J. Bacteriol. 176:6852–6860.
30. Lacour, S., and P. Landini. 2004. Sigma S-dependent gene expression at the
onset of stationary phase in Escherichia coli: function of sigma S-dependent
genes and identification of their promoter sequences. J. Bacteriol. 186:7186–
31. Leang, C., M. V. Coppi, and D. R. Lovley. 2003. OmcB, a c-type polyheme
cytochrome, involved in Fe(III) reduction in Geobacter sulfurreducens. J.
32. Leang, C., and D. R. Lovley. 2005. Regulation of two highly similar genes,
omcB and omcC, in a 10 kb chromosomal duplication in Geobacter sulfurre-
ducens. Microbiology 151:1761–1767.
33. Lin, W. C., M. V. Coppi, and D. R. Lovley. 2004. Geobacter sulfurreducens can
grow with oxygen as a terminal electron acceptor. Appl. Environ Microbiol.
34. Lloyd, J. R., C. Leang, A. L. Hodges Myerson, M. V. Coppi, S. Cuifo, B.
Methe ´, S. J. Sandler, and D. R. Lovley. 2003. Biochemical and genetic
characterization of PpcA, a periplasmic c-type cytochrome in Geobacter
sulfurreducens. Biochem. J. 369:153–161.
35. Loewen, P. C., B. Hu, J. Strutinsky, and R. Sparling. 1998. Regulation in the
rpoS regulon of Escherichia coli. Can. J. Microbiol. 44:707–717.
36. Lovley, D., E. J. P. Phillips, Y. A. Gorby, and E. R. Landa. 1991. Microbial
reduction of uranium. Nature 350:413–416.
37. Lovley, D. R., and F. H. Chapelle. 1995. Deep subsurface microbial pro-
cesses. Rev. Geophys. 33:365–381.
38. Lovley, D. R., and S. Goodwin. 1988. Hydrogen concentrations as an indi-
cator of the predominant terminal electron accepting reactions in aquatic
sediments. Geochim. Cosmochim. Acta 52:2993–3003.
39. Lovley, D. R., and E. J. P. Phillips. 1987. Competitive mechanism for inhi-
bition of sulfate reduction and methane production in zone of ferric iron
reduction in sediments. Appl. Environ Microbiol. 53:2636–2641.
40. Methe ´, B. A., K. E. Nelson, J. A. Eisen, I. T. Paulsen, W. Nelson, J. F.
Heidelberg, D. Wu, M. Wu, N. Ward, M. J. Beanan, R. J. Dodson, R.
Madupu, L. M. Brinkac, S. C. Daugherty, R. T. DeBoy, A. S. Durkin, M.
Gwinn, J. F. Kolonay, S. A. Sullivan, D. H. Haft, J. Selengut, T. M. Davidsen,
N. Zafar, O. White, B. Tran, C. Romero, H. A. Forberger, J. Weidman, H.
Khouri, T. V. Feldblyum, T. R. Utterback, S. E. Van Aken, D. R. Lovley, and
C. M. Fraser. 2003. Genome of Geobacter sulfurreducens: metal reduction in
subsurface environments. Science 302:1967–1969.
41. Methe ´, B. A., J. Webster, K. Nevin, J. Butler, and D. R. Lovley. 2005. DNA
microarray analysis of nitrogen fixation and Fe(III) reduction in Geobacter
sulfurreducens. Appl. Environ. Microbiol. 71:2530–2538.
42. Mukherjee, A., Y. Cui, W. Ma, Y. Liu, A. Ishihama, A. Eisenstark, and A. K.
VOL. 188, 2006 RpoS REGULON IN GEOBACTER SULFURREDUCENS2799
Chatterjee. 1998. RpoS (sigma-S) controls expression of rsmA, a global
regulator of secondary metabolites, harpin, and extracellular proteins in
Erwinia carotovora. J. Bacteriol. 180:3629–3634.
43. Mulvey, M. R., P. A. Sorby, B. L. Triggs-Raine, and P. C. Loewen. 1988.
Cloning and physical characterization of katE and katF required for catalase
HPII expression in Escherichia coli. Gene 73:337–345.
44. Nun ˜ez, C., L. Adams, S. Childers, and D. R. Lovley. 2004. The RpoS sigma
factor in the dissimilatory Fe(III)-reducing bacterium Geobacter sulfurredu-
cens. J. Bacteriol. 186:5543–5546.
45. O’Farrell, P. H. 1975. High-resolution two-dimensional electrophoresis of
proteins. J. Biol. Chem. 250:4007–4021.
46. Olsen, A., A. Jonsson, and S. Normark. 1989. Fibronectin binding mediated
by a novel class of surface organelles on Escherichia coli. Nature 338:652–
47. Ortiz-Bernad, I., R. T. Anderson, H. A. Vrionis, and D. R. Lovley. 2004.
Vanadium respiration by Geobacter metallireducens: novel strategy for in situ
removal of vanadium from groundwater. Appl. Environ. Microbiol. 70:3091–
48. Patten, C. L., M. G. Kirchhof, M. R. Schertzberg, R. A. Morton, and H. E.
Schellhorn. 2004. Microarray analysis of RpoS-mediated gene expression in
Escherichia coli K-12. Mol. Genet. Genomics 272:580–591.
49. Ramagli, L. S., and L. V. Rodriguez. 1985. Quantitation of microgram
amounts of protein in two-dimensional polyacrylamide gel electrophoresis
sample buffer. Electrophoresis 6:559–563.
50. Rockabrand, D., K. Livers, T. Austin, R. Kaiser, D. Jensen, R. Burgess, and
P. Blum. 1998. Roles of DnaK and RpoS in starvation-induced thermotol-
erance of Escherichia coli. J. Bacteriol. 180:846–854.
51. Roling, W. F., B. M. van Breukelen, M. Braster, B. Lin, and H. W. van
Verseveld. 2001. Relationships between microbial community structure and
hydrochemistry in a landfill leachate-polluted aquifer. Appl. Environ. Mi-
52. Rooney-Varga, J. N., R. T. Anderson, J. L. Fraga, D. Ringelberg, and D. R.
Lovley. 1999. Microbial communities associated with anaerobic benzene
degradation in a petroleum-contaminated aquifer. Appl. Environ. Microbiol.
53. Sadygov, R. G., J. K. En, E. Durr, A. Saraf, W. H. McDonald, M. J.
MadCoss, and J. R. Yates III. 2002. Code development to improve the
efficiency of automated MS/MS spectra interpretation. J. Proteome Res.
54. Schuster, M., A. C. Hawkins, C. S. Harwood, and E. P. Greenberg. 2004. The
Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sens-
ing. Mol. Microbiol. 51:973–985.
55. Snoeyenbos-West, O. L., K. P. Nevin, R. T. Anderson, and D. R. Lovley. 2000.
Enrichment of Geobacter species in response to stimulation of Fe(III) re-
duction in sandy aquifer sediments. Microb. Ecol. 39:153–167.
56. Stokes, N. R., H. D. Murray, C. Subramaniam, R. L. Gourse, P. Louis, W.
Bartlett, S. Miller, and I. R. Booth. 2003. A role for mechanosensitive
channels in survival of stationary phase: regulation of channel expression by
RpoS. Proc. Natl. Acad. Sci. USA 100:15959–15964.
57. Storey, J., and R. Tibshiri. 2003. Statistical significance for genome-wide
studies. Proc. Natl. Acad. Sci. USA 100:9440–9445.
58. Reference deleted.
59. Toulme, F., C. Mosrin-Huaman, J. Sparkowski, A. Das, M. Leng, and A. R.
Rahmouni. 2000. GreA and GreB proteins revive backtracked RNA poly-
merase in vivo by promoting transcript trimming. EMBO J. 19:6853–6859.
60. Tusher, V., R. Tibshiri, and C. Gilbert. 2001. Significance analysis of mi-
croarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci.
61. Vijayakumar, S. R., M. G. Kirchhof, C. L. Patten, and H. E. Schellhorn.
2004. RpoS-regulated genes of Escherichia coli identified by random lacZ
fusion mutagenesis. J. Bacteriol. 186:8499–8507.
62. Wosten, M. M. 1998. Eubacterial sigma-factors. FEMS Microbiol. Rev. 22:
63. Xu, J., and R. C. Johnson. 1995. Identification of genes negatively regulated
by Fis: Fis and RpoS comodulate growth-phase-dependent gene expression
in Escherichia coli. J. Bacteriol. 177:938–947.
2800NU ´N ˜EZ ET AL. J. BACTERIOL.