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Journal of Bacteriology, August 2004, p . 5543-5546, Vol . 186,
No . 16
The
RpoS Sigma Factor in the Dissimilatory Fe(III)-Reducing Bacterium Geobacter
sulfurreducens
Cinthia Núñez,1,2* Lorrie Adams,1
Susan Childers,1,3 and Derek R . Lovley1
Department of Microbiology, University of Massachusetts, Amherst,
Massachusetts 01003,1 Department of Geological Sciences, University
of Idaho, Moscow, Idaho 83844,3 Departamento de Microbiología
Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México,
Cuernavaca, Morelos, México 622102
Received 30 January 2004/ Accepted 10 May 2004
Geobacter sulfurreducens RpoS sigma factor was shown to contribute
to survival in stationary phase and upon oxygen exposure . Furthermore,
a mutation in rpoS decreased the rate of reduction of insoluble
Fe(III) but not of soluble forms of iron . This study suggests
that RpoS plays a role in regulating metabolism of Geobacter
under suboptimal conditions in subsurface environments .
The physiology of microorganisms in the family Geobacteraceae
in the
 -Proteobacteria
is of special interest, because these are commonly the predominant
organisms in sedimentary environments in which Fe(III) reduction is
important (1, 14, 31,
32, 35, 36) .
Their ability to oxidize aromatic contaminants with the reduction of
Fe(III) (24, 25), reductively precipitate
uranium (1, 26), or grow via
reductive dehalogenation (6, 16) suggests
that they can contribute to the bioremediation of a variety of
contaminants in subsurface environments, such as aromatic
hydrocarbons (2, 31, 32,
35) and uranium (1, 14) .
The rpoS gene encodes a subunit of the RNA polymerase which
is the master regulator of the general stress response in Escherichia
coli and related bacteria (8, 9) .
This response is observed when cells face a number of different
adverse or suboptimal growth conditions and is commonly accompanied
by a reduced growth rate or entry into stationary phase . Thus, in the
Geobacteraceae RpoS might be expected to play a role in
metabolic adaptation during growth in the subsurface, where it is
expected to be more analogous to the stationary phase of culture than
to the exponential phase . The goal of the present work was to
initiate the study of the role of RpoS in Geobacter sulfurreducens
in an attempt to understand the mechanisms of stress responses
in the Geobacteraceae . G . sulfurreducens serves as a model because
it is closely related to the Geobacter species that predominate
in subsurface environments, it can be cultured in laboratory
conditions, and its genome sequence (28) as well as a genetic
system is available (5) .
G . sulfurreducens genome contains an rpoS homologue
gene. A search of the G . sulfurreducens genome sequence (www.tigr.org),
using the E . coli rpoS gene as a query, revealed an open reading
frame encoding a protein which has 42% identity with the RpoS
protein of E . coli and consists of 328 amino acids with a molecular
mass of 38 kDa . It has 87 and 68% amino acid identity with putative
RpoS proteins of Geobacter metallireducens and Desulfuromonas
acetoxidans, two other members of the Geobacteraceae
(preliminary genome sequence data are available at http://www.jgi.doe.gov) .
The genetic arrangement of the G . sulfurreducens rpoS region
differs from that of E . coli and related bacteria . The nlpD
gene, encoding an outer membrane lipoprotein (17), is
absent, but this region contains a gene, downstream of rpoS,
whose deduced amino acid sequence has 47.5% identity to the adenine
phosphoribosyltransferase enzyme Apt of E . coli, which is
involved in the one-step salvage pathway of adenine to AMP (12) .
Reverse transcription-PCR analysis demonstrated that rpoS
forms part of the operon surE-pcm-rpoS-apt (data not shown) .
However, the presence of additional rpoS-containing
transcripts cannot be discounted . Primer extension analyses of the
surE-pcm-rpoS-apt operon were carried out, as described
previously (3), with total RNA extracted from the wild-type
strain DL1 (4) and rpoS mutant DLCN16( rpoS::Km)
grown in NBAF media (5) and with oligonucleotide
surEPE (5'-GCGTGAACACCATCATC-3') (which is complementary to the 5'
region of the surE gene) . As shown in Fig . 1,
the surE promoter contains –10 (TTTCGT) and –35 (TTTACA)
sequences which are similar to the consensus sequences recognized by
E . coli
 70 .
This promoter was found to be RpoS independent either in the
logarithmic or in the stationary phase of growth (Fig .
1) . This is similar to E . coli, in which the transcription
of surE, pcm, and rpoS is RpoS independent (10,
20) . The rpoS mutant DLCN16(rpoS::Km)
was constructed as described previously (18),
using the oligonucleotides rpoS1 (5'-CTTACATGGTCGCCCTGATG-3') and
rpoS2 (5'-CATGGAGATCTCCGTCGC-3') to amplify the upstream region of
rpoS, oligonucleotides rpoS5 (5'-CGAGGCCAAGTCTCTGG-3') and rpoS6
(5'-GCCGTATTCGAGCTGATAGG-3') to amplify the rpoS downstream
region, and oligonucleotides rpoS3
(5'-GCGACGGAGATCTCCATGACCTGGGATGAATGTCAGCTAC-3') and rpoS4
(5'-CCAGAGACTTGGCCTCGAGAAGGCGGCGGTGGAATCG-3') to amplify the
kanamycin cassette . This cassette was inserted in the same orientation
as that of rpoS transcription, resulting in a nonpolar mutation .
This was confirmed by reverse transcription-PCR analysis using
total RNA extracted from DLCN16 cultures and oligonucleotides
designed to amplify the apt gene (data not shown) .
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FIG . 1 . Primer extension analysis of surE in G . sulfurreducens
DL1 (lanes 1 and 3) and rpoS mutant DLCN16 (lanes 2 and 4) . Total
RNA was extracted from mid-log (lanes 1 and 2) or stationary-phase
(lanes 3 and 4) cultures . The transcription initiation site P1 is
indicated . (B) DNA sequence of the surE regulatory region . The
arrow indicates the transcription start point.
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Stationary-phase survival in the rpoS mutant, using NBAF medium
with limiting acetate (8 mM) as electron donor and excess fumarate
(37 mM) as electron acceptor (5), decreased at least
10-fold compared to that of the wild-type strain DL1, indicating that
RpoS is involved in stationary-phase survival of G . sulfurreducens
(Fig . 2) . This result is consistent with the fact that
G . sulfurreducens rpoS is cotranscribed with surE,
pcm, and apt, whose products are necessary for
stationary-phase survival in E . coli (7,
19, 38) .
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FIG . 2 . (A) Growth of G . sulfurreducens DL1 (triangles) and
rpoS mutant (circles) in media containing acetate as the electron
donor and fumarate as the electron acceptor . Data are means of
triplicates . The arrow indicates time zero of the survival curve . OD
600, optical density at 60 nm . (B) Stationary-phase survival of DL1
(triangles) and rpoS mutant DLCN16 (circles) . Percent viability
is expressed as the viable cell number at each time point divided by the
viable cell number at time zero . Similar results were obtained in three
experiments.
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The utilization of either fumarate or soluble Fe(III) [in the form of
Fe(III) citrate] as electron acceptor was not affected in the rpoS
mutant (Fig . 2A and 3A); in contrast, the
reduction of insoluble Fe(III) (Fig . 3B) was
significantly diminished in the rpoS mutant compared to that
of the wild-type strain . The same result was obtained with two
independent rpoS mutant isolates . This result suggests that
RpoS may regulate the expression of genes in G . sulfurreducens
that are specifically required for the reduction of insoluble Fe(III)
oxide, the primary form of Fe(III) in most sedimentary environments .
Preliminary studies have indicated that the pattern of c-type
cytochromes in the rpoS mutant is different from that in the
wild-type strain (data not shown), which is significant because c-type
cytochromes are involved in electron transfer to Fe(III) in G .
sulfurreducens (18, 22,
27) . The identity of such c-type cytochromes is
presently being investigated .
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FIG . 3 . Fe(III) citrate (A) or poorly crystalline Fe(III) oxide (B)
reduction in the wild-type strain DL1 (triangles) and rpoS mutant
(circles) in media containing acetate as electron donor . Determination
of Fe(II) was carried out as previously described (30) .
Data are means of triplicates.
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Although G . sulfurreducens was originally designated a strict
anaerobe (4), subsequent studies demonstrated that it can
tolerate long-term exposure to oxygen (21) . Oxygen
intrusions into sediments in which Geobacter species
predominate are common, and thus the ability to tolerate oxygen
exposure is an important feature in the survival of these organisms (21) .
As shown in Fig . 4, tolerance to oxygen was clearly
reduced in the rpoS mutant DLCN46(rpoS::Cm)
after 4 days of exposure and it was rescued in the rpoS-complemented
mutant strain DLCN52(rpoS::Cm/rpoS+) .
(The procedure for the construction of a
rpoS::Cm
mutant was the same as that for the DLCN16 mutant, except that
oligonucleotides rpoSCm-3 [5'-GCGACGGAGATCTCCATGACGGAAGATCACTTCGC-3']
and rpoSCm4 [5'-CCAGAGACTTGGCCTCGAGGGCAGCAATAACTGCC-3'] were used to
amplify the chloramphenicol cassette; the complementation was
achieved, as described before [29], by cointegration of
plasmid pCNDL17, a PCR 2.1-TOPO derivative [Kmr] [Invitrogen,
Carlsbad, Calif.] carrying the entire rpoS gene.) These results
indicate that RpoS is involved in the aerotolerance of G .
sulfurreducens, which is consistent with its role in
oxidative-stress resistance in other bacteria (15,
34, 37) . However, the fact that the
rpoS mutant of G . sulfurreducens was still viable after 4
days of oxygen exposure indicates that the mechanism for coping with
oxidative damage is only partially RpoS dependent .
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FIG . 4 . Aerotolerance of DL1 (wild type) (triangles), DLCN46(rpoS::Cm)
(circles), and DLCN52(rpoS::Cm/rpoS+)
(diamonds) after 0, 1, 2, and 4 days of air exposure . Data are means of
triplicates . Survival was estimated as the lag time of growth of a
culture derived from oxygen-exposed cells as described previously (21);
determination of CFU was not feasible due to the fact that G .
sulfurreducens forms cellular aggregates when exposed to oxygen, a
trait that was enhanced by the rpoS mutation . OD 600, optical
density at 600 nm.
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G . sulfurreducens RpoS had no apparent function in resistance
to high temperature (45°C for 7 days) or alkaline pH (pH 6 for 60
min) (data not shown), indicating that some stress response
mechanisms are not controlled by RpoS . In addition to RpoS and the
well-known sigma factors RpoD (70),
RpoH (32),
RpoF (28),
and RpoN (54),
G . sulfurreducens contains a sigma factor that belongs to the
family having extracytoplasmic functions (data not shown) (23) .
This sigma factor, designated RpoE, is likely to be involved in
resistance to oxidative stress and other adverse conditions in G .
sulfurreducens based on its role in E . coli and other
gram-negative bacteria (11, 13,
33); however, this remains to be investigated .
This first study of RpoS in a member of the
-Proteobacteria
illustrates some similarities and differences in gene organization
and function compared to those of other classes of previously
investigated Proteobacteria . The effect of the rpoS mutant on
survival in stationary phase and on reduction of Fe(III) oxide,
the primary electron acceptor supporting the growth of Geobacteraceae,
suggests that RpoS may play a role in controlling activity of
G . sulfurreducens in subsurface environments . Thus, it seems
likely that further investigation of the RpoS regulon will provide
insights into the mechanisms by which G . sulfurreducens and
related organisms function so effectively in Fe(III)-reducing
subsurface environments .
This research was funded by the Genomes to Life program, U.S .
Department of Energy (grant DE-FC02-02ER63446) . C.N . was the
recipient of a DGAPA/UNAM Postdoctoral Fellowship .
* Corresponding author . Mailing address: Departamento de
Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional
Autónoma de México, Av . Universidad 2001, Col . Chamilpa Cuernavaca, Morelos
62210, México . Phone: (52) 777 329-16-29 . Fax: (52) 777 317-23-88 . E-mail: cinthia@ibt.unam.mx .
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