Role of RelGsu in Stress Response and Fe(III) Reduction in Geobacter sulfurreducens

Geobacter species are key members of the microbial communityin many subsurface environments in which dissimilatory metalreduction is an important process. The genome of Geobacter sulfurreducenscontains a gene designated rel_Gsu, which encodes a RelA homologpredicted to catalyze both the synthesis and the degradationof guanosine 3',5'-bispyrophosphate (ppGpp), a regulatory moleculethat signals slow growth in response to nutrient limitationin bacteria. To evaluate the physiological role of Rel_Gsu inG. sulfurreducens, a rel_Gsu mutant was constructed and characterized,and ppGpp levels were monitored under various conditions inboth the wild-type and rel_Gsu mutant strains. In the wild-typestrain, ppGpp and ppGp were produced in response to acetateand nitrogen deprivation, whereas exposure to oxygen resultedin an accumulation of ppGpp alone. Neither ppGpp nor ppGp couldbe detected in the rel_Gsu mutant. The rel_Gsu mutant consistentlygrew to a higher cell density than the wild type in acetate-fumaratemedium and was less tolerant of oxidative stress than the wildtype. The capacity for Fe(III) reduction was substantially diminishedin the mutant. Microarray and quantitative reverse transcription-PCRanalyses indicated that during stationary-phase growth, proteinsynthesis genes were up-regulated in the rel_Gsu mutant and genesinvolved in stress responses and electron transport, includingseveral implicated in Fe(III) reduction, were down-regulatedin the mutant. The results are consistent with a role for Rel_Gsuin regulating growth, stress responses, and Fe(III) reductionin G. sulfurreducens under conditions likely to be prevalentin subsurface environments.

INTRODUCTION

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Introduction

Members of the family Geobacteraceae carry out a number of importantprocesses in sedimentary environments, but little is known aboutthe mechanisms regulating their metabolism in response to theenvironmental stresses typical of the subsurface. The hallmarkphysiological characteristic of Geobacteraceae is their abilityto oxidize organic compounds with the reduction of extracellularelectron acceptors, such as Fe(III) and Mn(IV) oxides (45),U(VI) (46), humic substances (43), and electrodes (6). Geobacteraceaeare important not only in the anaerobic oxidation of naturallyoccurring organic matter coupled to Fe(III) reduction (44) butalso in the degradation of organic contaminants in subsurfaceenvironments (40, 67, 68) and are useful agents in uranium-contaminatedsubsurface environments (1, 59, 79).

Geobacteraceae are likely to face suboptimal concentrationsof electron donors (1) and nutrients (33), as well as otherstresses, such as heavy metals and toxic organics, in subsurfaceenvironments. Many bacterial stress response systems that havebeen identified and characterized for other organisms are encodedin the genome of the Geobacteraceae model species, Geobactersulfurreducens (52). These include regulatory genes involvedin oxidative stress response (RpoS and PerR), heat shock (RpoH),and metal homeostasis (Fur, Zur, and IdeR), as well as manytwo-component regulatory system genes (52). Studies have confirmeda role for RpoS in stationary-phase survival and oxidative stressresponse in G. sulfurreducens (60), and preliminary studiesof several other regulators suggest their roles are analogousto those found in other organisms.

Another well-known response of microorganisms to suboptimalgrowth conditions is the stringent response (7, 13, 16, 34,47). In the stringent response, guanosine 3',5'-bispyrophosphate(ppGpp) and, in some species, triphosphate (21, 35, 62) andpentaphosphate (14, 20, 28) derivatives of this molecule areproduced in response to nutrient limitation. These stringentfactors interact with RNA polymerase to influence transcriptionof various genes. The hallmark of the stringent response isthe down-regulation of stable RNA molecules and translationmachinery, but this response also includes the up-regulationof stress response genes (26, 76). Intracellular levels of ppGppare regulated by two enzyme activities that act to synthesizeand degrade the molecule in response to various triggers inthe cell. In Escherichia coli, synthesis and degradation ofppGpp are catalyzed by two distinct but homologous proteins,RelA and SpoT (13). Several organisms, including many proteobacteriaand gram-positive organisms, contain a single rel-like genethat is predicted to perform both enzyme activities (31, 49,54, 80, 81).

Here we present evidence that G. sulfurreducens has a singleprotein, designated Rel_Gsu, for controlling levels of ppGp(p)and that this activity plays an important role in regulatingthe expression of genes necessary not only for adapting to environmentalstress but also for Fe(III) reduction.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains, plasmids, and culturing conditions. Escherichia coli strain DH5 (supE44 lacU169 ${phi}$ 80 lacZ M15 hsdR17recA1 endA1 gyrA96 thi-1 relA1) (84) was used for DNA manipulations.Strain DLLD1 (rel_Gsu::kan) was produced through targeted genedisruption of Geobacter sulfurreducens strain DL1 (ATCC 51573)(11, 17). G. sulfurreducens strains were routinely culturedanaerobically in acetate-fumarate (NBAF) medium or acetate-Fe(III)citrate medium in batch culture as previously described (17).The plasmid pBBR1MCS-2 (36) was obtained from Michael Kovach.

DNA manipulations and reagents. G. sulfurreducens genomic DNA was extracted with a MasterPurecomplete DNA and RNA purification kit (Epicenter Technologies,Madison, WI). Plasmid purification, PCR product purification,and gel extractions were performed with the following kits:a QIAprep Spin miniprep kit, a QIAquick PCR purification kit,and a QIAquick gel extraction kit (QIAGEN, Inc., Valencia, CA).Transformations into E. coli and other routine DNA manipulationswere carried out as outlined by Sambrook et al. (70). Restrictionenzymes were purchased from New England Biolabs (Beverly, MA).Ligations were carried out using either a rapid DNA ligationkit (Roche Applied Science, Basel, Switzerland) or a TOPO TAcloning kit (Invitrogen, Carlsbad, CA). Southern blotting wasperformed as previously described (17), and hybridization anddetection were performed with a Roche Applied Science digoxigenin-labelingand digoxigenin nucleic acid detection kit using the chromogenicmethod and nitroblue tetrazolium-BCIP (5-bromo-4-chloro-3-indolylphosphate)substrate according to the manufacturer's instructions. TaqDNA polymerase (QIAGEN, Inc.) was used for all PCR amplifications.Unless otherwise stated, chemicals were reagent grade or betterand were purchased from Sigma Chemical Co. (St. Louis, MO).

Construction of Rel_Gsu-deficient strain via single-step gene replacement. To construct a Rel_Gsu-deficient mutant, recombinant PCR (55)was used to construct a linear DNA fragment consisting of akanamycin resistance cassette flanked by homologous sequencefrom the 5' and 3' ends of the rel_Gsu gene. Three primary PCRswere carried out: (i) amplification of the 5' end of the linearfragment with primers RelA.1 (GGT GCT GGA TGC GGT TTC) and RelA.2(GGA CCT TTG CAC AGT AGA C), (ii) amplification of the kanamycinresistance cassette from pBBR1MCS-2 (36) with primers RelA.3(GTC TAC TGT GCA AAG GTC C/ACC TGG GAT GAA TGT CAG CTA C) andRelA.4 (CGT GAC AGG AAA CTC GG/AGA AGG CGG CGG TGG AAT CG),and (iii) amplification of the 3' end of the linear fragmentwith primers RelA.5 (CCG AGT TTC CTG TCA CG) and RelA.6 (GTCCAT TAC GCG CAT CG). Following recombinant PCR with the threeprimary PCR products serving both as templates and as primers,the final fragment was amplified with the distal primers RelA.1and RelA.6. PCR conditions were as follows: 94°C for 15s, followed by 30 cycles of 94°C for 30 s, 55°C for1 min, and 72°C for 2 min, and a final 10-min extensionstep at 72°C. The same amount of primer (20 pmol) was usedfor each reaction. Electroporation, mutant isolation, and genotypeconfirmation were performed as described by Coppi et al. (17)and Lloyd et al. (41). One of the resulting mutants was chosenas the representative strain.

Expression of rel_Gsu in trans. The complete coding sequence for rel_Gsu was amplified usingprimers containing either an EcoRI (GCATGAATTC CAA TCT CTT TCATGC TCC) or a BamHI (GTATGGATCC GCT AAT CAC AG ACC TCC) site.PCR amplification conditions were the same as those describedabove. The amplicon was digested with BamHI and EcoRI and insertedinto the corresponding sites of the broad-host-range expressionvector pRG5 (9). The insert was then sequenced to screen forPCR artifacts. Following electroporation of the rel_Gsu mutantstrain with the appropriate vector, spectinomycin-resistanttransformants were isolated. The simultaneous presence of complementationvector and the original mutation in the resulting strain wasconfirmed by PCR screening and plasmid isolation.

Nutrient shutdown experiments and quantitation of guanosine phosphates. Wild-type and Rel_Gsu-deficient strains of G. sulfurreducenswere initially cultured in NBAF medium (17) supplemented with1 mM cysteine. Log-phase (optical density at 600 nm [OD₆₀₀]of 0.4 to 0.5) acetate-fumarate cultures (100, 300, or 500 ml)were harvested by centrifugation, washed, and resuspended in500 ml freshwater acetate-fumarate (FWAF) medium (17) containingeither acetate (15 mM) and ammonium (6 mM) or lacking eitheracetate or ammonium. FWAF differs from NBAF mainly in bufferingcapacity, trace element content, and fumarate concentration(27.7 mM for FWAF versus 40 mM for NBAF). Initial culturingin NBAF was required to obtain sufficient biomass for detectionof guanosine phosphates. FWAF medium was used for subsequentsteps, because use of this medium resulted in a cleaner lysateand significantly prolonged the life of the column used to quantitateguanosine phosphate content. At 20-min intervals, 100-ml aliquotsof the various cultures were filtered through a 90-mm Milliporenitrocellulose prefilter (Bedford, MA) and then extracted in15 ml of 1 N formic acid as previously described (25). Extractswere freeze-dried using a Labconco lyophilizer (Kansas City,KS) and stored at –20°C.

Guanosine phosphate derivatives (ppGp and ppGpp) were quantitatedby high-pressure liquid chromatography (HPLC) on a 250-mm by4.6-mm Partisil SAX 10-µm column (Alltech, Deerfield,IL) as described by Jones et al. (35), using an LC-10AT high-pressureliquid chromatograph (Shimadzu, Kyoto, Japan) and a 20-µlinjection volume. Immediately prior to injection, samples wereresuspended in either 400 µl or 500 µl H₂O. ppGppstandards were obtained from TriLink Biotechnologies (San Diego,CA), and ppGp standards were a gift from Mercian Corporation,Japan.

Analytical techniques. Growth of fumarate cultures was assessed by measuring turbidityat 600 nm. Fe(II) concentrations were determined by ferrozineassay (45). Cell densities of Fe(III)-grown cultures were determinedby epifluorescence microscopy using acridine orange staining(45). The protein content of cell fractions was determined bythe bicinchoninic acid method, with bovine serum albumin asthe standard (74). Pairwise alignments were performed usingthe Needleman and Wunsch algorithm (57).

RNA isolation. Cells were harvested as previously described (51). Briefly,cultures were centrifuged at 4°C for 15 min and pelletswere flash frozen and stored at –80°C. To extracttotal RNA, cells were mechanically disrupted using a FastPrepinstrument (Qbiogene, Inc., Irvine, CA) with lysing matrix B(Qbiogene) and nucleic acids were extracted with TRIzol reagent(Invitrogen, Carlsbad, CA), a monophasic solution of phenoland guanidine isothiocyanate. Residual DNA was removed usingRNase-free DNase (Ambion, Inc., Austin, TX) according to themanufacturer's instructions. The treated RNA was subsequentlycleaned and concentrated with RNeasy minicolumns (QIAGEN, Inc.,Valencia, CA). Quality of total RNA was assessed by agarose-formaldehydegel electrophoresis, and the concentration was determined usinga NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies,Inc., Wilmington, DE) (51).

DNA microarray hybridization and data analysis. Total RNA was isolated from three sets of identically treated,early-stationary-phase, 100-ml NBAF batch cultures of both thewild-type and the Rel_Gsu-deficient strains. DNA microarray hybridizationand data analyses were performed as described previously (51).Briefly, approximately 5 µg of total RNA was used forindirect labeling with either cyanine 3 or cyanine 5 (Cy3/Cy5)fluorescent dyes, leading to production of approximately 4 to5 µg of cDNA with greater than 200 pmol, respectively,of each dye molecule incorporated per microgram of cDNA synthesized.Triplicate control and treatment stationary-phase cultures wereextracted for each experiment so that extracted RNA could bepaired to produce three biological replicates from which hybridizationscould be repeated (technical replicates). Following hybridizations,slides were promptly scanned at a 10-µm resolution usingan Axon 4000B scanner with GenePix 4.0 software.

Processing of 16-bit TIFF images from hybridized arrays wasdone using the TIGR TM4 package (www.tigr.org/software). Intensityvalues for Cy3 and Cy5 channels were obtained using TIGR-Spotfindersoftware. Normalization was performed using the LOWESS algorithmavailable in TIGR-MIDAS using block mode and a smooth parameterof 0.33. All intensity values less than two times greater thanbackground were removed from subsequent analysis, and replicatereporter intensities on one slide (one technical replicate)were reduced to a single value by computing the geometric mean.Four hybridizations were performed from each of three biologicalreplicate stationary-phase pairs (control and treatment). Halfof the technical replicate dye labelings were dye swaps (flipdyes) performed as part of overall quality assurance.

Measurement of relative transcript levels using quantitative reverse transcription-PCR (RT-PCR). Total RNA was isolated as described in "RNA isolation" above.Single-stranded cDNA was generated by the reverse transcriptionof 2 µg of total RNA in a 100-µl reaction volumeusing TaqMan reverse transcription reagents (Applied Biosystems,Foster City, CA). The cDNA was then subjected to quantitativePCR using SYBR green PCR master mix (Applied Biosystems, FosterCity, CA). Forward and reverse primers were added to the reactionmixture at a final concentration of 200 nM along with 1 µlof the cDNA reaction mixture. The incorporation of SYBR greendye into the PCR products was detected in real time on an ABIPrism 7900HT sequence detection system. ROX (6-carboxyl-X-rhodamine)passive reference dye was used to factor in well and pipettingvariability. The incorporation of SYBR green resulted in thedetermination of the cycle threshold, which identifies the PCRcycle at which exponential growth of the PCR products begins.Standard curves were established for each cDNA sample beinganalyzed by use of primers for a gene, DNA polymerase III, betasubunit (GSU0001), showing unchanged expression levels in priormicroarray analyses. The standard curves were normalized toeach other through the control gene (GSU0001), and quantitationwas subsequently determined. Primers used for amplificationare listed in Table 1.

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TABLE 1. Sequences of primers used in quantitative RT-PCR

Microarray data 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 A-TIGR-20 and E-TIGR-5000.

Nucleotide sequence accession numbers. The GenBank accession numbers for the proteins described inthis report are as follows: for Rel_Gsu, accession number AAR35612;for Geobacter metallireducens Rel, accession number ABB32550.1;for Pelobacter propionicus Rel, accession number ZP_00677757.1;and for Pelobacter carbinolicus Rel, accession number ABA88536.1.

RESULTS

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Results

Rel_Gsu protein and operon characteristics. A RelA/SpoT homolog was identified in the Geobacter sulfurreducensgenome and designated rel_Gsu. Rel_Gsu appears to consist of fourdomains, each of which is homologous to those commonly foundin bifunctional RelA/SpoT proteins (Fig. 1). These domains includea RelA/SpoT domain, which is the source of ppGpp synthetaseactivity (4, 31, 48, 49); a metal-dependent hydrolase domainimplicated in hydrolysis of ppGpp (3, 4, 31, 48, 49); an ACT(aspartokinase, chorismate mutase, TyrA, or prephenate dehydrogenase)domain (2, 72), considered to have a regulatory role involvingamino acid binding; and a TGS (ThrRS, GTPase, and SpoT) domain,predicted to function in nucleotide binding (2, 71, 72, 82)(Fig. 1). In contrast, most monofunctional RelA homologs lackthe hydrolase domain, and SpoT homologs lack the ACT domain(NCBI, CDART [http://www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi]).In addition, this homolog is more similar to bifunctional RelA/SpoTproteins (5, 48, 81), such as that found in Bacillus subtilis,with which it shares 69% similarity (81), than to individualRelA and SpoT proteins of E. coli (53), with which it shares58% and 59% similarity, respectively. Thus, Rel_Gsu is likelyto catalyze both the synthesis and the hydrolysis of ppGpp inresponse to multiple signals in the cell.

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FIG. 1. Structure of putative rel_Gsu operon and domain architecture of Rel_Gsu protein.

Genes with high similarity to Rel_Gsu are present in other Geobacterspecies, including Geobacter metallireducens, with 95.8% similarity,Geobacter uraniumreducens, with 93.3% similarity, Pelobacterpropionicus, with 89.7% similarity, and Pelobacter carbinolicus,with 77.5% similarity. Homologs with high percent similarityare also found in other ${delta}$ -proteobacteria, such as Myxococcusxanthus, with 72.9% similarity (28), and Desulfovibrio species,with 69 to 71% similarity (29). Because all of these genes appearto show high homology to a predicted bifunctional RelA/SpoTprotein, it suggests that ${delta}$ -proteobacteria utilize a bifunctionalRelA/SpoT protein for the regulation of ppGpp levels ratherthan the two different proteins, RelA and SpoT, found in manyother bacteria.

rel_Gsu is predicted to be in an operon with four open readingframes (86). The genes include guanylate kinase; the RNA polymeraseomega subunit rpoZ, which has recently been determined to benecessary for ppGpp binding to RNA polymerase (78); rel_Gsu;and endoribonuclease liver perchloric acid-soluble protein (Fig.1). The structure of this putative operon is similar to spoToperons of several ${gamma}$ -proteobacteria, such as Shewanella oneidensisand Pseudomonas aeruginosa, and is conserved throughout theGeobacteraceae (http://microbesonline.org). Similar gene clustersare not found in the genomes of other families of ${delta}$ -proteobacteria,such as Desulfovibrio spp., in which the rel gene lies in aputative operon with an ABC dipeptide transport protein (http://microbesonline.org).

Production of guanosine phosphate derivatives ppGpp and ppGp in response to nutrient deprivation and oxidative stress. In order to evaluate whether nutrient limitation triggered accumulationof guanosine phosphates in G. sulfurreducens, levels of ppGppand its derivatives were measured during growth in nutrient-deprivedconditions. Mid-log-phase NBAF cultures were washed and resuspendedin FW medium containing fumarate but no acetate. This resuspensionmedium differed from the growth medium, containing less bufferingcapacity and fewer trace minerals, as described in Materialsand Methods, likely resulting in a short-term increase in detectableguanosine phosphates even in the presence of acetate (Fig. 2A,FW control). However, levels of ppGpp were substantially higherin the absence of acetate (Fig. 2A), and levels of ppGp, a secondguanosine phosphate derivative detected in these cultures, alsoincreased in the absence of acetate (Fig. 2B). Omitting fixednitrogen, in the form of ammonium, from the resuspension mediumalso resulted in elevated levels of ppGpp and ppGp (Fig. 2).ppGpp and ppGp were the only two guanosine phosphate derivativesdetected in this analysis. A derivative that is commonly foundin other organisms, pppGpp (14, 28), was not detected.

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FIG. 2. Production of ppGp(p) in response to nutrient deprivation. Effect of nutrient deprivation on accumulation of (A) ppGpp and (B) ppGp in wild-type G. sulfurreducens and the rel_Gsu mutant. Wild-type and mutant cells were grown in NBAF medium to mid-log phase, harvested by centrifugation, and transferred to FW medium containing acetate and ammonium (FW control), FW medium without acetate [(–) acetate], or FW medium without ammonium [(–) nitrogen]. All wild-type data points represent averages of duplicate experiments. No ppGp(p) was detected in the rel_Gsu mutant ( ${Delta}$ rel_Gsu) in media lacking acetate or in media lacking ammonia. DCW, dry cell weight.

To examine ppGp(p) levels induced during other stress responses,ppGp(p) was measured during oxidative stress by exposing cellsto oxygen. Control cultures growing in NBAF medium typicallyhad concentrations of ppGpp of less than 10 pmol/mg cells (dryweight) (Fig. 3), while levels of ppGp in 20 µl of injectedcell extract were never above the HPLC detection limit of 3pmol (data not shown). When 6% oxygen was added to the headspaceof mid-log-phase NBAF cultures, ppGpp accumulated (Fig. 3),but ppGp remained undetectable (data not shown), indicatingthat ppGpp is the only guanosine phosphate derivative producedat detectable levels in G. sulfurreducens in response to oxidativestress.

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FIG. 3. Production of ppGpp in response to oxidative stress. Wild-type cells were grown in NBAF medium to mid-log phase, and then 6% oxygen was added to the headspace. Samples were taken every 20 min after the addition of oxygen. Detection of ppGp(p) was monitored using HPLC as described in Materials and Methods. Production of ppGp was not detected. Data points are representative values from duplicate experiments. DCW, dry cell weight.

Phenotypic characterization of a Rel_Gsu-deficient mutant. A rel_Gsu mutant was constructed by homologous recombination(see Materials and Methods). Neither ppGpp nor ppGp levels,in a 20-µl injection volume, were detected above the HPLCdetection limit of 14 pmol or 3 pmol, respectively, in mutantcultures subjected to the same nutrient-deprived conditionsthat yielded accumulation of ppGpp and ppGp in the wild-typestrain (Fig. 2). This result indicates that the rel_Gsu mutantcannot produce significant levels of ppGp(p), consistent withRel_Gsu being involved in the production of both compounds inG. sulfurreducens.

The rel_Gsu mutant cells grew at a rate similar to that of wild-typecells during exponential growth in NBAF medium (Fig. 4). However,the mutant consistently achieved a higher cell density (OD₆₀₀of 0.86) than the wild type (OD₆₀₀ of 0.72), and this highercell density was most apparent when the cells were approachingand entering stationary phase (Fig. 4). Complementing the mutantwith the rel_Gsu gene expressed in trans restored the wild-typegrowth pattern (Fig. 4).

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FIG. 4. Growth of the rel_Gsu mutant in acetate-fumarate medium. Wild-type (WT), ${Delta}$ rel_Gsu mutant, and complemented ${Delta}$ rel_Gsu ( ${Delta}$ rel_Gsu::pRG-rel) strains were grown in NBAF medium. Values represent the means ± standard errors of triplicate cultures.

Cysteine (1 mM) is typically added as a reductant to NBAF mediumbecause it reduces the length of the lag phase and increasesthe maximum density of the cultures. Although the rel_Gsu mutantgrew at least as well as the wild type in the typical NBAF medium(see above), when cysteine was omitted, raising the redox potentialof the medium, the mutant cells had a lag phase that was nearlydouble that of the wild-type cells (Fig. 5A). Cells of the complementedstrain had a lag phase in cysteine-free medium comparable tothat of the wild-type cells (Fig. 5A).

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FIG. 5. Growth of the rel_Gsu mutant under oxidative stress. (A) Wild-type (WT), ${Delta}$ rel_Gsu mutant, and complemented ${Delta}$ rel_Gsu ( ${Delta}$ rel_Gsu::pRG-rel) strains were grown in NBAF medium without cysteine. Values represent means ± standard errors of triplicate cultures. (B) Effect of exposure to air on wild-type and ${Delta}$ rel_Gsu strains. Strains were inoculated into pressure tubes containing 2% air in the headspace and allowed to grow to stationary phase. The arrow indicates aggregates present in the wild-type culture that were absent in the ${Delta}$ rel_Gsu culture.

Further evidence for an impaired oxidative stress response inrel_Gsu mutants became evident when cysteine-free cultures weregrown to stationary phase after the introduction of air intothe headspace. The wild-type strain was capable of growth inthe presence of 2% air but formed distinct aggregates, a phenomenonseen previously and believed to be an adaptive response to oxidativestress in G. sulfurreducens (60) (Fig. 5B). Under the same conditions,the rel_Gsu mutant failed to aggregate and grew as a uniformsuspension (Fig. 5B).

The rel_Gsu mutant also demonstrated defects in Fe(III) reduction,reducing Fe(III) nearly threefold more slowly than the wildtype in medium with acetate as the electron donor and Fe(III)citrate as the electron acceptor. The rel_Gsu mutant cell yieldwas also only 60% of that seen with the wild type (Fig. 6).The complemented strain reduced Fe(III) at wild-type rates.

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FIG. 6. Growth of the wild-type (WT), rel_Gsu mutant, and complemented ${Delta}$ rel_Gsu ( ${Delta}$ rel_Gsu::pRG-rel) strains in acetate-Fe(III) citrate medium. (A) Fe(III) citrate reduction and (B) cell growth. Data are means ± standard errors of triplicate cultures.

Microarray analysis of the G. sulfurreducens rel_Gsu mutant versus the wild type. To identify genes that were differentially regulated in theabsence of Rel_Gsu activity, levels of gene expression in rel_Gsumutant and wild-type cultures grown to stationary phase in NBAFmedium supplemented with cysteine were compared using a whole-genomemicroarray. Stationary-phase cells were harvested after approximately35 h of growth, when cell density increases slowed. A subsetof genes that were differentially expressed in the microarrayanalysis were subsequently validated using quantitative RT-PCR(51). Stationary-phase cells were chosen for this analysis becausedepletion of nutrients that occurs during this stage has beenshown to induce Rel activity in other organisms (7, 16) andbecause growth characteristics were shown to differ betweenthe wild type and the rel_Gsu mutant during this stage (Fig.4). For microarray results, differentially expressed genes wereidentified by the application of the SAM algorithm (77) to replicatehybridizations performed with RNA from individual identicallytreated biological samples. Genes showing significant differentialexpression under these criteria, as defined by Methéet al. (51), were then further selected according to whetherdifferential expression occurred in at least two biologicalreplicates. Application of these criteria resulted in identificationof 332 genes that were differentially expressed in the rel_Gsumutant during stationary-phase growth. Of the genes found tobe differentially expressed, 162 genes were up-regulated, and170 genes were down-regulated (see Tables S1 and S2 in the supplementalmaterial). As expected, rel_Gsu was among the down-regulatedgenes.

The differentially expressed genes were assigned to 12 functionalcategories based on the published annotation of the G. sulfurreducensgenome (52) (Fig. 7; also see Tables S1 and S2 in the supplementalmaterial). The largest category of differentially expressedgenes consisted of genes of unknown function, including bothhypothetical genes and conserved genes of unknown function.The next-largest category of differentially expressed genesincluded those involved in electron transport. The majorityof these genes were down-regulated in the mutant (Table 3; Fig.7). Another large category of differentially expressed genesincluded those involved in protein biosynthesis, which wereprimarily up-regulated in the mutant (see Table S1 in the supplementalmaterial). These genes included many encoding proteins involvedin sulfur assimilation, which were highly up-regulated, as wellas ribosomal proteins and tRNA synthetases. Four genes fromthis group were confirmed to be up-regulated in the mutant byuse of quantitative RT-PCR (Table 2) . Many signaling and transportgenes were also differentially regulated under these conditions(see Tables S1 and S2 in the supplemental material). Many genesinvolved in stress response were down-regulated in the mutant,including a universal stress protein (56), an RND efflux transporter(58), and desulfoferredoxin (42), as well as several redox proteinsimplicated in combating oxidative stress, such as cytochromec₅₅₁ peroxidase (12, 73), Hcp1, or prismane (8), and rubrerythrin(75) (see Table S2 in the supplemental material). Cytochromec₅₅₁ peroxidase and rubrerythrin were confirmed by quantitativeRT-PCR to be down-regulated in the mutant (Table 2).

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FIG. 7. Functional categorization of genes that are differentially expressed in the rel_Gsu mutant in stationary-phase NBAF cultures, identified by whole-genome microarray analysis as described in Materials and Methods. Functional assignments were obtained from the G. sulfurreducens genome page at the TIGR website (http://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=ggs). Genes of unknown function constituted the largest group of differentially expressed genes and included 54 up-regulated genes and 128 down-regulated genes. This functional group was not included in the figure in order to better compare the remaining groups. Gray bars indicate up-regulated genes, while black bars indicate down-regulated genes.

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TABLE 3. Down-regulation of energy metabolism genes in the ${Delta}$ rel_Gsu mutant

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TABLE 2. Differential expression of genes, shown by both quantitative RT-PCR and microarray analyses

The large number of differentially expressed genes involvedin energy metabolism was of particular interest, as this processis significant to Geobacter physiology. A greater number ofthese genes were down-regulated than up-regulated in the rel_Gsumutant. Up-regulated genes included a putative NADH dehydrogenaseoperon, an ATP synthase operon, and several uncharacterizedc-type cytochromes (see Table S1 in the supplemental material).Down-regulated genes included those encoding c-type cytochromesknown to be involved in electron transport to Fe(III), suchas OmcB (37), OmcS (32, 50), and MacA (10), as well as the outermembrane nanowire component PilA (66) (Table 3). In addition,other respiratory genes were down-regulated in the mutant, suchas those encoding HyB hydrogenase, ferredoxin, cytochrome dubiquinol oxidase, cytochrome b, and cytochrome c oxidase, aswell as many uncharacterized c-type cytochromes (Table 3). OmcB,OmcS, MacA, and PilA were all confirmed by quantitative RT-PCRto be down-regulated in the mutant (Table 2).

DISCUSSION

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Discussion

These results demonstrate that Rel_Gsu, the RelA/SpoT homologin G. sulfurreducens, plays a role in the response of G. sulfurreducensto nutrient deprivation and oxidative stress, conditions oftenpresent in subsurface environments where these organisms arefound. In addition, this study illustrates that the G. sulfurreducensstringent response regulates Fe(III) reduction, the primarymode of respiration for Geobacteraceae in their environment.The fact that a similar Rel homolog is found in all other membersof the Geobacteraceae suggests that ppGpp and ppGp may be importantregulators of growth and respiration in the subsurface Geobacteraceaecommunity.

Guanosine phosphate derivatives in G. sulfurreducens. Several previous studies of other organisms have found two guanosinephosphate derivatives produced in response to nutrient deprivation,as was seen with G. sulfurreducens. However, in most organisms,a pentaphosphate derivative, pppGpp, was commonly found alongwith ppGpp (13, 14, 28, 31). Our experiments did not detectpppGpp but instead detected a triphosphate derivative that israrely seen with other organisms (Fig. 2). The rarity of ppGpmay simply be a consequence of the methods used for detection,as the most commonly used method, thin-layer chromatography,does not allow differentiation between different guanosine triphosphates.The HPLC-based technique used in this study allows distinctionbetween ppGp and GTP, and both ppGpp and ppGp compounds havebeen detected in other studies using this technique (21, 35).It remains to be investigated how these individual stringentfactors might exert specific effects on G. sulfurreducens.

Role of Rel_Gsu is comparable to that of RelA/SpoT homologs in other organisms. In some ways, the role of Rel_Gsu in G. sulfurreducens appearsto be similar to that in other organisms. For example, in manybacteria, the stringent response slows growth in response tonutrient deprivation (7, 16). Both microarray and phenotypicanalyses of the rel_Gsu mutant suggest a role for Rel_Gsu in inducingslow growth. Comparison of levels of gene expression betweenthe Rel_Gsu-deficient mutant and the wild type during stationaryphase demonstrated that transcript levels for multiple genesinvolved in protein synthesis were higher in the rel_Gsu mutant.These genes included those encoding ribosomal proteins, tRNAsynthetases, chaperones, and enzymes involved in amino acidbiosynthesis. In addition, several genes involved in nucleotideand cell membrane biosynthesis were also higher in the mutant(see Table S1 in the supplemental material). These results arecomparable to results from microarray analyses of the stringentresponse in other organisms (22, 23, 69). In addition, in theabsence of Rel_Gsu, G. sulfurreducens reached higher cell densitiesbefore the onset of stationary phase than the wild type (Fig.4), suggesting the wild-type decrease in growth rate upon nutrientdeprivation may be Rel dependent. Mutant cells subsequentlyexhibited wild-type growth characteristics when Rel_Gsu was reintroduced.These data are consistent with a role for Rel_Gsu and ppGp(p)in slowing growth rate in response to nutrient deficiency.

Interestingly, the increased growth seen in the rel_Gsu mutantis not commonly found in Rel mutants from other bacteria. Severalother microorganisms in which Rel activity was knocked out andno ppGpp was produced demonstrated growth defects. In E. coli,a double RelA/SpoT mutant demonstrated slower growth than thewild type did (30). In Myxococcus xanthus, loss of ppGpp, causedby a Rel mutation, resulted in developmental arrest (28). Oneexample where the loss of ppGpp resulted in faster growth wasreported for Mycobacterium tuberculosis (65). An M. tuberculosisRel mutant had a higher growth rate as well as a higher cellyield when grown under certain growth conditions, but the mutantshowed growth defects under several other conditions (65). Asit is widely understood that production of ppGpp induces slowgrowth in bacteria (7, 13, 16), it follows that the lack ofppGpp in a Rel mutant might lead to increased growth rates.More observations of slower growth, as opposed to faster growth,caused by rel mutations can be explained by findings that ppGppand rel signaling systems affect many cellular processes, anddefects in these other processes, due to altered ppGpp levels,may result in defects in growth.

In addition to slowing growth, the stringent response is knownto increase stress tolerance in other organisms (24, 26, 39,85). In G. sulfurreducens, ppGpp was produced in response tooxygen exposure (Fig. 3), and deleting the rel_Gsu gene increasedan oxidative stress-dependent growth lag in medium lacking thereductant cysteine (Fig. 5). Furthermore, Rel_Gsu deficiencyinhibited a cell aggregation response found in the presenceof oxygen that occurs in the wild type under the same conditionsand is believed to be an adaptive response to oxidative stress(60) (Fig. 5). Transcript levels for several genes consideredto be involved in oxidative stress response were lower in rel_Gsumutant cells than in wild-type cells (Tables 2 and 3; also seeTable S2 in the supplemental material). In addition, there wasa substantial overlap between the rel_Gsu regulon, determinedwith the microarray analysis reported here, and the regulonof RpoS (61), a well-characterized sigma factor shown to functionin stress response in many organisms, including in G. sulfurreducens(60). Together, these findings are consistent with a role forRel_Gsu and ppGpp in oxidative stress response in G. sulfurreducens.

Role of Rel_Gsu in regulating Fe(III) respiration. The results also indicate an important role for Rel_Gsu in regulatingkey genes required for Fe(III) reduction. The rel_Gsu mutantwas severely limited in its ability to reduce Fe(III), and thecapacity for Fe(III) reduction was restored when rel_Gsu wasexpressed in trans (Fig. 6). Transcript levels for many electrontransport genes were lower in the rel_Gsu mutant. These includedgenes for several c-type cytochromes known to be necessary foroptimal Fe(III) reduction, as well as the electricity conductivepili that are essential for Fe(III) oxide reduction (66) (Tables2 and 3).

The stringent response has often been found to mediate responsesthat are related to diverse cellular processes specific to anindividual organism's growth requirements. This is the casefor Myxococcus xanthus, where A-factor production and fruitingbody formation are under stringent control (28), and for severalpathogenic bacteria, where virulence is under stringent control(27, 39, 63). However, to our knowledge the results presentedhere are the first indication that the stringent response resultsin the increased expression of genes involved in respiration.In fact, studies of transient growth arrest and stationary-phasegrowth in E. coli have suggested that aerobic electron transportchain gene expression decreases under stringent control (15).

Conclusions. These results suggest that Rel_Gsu activity regulates the expressionof genes involved in Fe(III) reduction, as well as responsesto several common environmental stresses in G. sulfurreducens.Thus, the stringent response is likely to play an importantrole in balancing growth in subsurface environments where nutrientsare low and stresses are high. Further examination of this responsemay provide insight into strategies for optimizing practicalapplications of Geobacter species during bioremediation of subsurfacecontaminants and in harvesting energy from the environment.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology, University of Massachusetts, Morrill Science Center, 639 N. Pleasant St. Amherst, MA 01003. Phone: (413) 577-2439. Fax: (413) 545-1578. E-mail: lauried{at}microbio.umass.edu.

Published ahead of print on 13 October 2006.

Supplemental material for this article may be found at http://jb.asm.org/.

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