The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens
© Aklujkar et al; licensee BioMed Central Ltd. 2009
Received: 11 December 2008
Accepted: 27 May 2009
Published: 27 May 2009
The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences.
The experimentally observed greater metabolic versatility of G. metallireducens versus G. sulfurreducens is borne out by the presence of more numerous genes for metabolism of organic acids including acetate, propionate, and pyruvate. Although G. metallireducens lacks a dicarboxylic acid transporter, it has acquired a second putative succinate dehydrogenase/fumarate reductase complex, suggesting that respiration of fumarate was important until recently in its evolutionary history. Vestiges of the molybdate (ModE) regulon of G. sulfurreducens can be detected in G. metallireducens, which has lost the global regulatory protein ModE but retained some putative ModE-binding sites and multiplied certain genes of molybdenum cofactor biosynthesis. Several enzymes of amino acid metabolism are of different origin in the two species, but significant patterns of gene organization are conserved. Whereas most Geobacteraceae are predicted to obtain biosynthetic reducing equivalents from electron transfer pathways via a ferredoxin oxidoreductase, G. metallireducens can derive them from the oxidative pentose phosphate pathway. In addition to the evidence of greater metabolic versatility, the G. metallireducens genome is also remarkable for the abundance of multicopy nucleotide sequences found in intergenic regions and even within genes.
The genomic evidence suggests that metabolism, physiology and regulation of gene expression in G. metallireducens may be dramatically different from other Geobacteraceae.
Geobacter metallireducens is a member of the Geobacteraceae, a family of Fe(III)-respiring Delta-proteobacteria that are of interest for their role in cycling of carbon and metals in aquatic sediments and subsurface environments as well as the bioremediation of organic- and metal-contaminated groundwater and the harvesting of electricity from complex organic matter [1, 2]. G. metallireducens is of particular interest because it was the first microorganism found to be capable of a number of novel anaerobic processes including: (1) conservation of energy to support growth from the oxidation of organic compounds coupled to the reduction of Fe(III) or Mn(IV) [3, 4]; (2) conversion of Fe(III) oxide to ultrafine-grained magnetite ; (3) anaerobic oxidation of an aromatic hydrocarbon [5, 6]; (4) reduction of U(VI) ; (5) use of humic substances as an electron acceptor ; (6) chemotaxis toward metals ; (7) complete oxidation of organic compounds to carbon dioxide with an electrode serving as the sole electron acceptor (; and (8) use of a poised electrode as a direct electron donor . Although the complete genome sequence of the closely related Geobacter sulfurreducens is available  and can provide insights into some of the common metabolic features of Geobacter species, G. metallireducens and G. sulfurreducens are significantly different in many aspects of their physiology. G. sulfurreducens is known to use only four carbon sources: acetate, formate, lactate (poorly) and pyruvate (only with hydrogen as electron donor), whereas G. metallireducens uses acetate, benzaldehyde, benzoate, benzylalcohol, butanol, butyrate, p-cresol, ethanol, p-hydroxybenzaldehyde, p-hydroxybenzoate, p-hydroxybenzylalcohol, isobutyrate, isovalerate, phenol, propionate, propanol, pyruvate, toluene and valerate .
Therefore, in order to gain broader insight into the physiological diversity of Geobacter species, the genome of G. metallireducens was sequenced and compared to that of Geobacter sulfurreducens . Both genome annotations were manually curated with the addition, removal and adjustment of hundreds of protein-coding genes and other features. Phylogenetic analyses were conducted to validate the findings, including homologs from the finished and unfinished genome sequences of more distantly related Geobacteraceae. This paper presents insights into the conserved and unique features of two Geobacter species, particularly the metabolic versatility of G. metallireducens and the numerous families of multicopy nucleotide sequences in its genome, which suggest that regulation of gene expression is very different in these two species.
Results and Discussion
Contents of the two genomes
The automated annotation of the G. metallireducens genome identified 3518 protein-coding genes on the chromosome of 3997420 bp and 13 genes on the plasmid (designated pMET1) of 13762 bp. Manual curation added 59 protein-coding genes plus 56 pseudogenes to the chromosome and 4 genes to the plasmid. Ten of the chromosomal genes were reannotated as pseudogenes and another 22 were removed from the annotation. In addition to the 58 RNA-coding genes in the automated annotation, manual curation identified 479 conserved nucleotide sequence features. Likewise, to the 3446 protein-coding genes in the automated annotation of the G. sulfurreducens genome , manual curation added 142 protein-coding genes and 19 pseudogenes. Five genes were reannotated as pseudogenes and 103 genes were removed from the annotation. In addition to the 55 RNA-coding genes in the automated annotation, manual curation identified 462 conserved nucleotide sequence features. Of the 3629 protein-coding genes and pseudogenes in G. metallireducens, 2403 (66.2%) had one or more full-length homologs in G. sulfurreducens.
The nucleotide composition of the 3563 intact protein-coding genes of G. metallireducens was determined in order to identify some of those that were very recently acquired. The average G+C content of the protein-coding genes was 59.5%, with a standard deviation of 5.9%. Only three genes had a G+C content more than two standard deviations above the mean (> 71.2%), but 146 genes had a G+C content more than two standard deviations below the mean (< 47.7%), most of which lack homologs in G. sulfurreducens and may be recent acquisitions (Additional file 1: Table S1). Clusters of such genes (shaded in Additional file 1: Table S1) were often interrupted or flanked by transposons with higher G+C content. The functions of most of these genes cannot be assigned at present, but 23 of them are predicted to act in cell wall biogenesis.
Plasmid pMET1 of G. metallireducens consists of a series of six predicted transcriptional units on one strand, tentatively attributed to the mobilization (Gmet_A3575-Gmet_A3574-Gmet_A3573-Gmet_A3572-Gmet_A3643), entry exclusion (Gmet_A3571), addiction (Gmet_A3570-Gmet_A3579-Gmet_A3642), partition (Gmet_A3568-Gmet_A3641), transposition (Gmet_A3567), and replication (Gmet_A3566-Gmet_A3565) functions of the plasmid, and one operon on the opposite strand, comprised of three genes of unknown function (Gmet_A3576-Gmet_A3577-Gmet_A3644). The predicted origin of replication, located 3' of the repA gene (Gmet_A3565), includes four pairs of iterons and a set of six hairpins, suggesting that pMET1 replicates by a rolling-circle mechanism, although it is significantly larger than most such plasmids . Among the fifteen other nucleotide sequence features identified on the plasmid during manual curation was a palindromic putative autoregulatory site (TTTGTTATACACGTATAACAAA) located 5' of the addiction module. Other than the potential toxicity of the addiction module, the impact of pMET1 on the physiology of G. metallireducens is unknown.
Metabolism of acetate and other carbon sources
Three enzymes distantly related to the succinyl:acetate CoA-transferases are encoded by Gmet_2054, Gmet_3294, and Gmet_3304, for which there are no counterparts in G. sulfurreducens. All three of these proteins closely match the characterized butyryl:4-hydroxybutyrate/vinylacetate CoA-transferases of Clostridium species . However, their substrate specificities may be different because the G. metallireducens proteins and the Clostridium proteins cluster phylogenetically with different CoA-transferases of Geobacter strain FRC-32 and Geobacter bemidjiensis (data not shown). The presence of these CoA-transferases indicates that G. metallireducens has evolved energy-efficient activation steps for some unidentified organic acid substrates that G. sulfurreducens cannot utilize.
Numerous other enzymes of acyl-CoA metabolism are predicted from the genome of G. metalllireducens but not that of G. sulfurreducens (Additional file 2: Table S2), including six gene clusters, three of which have been linked to degradation of aromatic compounds that G. metallireducens can utilize [6, 21–23] but G. sulfurreducens cannot . All seven acyl-CoA synthetases of G. sulfurreducens have orthologs in G. metallireducens, but the latter also possesses acetyl-CoA synthetase, benzoate CoA-ligase (experimentally validated ), and seven other acyl-CoA synthetases of unknown substrate specificity. The G. metallireducens genome also includes eleven acyl-CoA dehydrogenases, three of which are specific for benzylsuccinyl-CoA (69% identical to the Thauera aromatica enzyme ), glutaryl-CoA (experimentally validated ) and isovaleryl-CoA (69% identical to the Solanum tuberosum mitochondrial enzyme ), whereas none can be identified in G. sulfurreducens. G. metallireducens also has nine pairs of electron transfer flavoprotein genes (seven of which are adjacent to genes encoding iron-sulfur cluster-binding proteins) that are hypothesized to connect acyl-CoA dehydrogenases to the respiratory chain, whereas G. sulfurreducens has only one. None of the seventeen enoyl-CoA hydratases of G. metallireducens is an ortholog of GSU1377, the sole enoyl-CoA hydratase of G. sulfurreducens. G. metallireducens also possesses eleven acyl-CoA thioesterases, of which G. sulfurreducens has orthologs of five plus the unique thioesterase GSU0196. Of the ten acyl-CoA thiolases of G. metallireducens, only Gmet_0144 has an ortholog (GSU3313) in G. sulfurreducens. BLAST searches and phylogenetic analyses demonstrated that several of these enzymes of acyl-CoA metabolism have close relatives in G. bemidjiensis, Geobacter FRC-32, Geobacter lovleyi and Geobacter uraniireducens, indicating that their absence from G. sulfurreducens is due to gene loss, and that this apparent metabolic versatility is largely the result of expansion of enzyme families within the genus Geobacter (data not shown). The ability of G. metallireducens and other Geobacteraceae to utilize carbon sources that G. sulfurreducens cannot utilize may be due to stepwise breakdown of multicarbon organic acids to simpler compounds by these enzymes.
Growth of G. metallireducens on butyrate may be attributed to reversible phosphorylation by either of two butyrate kinases (Gmet_2106 and Gmet_2128), followed by reversible CoA-ligation by phosphotransbutyrylase (Gmet_2098), a pathway not present in G. sulfurreducens, which cannot grow on butyrate . These gene products are 42–50% identical to the enzymes characterized in Clostridium beijerinckii and Clostridium acetobutylicum [28, 29].
Gmet_0149 (GSU3448) is a homolog of acetate kinase that does not contribute sufficient acetate kinase activity to sustain growth of G. sulfurreducens  and has a closer BLAST hit to propionate kinase of E. coli (40% identical sequence) than to acetate kinase of E. coli. Although it does not cluster phylogenetically with either of the E. coli enzymes, its divergence from acetate kinase (Gmet_1034 = GSU2707) is older than the last common ancestor of the Geobacteraceae (data not shown). This conserved gene product remains to be characterized as a propionate kinase or something else.
The proposed pathway for growth of G. metallireducens on propionate (Figure 2) is contingent upon its experimentally established ability to grow on pyruvate . G. sulfurreducens cannot utilize pyruvate as the carbon source unless hydrogen is provided as an electron donor . Oxidation of acetyl-CoA derived from pyruvate in G. sulfurreducens may be prevented by a strict requirement for the succinyl:acetate CoA-transferase reaction (thermodynamically inhibited when acetyl-CoA exceeds acetate) to complete the TCA cycle in the absence of detectable activity of succinyl-CoA synthetase (GSU1058-GSU1059) . With three sets of succinyl-CoA synthetase genes (Gmet_0729-Gmet_0730, Gmet_2068-Gmet_2069, and Gmet_2260-Gmet_2261), G. metallireducens may produce enough activity to complete the TCA cycle.
G. sulfurreducens and G. metallireducens may interconvert malate and pyruvate through a malate oxidoreductase fused to a phosphotransacetylase-like putative regulatory domain (maeB; Gmet_1637 = GSU1700), which is 51% identical to the NADP+-dependent malic enzyme of E. coli . G. sulfurreducens has an additional malate oxidoreductase without this fusion (mleA; GSU2308) that is 53% identical to an NAD+-dependent malic enzyme of B. subtilis , but G. metallireducens does not.
Evidence of recent fumarate respiration in G. metallireducens
Nitrate respiration and loss of the modE regulon from G. metallireducens
Genes of molybdenum cofactor biosynthesis in G. sulfurreducens and G. metallireducens.
Gene in G. sulfurreducens
Gene in G. metallireducens
regulation of molybdate-responsive genes
inner membrane protein, possible quinolinate phosphoribosyltransferase
molybdate transport (periplasmic component)
molybdate transport (membrane component)
molybdate transport (ATP-binding component)
dithiolene addition to molybdopterin (molybdopterin synthase small subunit)
molybdopterin synthase sulfurylase
dithiolene addition to molybdopterin (molybdopterin synthase large subunit)
Gmet_1038; Gmet_0336; Gmet_1804
molybdopterin precursor Z synthesis
molybdopterin precursor Z synthesis
GSU3147 N-terminal domain
Gmet_0300 N-terminal domain
attachment of molybdopterin to guanosine
GSU3147 C-terminal domain
Gmet_0300 C-terminal domain
attachment of molybdopterin to guanosine
Gmet_0301; Gmet_0337; Gmet_2095
molybdopterin precursor Z synthesis
possible 4-hydroxybenzoyl-CoA reductase molybdenum cofactor biosynthesis protein
possible 4-hydroxybenzoyl-CoA reductase molybdenum cofactor biosynthesis protein
uncharacterized protein related to MobA
In G. sulfurreducens, putative binding sites for the molybdate-sensing ModE protein (GSU2964) have been identified by the ScanACE software [41, 42] in several locations, and the existence of a ModE regulon has been predicted . The genes in the predicted ModE regulon (Additional file 3: Table S3) include one of the two succinyl:acetate CoA-transferases, a glycine-specific tRNA (anticodon CCC, corresponding to 26% of glycine codons), several transport systems, and some nucleases. In G. metallireducens, there is no full-length modE gene, but a gene encoding the C-terminal molybdopterin-binding (MopI) domain of ModE (Gmet_0511) is present in the same location (Figure 6). Phylogenetic analysis shows that the Gmet_0511 gene product is the closest known relative of G. sulfurreducens ModE, and that it has evolved out of the Geobacteraceae/Chlorobiaceae cluster of full-length ModE proteins by loss of the N-terminal ModE-specific domain (data not shown). The ScanACE software detected only one of the ModE-binding sites of G. sulfurreducens at the corresponding location in the G. metallireducens genome, but some vestigial sites were apparent when other syntenous locations were visually inspected (Additional file 3: Table S3), indicating that the ModE regulon once existed in G. metallireducens, but recent loss of the ModE N-terminal domain is allowing the regulatory sites to disappear gradually over the course of genome sequence evolution due to the absence of selective pressure for these sites to remain conserved. Thus, genes that may be controlled globally by ModE in G. sulfurreducens and other Geobacteraceae to optimize molybdenum cofactor-dependent processes have recently acquired independence in G. metallireducens.
Amino acid biosynthesis and its regulation
The two genomes differ in several aspects of amino acid biosynthesis and its regulation. To make aspartate from oxaloacetate, a homolog of Bacillus circulans aspartate aminotransferase  is present in G. metallireducens (Gmet_2078; 65% identical), whereas a homolog of the Sinorhizobium meliloti enzyme  is found in G. sulfurreducens (GSU1242; 52% identical). Both species possess asparagine synthetase (Gmet_2172 = GSU1953 and Gmet_2024, 30% and 24% identical to asnB of B. subtilis ) and glutamine synthetase (Gmet_1352 = GSU1835, 61% identical to glnA of Fremyella diplosiphon ), as well as an aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase operon (Gmet_0076, Gmet_0075, Gmet_0073 = GSU3383, GSU3381, GSU3380, 36–53% identical to the homologous subunits in B. subtilis ) that includes glutamine synthetase adenylyltransferase (glnE; Gmet_0071 = GSU3378). The G. sulfurreducens glnE gene may be inactive due to a deletion of ~ 45 codons in the C-terminal domain.
For biosynthesis of lysine, threonine and methionine, G. metallireducens and other Geobacteraceae possess a linked pair of aspartate-4-semialdehyde dehydrogenase genes: Pseudomonas aeruginosa-type Gmet_0603 (69% identity)  and Mycobacterium bovis-type Gmet_0604 (47% identity) , but G. sulfurreducens has only the former (GSU2878). A haloacid dehalogenase family protein (Gmet_1630 = GSU1694) encoded between two genes of the threonine biosynthesis pathway could be the enzyme required to complete the pathway, a phosphoserine:homoserine phosphotransferase analogous to that of P. aeruginosa , and may overlap functionally with the unidentified phosphoserine phosphatase required to complete the biosynthetic pathway of serine.
Conserved nucleotide sequences 5' of biosynthetic operons.
Locus tag and sequence coordinates
aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase (gatCAB-mtnA-glnE-nth)
aromatic amino acids (aroG-2)
cobalamin (cobUTSCB-thiC-2; cbiM-1-cbiQ-1-cbiO-1-cbiX-cobH-cbiD-cobLIM-cbiG-cobQ-cbiB-cobD)
methionine (metC-1-metC-2; metX)1
coenzyme A (panBC)
purines, pyrimidines (purMN, rimI-pyrKD)
thiamin (thiE/D-thiC-1; thiS-1-thiG-tenI)3
S-adenosylmethionine (SAM)-responsive riboswitches (Table 2) may regulate homoserine O-acetyltransferase (Gmet_2783 = GSU2462, 45% identical to the Leptospira meyeri enzyme ), the first dedicated enzyme of methionine biosynthesis, and also two linked cystathionine-γ-synthase/cystathionine-β-lyase genes (Gmet_0698 = GSU0944; Gmet_0699 = GSU0945, 49% and 51% identical to the Lactococcus lactis lyase [56, 57]). Phylogenetic analysis could not distinguish the synthase from the lyase (data not shown), but their presence suggests that homocysteine can be made by transsulfuration of homoserine with cysteine, and not only by the putative O-acetylhomoserine sulfhydrylases (Gmet_0819 = GSU2425, Gmet_2390 = GSU1183 and Gmet_1566, 47%, 56% and 38% identical to the Emericella nidulans enzyme , respectively). In G. metallireducens, transsulfuration may also be controlled by a GC-rich element between Gmet_0698 and Gmet_0699, which contains four tandem repeats of the heptanucleotide GGGACCG and is found in 49 intergenic and intragenic locations in the genome (Additional file 6: Figure S2, Additional file 5: Table S4).
The leucine pathway-specific leuA gene (2-isopropylmalate synthase; Gmet_1265 = GSU1906, 49% identical to the E. coli enzyme ) may be controlled by feedback inhibition through a T-box  predicted to form an antiterminator structure in response to uncharged leucine-specific tRNA having the GAG anticodon (Gmet_R0037 = GSUR030) (Table 2), putatively the only tRNA capable of recognizing 55% of leucine codons in G. metallireducens and 48% in G. sulfurreducens (CTC and CTT).
There are three 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase isoenzymes to catalyze the first step of aromatic amino acid biosynthesis: one similar to aroF of E. coli (Gmet_2375 = GSU2291, 55% identity , but with a P148T substitution incompatible with feedback inhibition by tyrosine ) and two Thermotoga maritima-type enzymes (Gmet_0024 = GSU3333; Gmet_0346 = GSU3142, 51% and 46% identity , respectively). As one chorismate mutase is fused to prephenate dehydratase (pheA; Gmet_0862 = GSU2608, 41% identical to the Pseudomonas stutzeri fusion protein ), the other (Gmet_1955 = GSU1828, 30% identical to the chorismate mutase domain of the P. stutzeri fusion protein) may function predominantly in tyrosine biosynthesis, possibly regulated by the adjacent gene product (Gmet_1956 = GSU1829) that resembles the phenylalanine/tyrosine-responsive domain of T. maritima DAHP synthase . Gmet_1956 orthologs phylogenetically cluster with the regulatory domains of Gmet_0024 orthologs (data not shown), suggesting that Gmet_0024 may be a tyrosine-inhibited DAHP synthase and Gmet_0346 may be inhibited by another end product such as phenylalanine. A predicted short RNA element (Gmet_R0069 = GSUR082, Table 2), found 5' of Gmet_0346 and its orthologs in several Geobacteraceae, may participate in regulation of this isoenzyme's expression.
In all non-Geobacteraceae that possess an indole-scavenging tryptophan synthase β 2 protein, it is encoded apart from the trp operon containing the trpAB1 genes for the α (indole-producing) and β 1 (indole-consuming) subunits of tryptophan synthase . In G. metallireducens and G. sulfurreducens, however, the β 2 gene trpB2 (Gmet_2493 = GSU2379, 60% identical to the T. maritima protein ) is the penultimate gene of the predicted trp operon and the trpB1 (Gmet_2482 = GSU2375, 66% identical to the Acinetobacter calcoaceticus protein ) and trpA (Gmet_2477 = GSU2371, 47% identical to the Azospirillum brasilense protein ) genes are separated from the 3' end of the operon and from each other by three or more intervening genes, most of which are not conserved between the two genomes (not shown). Next to the trpB2 gene of G. metallireducens is one of 24 pairs of a conserved nucleotide motif (Additional file 7: Figure S3, Additional file 5: Table S4) hypothesized to bind an unidentified global regulator protein. Other, evolutionarily related paired sites where another unidentified global regulator may bind (Additional file 8: Figure S4, Additional file 5: Table S4) are found in 21 locations. Between the proBA genes of G. metallireducens, encoding the first two enzymes of proline biosynthesis (Gmet_3198-Gmet_3199 = GSU3212-GSU3211, 41% and 45% identical to the E. coli enzymes ), is one of eight pairs of predicted binding sites for yet another unidentified global regulator (Additional file 9: Figure S5, Additional file 5: Table S4). In G. sulfurreducens, the space between proBA is occupied by a different conserved nucleotide sequence (not shown), found only in four other places in the same genome. Overall, a comparison of the two genomes offers insight into unique features of amino acid biosynthesis and its regulation that deserve further study.
Differences in nucleotide metabolism were identified in the two genomes. G. metallireducens has acquired a possibly redundant large subunit of carbamoyl-phosphate synthetase (Gmet_0661, 50% identical to the P. aeruginosa protein ) in addition to the ancestral gene (Gmet_1774 = GSU1276, 65% identity to P. aeruginosa), Both genomes encode a second putative thymidylate kinase (Gmet_3250 = GSU3301) distantly related to all others, in addition to the one found in other Geobacteraceae (Gmet_2318 = GSU2229, 41% identical to the E. coli enzyme ). G. sulfurreducens has evidently lost the purT gene product of G. metallireducens and several other Geobacteraceae (Gmet_3193, 58% identical to the E. coli enzyme ), which incorporates formate directly into purine nucleotides instead of using the folate-dependent purN gene product (Gmet_1845 = GSU1759, 46% identical to the E. coli enzyme ).
Comparative genomics indicates that, similar to most Geobacter species, G. metallireducens possesses two glyceraldehyde-3-phosphate dehydrogenase isoenzymes: Gmet_1211 and Gmet_1946 (59% and 56% identical to gluconeogenic GapB and glycolytic GapA of Corynebacterium glutamicum , respectively), but G. sulfurreducens has an ortholog of only the latter (GSU1629). G. metallireducens also has a putative fructose 6-kinase (Gmet_2805, 39% identical to the E. coli enzyme ) that is not present in G. sulfurreducens. Remarkably, G. metallireducens possesses two isoenzymes each of UDP-glucose 4-epimerase (Gmet_1486; Gmet_2329 = GSU2240, 50% and 54% identical to the A. brasilense enzyme ), glutamine:fructose-6-phosphate aminotransferase (Gmet_1487; Gmet_0104 = GSU0270, 55% and 53% identical to the Thermus thermophilus enzyme ), GDP-mannose 4,6-dehydratase (Gmet_1488 = GSU0626; Gmet_1311, 61% and 72% identical to the E. coli enzyme ) and UDP-N-acetylglucosamine 2-epimerase (Gmet_1489 = GSU2243, 61% identical to the E. coli enzyme ; Gmet_1504, 39% identical to the Methanococcus maripaludis enzyme ). G. metallireducens has evolved a gene cluster of the four enzyme activities (Gmet_1486-Gmet_1489) from both ancestral gene duplication and lateral gene transfer (data not shown). The reason for this emphasis on interconversion of hexoses in G. metallireducens versus G. sulfurreducens is unknown.
Unlike the genomes of G. sulfurreducens and most other Geobacteraceae, which encode the enzymes of only the non-oxidative branch of the pentose phosphate pathway, the G. metallireducens genome includes a cluster of oxidative pentose phosphate pathway enzyme genes: 6-phosphogluconolactonase (Gmet_2618, 30% identical to the Pseudomonas putida enzyme ), glucose-6-phosphate dehydrogenase (Gmet_2619, 50% identical to the Nostoc punctiforme enzyme ), and 6-phosphogluconate dehydrogenase (Gmet_2620, 36% identical to YqeC of B. subtilis ), along with two ribose-5-phosphate isomerase isoenzymes (Gmet_2621 and Gmet_1604 = GSU1606, 39% and 44% identical to RpiB of E. coli ). Thus, G. metallireducens apparently generates biosynthetic reducing equivalents in the form of NADPH from carbohydrates. The NADPH supply of G. sulfurreducens, in contrast, may derive from the electron transfer chain via a ferredoxin:NADP+ reductase (GSU3058-GSU3057, each 52% identical to its Pyrococcus furiosus homolog ) that is found in other Geobacteraceae, but not in G. metallireducens.
Both G. sulfurreducens and G. metallireducens may protect themselves from desiccation by making trehalose from glucose storage polymers via maltooligose in three steps catalyzed by an alpha-amylase domain protein (Gmet_3469 = GSU2361), maltooligosyltrehalose synthase (Gmet_3468 = GSU2360, 35% identical to the Rhizobium leguminosarum enzyme ), and maltooligosyltrehalose trehalohydrolase (Gmet_3467 = GSU2358, 44% identical to the Arthrobacter strain Q36 enzyme ). G. sulfurreducens, P. propionicus and G. lovleyi may also make trehalose from glucose-6-phosphate by the sequential action of trehalose-6-phosphate synthase (GSU2337, containing a domain 37% identical to the Mycobacterium tuberculosis enzyme ) and trehalose-6-phosphatase (GSU2336, 29% identical to the E. coli enzyme ), which are missing in G. metallireducens. Thus, G. sulfurreducens is capable of achieving osmotolerance without consuming carbohydrate storage polymers, but G. metallireducens is not.
Biogenesis of c-type cytochromes and pili
The genome of G. metallireducens encodes 91 putative c-type cytochromes, of which 65 have homologs among the 103 c-type cytochromes of G. sulfurreducens. Of the c-type cytochrome genes implicated in Fe(III) and U(VI) reduction in G. sulfurreducens, those conserved in G. metallireducens are macA (Gmet_3091 = GSU0466) [91–93] and ppcA (Gmet_2902 = GSU0612) , whereas different c-type cytochrome sequences are found in syntenous locations where one would expect omcB and omcC (Gmet_0910 ≠ GSU2737; Gmet_0913 ≠ GSU2731) , and omcE (Gmet_2896 ≠ GSU0618) . The G. metallireducens genome contains no genes homologous to omcS (GSU2504) and omcT (GSU2503) , and only a paralog (Gmet_0155 = GSU2743) of omcF (GSU2432) . This lack of conservation is being investigated further (J. Butler, personal communication).
Notable differences between G. metallireducens and G. sulfurreducens are apparent in the biogenesis of c-type cytochromes, in biosynthesis of the heme group, and in reduction of disulfide bonds to allow covalent linkage to heme. In addition to the membrane-peripheral protoporphyrinogen IX oxidase of G. sulfurreducens and other Geobacteraceae, encoded by the hemY gene (Gmet_3551 = GSU0012, 38% identical to the Myxococcus xanthus enzyme ), G. metallireducens has a membrane-integral isoenzyme encoded by hemG (Gmet_2953, 43% identical to the E. coli enzyme ), with a homolog in Geobacter FRC-32. These two species also possess a putative disulfide bond reduction system not found in G. sulfurreducens and other Geobacteraceae, comprised of DsbA, DsbB, DsbE and DsbD homologs (Gmet_1380, Gmet_1381, Gmet_1383, Gmet_1384), encoded in a cluster alongside a two-component signalling system (Gmet_1378-Gmet_1379), an arylsulfotransferase (Gmet_1382), and a conserved protein of unknown function (Gmet_1385). Transcription of dsbA and dsbB is diminished during growth on benzoate , and phylogenetic analysis indicates that these DsbA and DsbB proteins belong to subfamilies distinct from those that have been characterized (R. Dutton, personal communication). Located apart from this cluster, DsbC/DsbG (Gmet_2250) of G. metallireducens has homologs in several Geobacteraceae, but not in G. sulfurreducens. However, CcdA/DsbD (Gmet_2451 = GSU1322) is present in both. Thus, the pathways of c-type cytochrome biogenesis may be significantly different in the two species and somehow linked to the degradation of aromatic compounds by G. metallireducens.
In both G. sulfurreducens and G. metallireducens, there are four c-type cytochrome biogenesis genes related to ResB of B. subtilis , each predicted to be co-transcribed with a gene encoding a ResC/HemX-like protein (hypothesized to be a heme transporter with eight predicted transmembrane segments)  and several multiheme c-type cytochrome genes (Additional file 10: Table S5). One more protein of the ResC/HemX-like family (Gmet_3232 = GSU3283) is encoded among enzymes of heme biosynthesis in both genomes. These gene arrangements suggest that each pair of c-type cytochrome biogenesis proteins may be dedicated to the efficient expression of the cytochromes encoded nearby. Two of the pairs are orthologously conserved (Gmet_2901-Gmet_2900 = GSU0613-GSU0614; Gmet_0592..Gmet_0594 = GSU2891-GSU2890); the other two pairs (Gmet_0572-Gmet_0573; Gmet_0578-Gmet_0579; GSU0704-GSU0705; GSU2881.1-GSU2880), which appear to derive from expansion of ancestral genes, may be relevant to the diversified c-type cytochrome repertoire of the two species. Interestingly, three of these gene pairs in G. metallireducens are arranged in proximity to each other in a cluster of ten operons with the same coding DNA strand (Gmet_0571 to Gmet_0601), suggesting that their expression may be co-ordinated by transcriptional readthrough (Additional file 10: Table S5). The purposes of various pairs of c-type cytochrome biogenesis proteins in Geobacteraceae remain to be determined.
The pili of G. sulfurreducens have been implicated in electron transfer [101, 102] and biofilm formation . Most genes attributed to pilus biogenesis in G. sulfurreducens have orthologs in G. metallireducens, suggesting that these roles of pili may be conserved. However, instead of the ancestral pilY1 gene found in G. sulfurreducens (GSU2038) and other Geobacteraceae, which may encode a pilus tip-associated adhesive protein , G. metallireducens possesses a phylogenetically distinct pilY1 gene in the same location (Gmet_0967; data not shown), surrounded by different genes of unknown function within a cluster of pilus biogenesis genes. Therefore, it remains possible that structural and functional differences between the pili of the two species will be identified in future.
Solute transport systems
Although the substrates of most solute transport systems of G. metallireducens and G. sulfurreducens are unknown, several features distinguish the two species (Additional file 11: Table S6). One of two predicted GTP-dependent Fe(II) transporters of the Geobacteraceae (feoB-1 Gmet_2444 = GSU1380), located next to the ferric uptake regulator gene (fur Gmet_2445 = GSU1379), is present in G. metallireducens; the other (feoB-2 GSU3268), with two feoA genes on its 5' side (GSU3268.1, GSU3270) potentially encoding an essential cytosolic component of the transport system , is not. Phylogenetic analysis showed that the FeoB-2 proteins of Geobacteraceae are closely related to the characterized Fe(II)-specific FeoB proteins of Porphyromonas gingivalis  and Campylobacter jejuni , whereas the FeoB-1 proteins of Geobacteraceae cluster apart from them (data not shown). FeoB-1 proteins are not closely related to the manganese-specific FeoB of P. gingivalis  either, and so their substrate specificity cannot be assigned at present.
In G. metallireducens, duplicate kup genes, predicted to encode low-affinity potassium/proton symporters, are found in one place (Gmet_0038 = GSU3342; Gmet_0039 = GSU2485, 29% and 31% identical to the E. coli protein ), apart from the kdpABCDE genes (Gmet_2433-Gmet_2437 = GSU2480-GSU2484, 38–49% identical to the homologs in E. coli [109, 110]) encoding an osmosensitive potassium-translocating ATPase complex. In G. sulfurreducens, one of these kup genes (GSU2485) is located 3' of the kdp gene cluster, apparently under control of an osmosensitive riboswitch (GSU2484.1, sequence coordinates 2728254 to 2728393), and there is a third kup gene (GSU2350, 49% identity to E. coli) not found in other Geobacteraceae. G. sulfurreducens also has at least two potassium/proton antiporters (GSU1203, 34% identical to CvrA of Vibrio parahaemolyticus ; GSU2759, 31% identical to KefB of E. coli ) and a sodium/proton antiporter complex (mrpABCDEFG GSU2344-GSU2338, 29–48% identical to the homologs in B. subtilis ) that are not found in G. metallireducens. Three mechanosensitive ion channels are common to the two species (Gmet_1942 = GSU1633; Gmet_2581 = GSU2316; and Gmet_2522 = GSU2794); two more are unique to G. sulfurreducens (GSU1557; GSU1723). Thus, control of monovalent cation homeostasis appears to be more complex in G. sulfurreducens.
Several heavy metal efflux pumps are conserved between the two species, but their substrate specificity is uncertain. Transporters present in G. sulfurreducens but not G. metallireducens include that for uracil (GSU0932, 48% identical to the Bacillus caldolyticus protein ). Transporters present in G. metallireducens but not G. sulfurreducens include those for nitrate/nitrite (Gmet_0333-Gmet_0334) and chromate (Gmet_2732-Gmet_2731), which are each present as two paralogous genes rather than gene fusions such as their homologs that have been characterized in other bacteria [36, 115].
Signalling, chemotaxis and global regulation
Sigma factors of G. metallireducens and G. sulfurreducens.
G. metallireducens gene
G. sulfurreducens gene
RNA polymerase sigma-32 factor
RNA polymerase sigma-24 factor, putative
RNA polymerase sigma-54 factor
RNA polymerase sigma-70 factor RpoD
RNA polymerase sigma-38 factor, stationary phase
RNA polymerase sigma-28 factor for flagellar operon
RNA polymerase sigma-Z factor
The G. metallireducens genome encodes 83 putative sensor histidine kinases containing HATPase_c domains (Additional file 12: Table S7), of which 45 (54%) have orthologs among the 95 such proteins of G. sulfurreducens. There are 94 proteins with response receiver (REC) domains in G. metallireducens (Additional file 12: Table S7), out of which 66 (70%) have orthologs among the 110 such proteins of G. sulfurreducens. Twenty-seven of the REC domain-containing proteins and another 101 genes and four pseudogenes (Additional file 12: Table S7) were predicted to be transcriptional regulators in G. metallireducens. There are 20 putative diguanylate cyclases containing GGDEF domains, of which 16 (80%) have orthologs among the 29 putative diguanylate cyclases of G. sulfurreducens (Additional file 13: Table S8). Overall, the portion of the genome dedicated to signalling and transcriptional regulation in G. metallireducens is slightly less than in G. sulfurreducens, but still considerable and significantly different in content.
Several protein factors involved in chemotaxis-type signalling pathways are conserved between the two genomes: G. sulfurreducens and G. metallireducens each possess four or five CheA sensor kinases and ten CheY response receivers, almost all of which are orthologous pairs (Additional file 14: Table S9). In contrast, 17 of the 34 methyl-accepting chemotaxis proteins (MCPs) of G. sulfurreducens have no full-length matches in G. metallireducens (Additional file 14: Table S9). Due to apparent gene family expansion in G. sulfurreducens, its remaining 17 MCPs correspond to only 13 MCPs of G. metallireducens (Additional file 14: Table S9). The other five MCPs of G. metallireducens lack full-length matches in other Geobacteraceae (Additional file 14: Table S9). Whereas G. sulfurreducens may use its closely related MCPs to fine-tune its chemotactic responses, G. metallireducens may accomplish response modulation by having twice as many MCP methyltransferases (CheR) and methylesterases (CheB) as G. sulfurreducens (Additional file 14: Table S9).
Integration host factor (IHF) and histone-like (HU) genes of G. metallireducens and G. sulfurreducens.
G. metallireducens gene
G. sulfurreducens gene
Although no quorum sensing through N-acylhomoserine lactones (autoinducers) has ever been demonstrated for any Geobacteraceae, this kind of signalling may be possible for G. metallireducens because it possesses a LuxR family transcriptional regulator with an autoinducer-binding domain (Gmet_1513), and two divergently transcribed genes with weak sequence similarity to autoinducer synthetases (Gmet_2037 and Gmet_2038). Both Gmet_2037 and Gmet_2038 have atypically low G+C content (Additional file 1: Table S1) and may have been recently acquired by G. metallireducens. The presence of a conserved nucleotide sequence on the 5' side of Gmet_2037 and in 15 other locations on the chromosome (Additional file 16: Figure S7, Additional file 5: Table S4) suggests that Gmet_2037 may be an unusual autoinducer synthetase that is regulated by a riboswitch rather than an autoinducer-binding protein. This conserved sequence is also found on the 5' side of many genes (frequently c-type cytochromes) in the genomes of G. sulfurreducens, G. uraniireducens, and P. propionicus, and overlaps with predicted cyclic diguanylate-responsive riboswitches .
The genomes of G. metallireducens and G. sulfurreducens differ in several other aspects of regulation. Nine pairs of potential toxins and antitoxins were identified in the G. metallireducens genome (Additional file 17: Table S10), which may poison vital cellular processes in response to stimuli that interfere with their autoregulation. Only one of these was similar to one of the five potential toxin/antitoxin pairs of G. sulfurreducens. Both the CRISPR1 and CRISPR2 (clustered regularly interspaced short palindromic repeat) loci of G. sulfurreducens, thought to encode 181 short RNAs that may provide immunity against infection by unidentified phage and plasmids [121, 122], have no parallel in G. metallireducens, which has CRISPR3 (also found in G. uraniireducens) instead, encoding only twelve putative short RNAs of more variable length and unknown target specificity (Additional file 18: Table S11). Another difference in RNA-level regulation is that a single-stranded RNA-specific nuclease of the barnase family (Gmet_2616) and its putative cognate inhibitor of the barstar family (Gmet_2617) are present in G. metallireducens but not G. sulfurreducens.
Several conserved nucleotide sequences were identified by comparison of intergenic regions between the G. sulfurreducens and G. metallireducens genomes, and those that are found in multiple copies (Additional file 19: Figure S8, Additional file 5: Table S4) may give rise to short RNAs with various regulatory or catalytic activities.
Inspection of the G. metallireducens genome indicates that this species has many metabolic capabilities not present in G. sulfurreducens, particularly with respect to the metabolism of organic acids. Many biosynthetic pathways and regulatory features are conserved, but several putative global regulator-binding sites are unique to G. metallireducens. The complement of signalling proteins is significantly different between the two genomes. Thus, the genome of G. metallireducens provides valuable information about conserved and variable aspects of metabolism, physiology and genetics of the Geobacteraceae.
Sequence analysis and annotation
The genome of G. metallireducens GS-15  was sequenced by the Joint Genome Institute from cosmid and fosmid libraries. Two gene modeling programs – Critica (v1.05), and Glimmer (v2.13) – were run on both replicons [GenBank:NC007517, GenBank:NC007515], using default settings that permit overlapping genes and using ATG, GTG, and TTG as potential starts. The results were combined, and a BLASTP search of the translations vs. Genbank's non-redundant database (NR) was conducted. The alignment of the N-terminus of each gene model vs. the best NR match was used to pick a preferred gene model. If no BLAST match was returned, the longest model was retained. Gene models that overlapped by greater than 10% of their length were flagged for revision or deletion, giving preference to genes with a BLAST match. The revised gene/protein set was searched against the Swiss-Prot/TrEMBL, PRIAM, Pfam, TIGRFam, Interpro, KEGG, and COGs databases, in addition to BLASTP vs. NR. From these results, product assignments were made. Initial criteria for automated functional assignment set priority based on PRIAM, TIGRFam, Pfam, Interpro profiles, pairwise BLAST vs. Swiss-Prot/TrEMBL, KEGG, and COG groups. tRNAs were annotated using tRNAscan-SE (v1.23). rRNAs were annotated using a combination of BLASTN and an rRNA-specific database. The srpRNA was located using the SRPscan website. The rnpB and tmRNA were located using the Rfam database and Infernal. Riboswitches and other noncoding RNAs predicted in the G. sulfurreducens genome [GenBank:NC00293] were retrieved from the Rfam database  and used to annotate the corresponding sequences in G. metallireducens.
Operon organization was predicted using the commercial version of the FGENESB software (V. Solovyev and A. Salamov, unpublished; Softberry, Inc; 2003–2007), with sequence parameters estimated separately from the G. sulfurreducens and G. metallireducens genomes. Default parameters were used in operon prediction, including minimum ORF length of 100 bp.
Binding sites of the global regulator ModE (consensus AT CGC TATATANNNNNN TATATAA CGAT) were predicted using ScanACE software [41, 42] using the algorithm of Berg and von Hippel  and the footprinted matrix of E. coli ModE-regulated sites from the Regulon DB database v 4.0 . Functional annotations of transport proteins were evaluated by referring to TCDB http://www.tcdb.org, and PORES http://garlic.mefos.hr/pores was used to annotate porins. Transposase families were assigned ISGme numbers for inclusion in the ISFinder database http://www-is.biotoul.fr.
The automated genome annotation of G. metallireducens was queried with the protein BLAST algorithm  using all predicted proteins in the automated annotation of the G. sulfurreducens genome  to identify conserved genes that aligned over their full lengths. The coordinates of numerous genes in both genomes were adjusted according to the criteria of full-length alignment, plausible ribosome-binding sites, and minimal overlap between genes on opposite DNA strands. The annotations of all other genes in G. metallireducens were checked by BLAST searches of NR. Discrepancies in functional annotation of conserved genes between the two genomes were also resolved by BLAST of NR and of the Swiss-Prot database. All hypothetical proteins were checked for similarity to previously identified domains, conservation among other Geobacteraceae, and absence from species other than Geobacteraceae. Genes that had no protein-level homologs in NR were checked (together with flanking intergenic sequences) by translated nucleotide BLAST in all six reading frames, and by nucleotide BLAST to ensure that conserved protein-coding or nucleotide features had not been missed. All intergenic regions of 120 bp or larger were also checked, which led to the annotation of numerous conserved nucleotide sequences numbered as follows: Gmet_R#### (for predicted RNAs and miscellaneous conserved sequences, a nonzero first digit indicating membership in a group of four or more sequences); Gmet_P#### (for conserved, putative regulatory sequences 5' of predicted operons, numbers corresponding to the first gene of the operon); Gmet_I[1-4]## [A, B] (for the four classes of putative global regulator binding sites, mostly found in pairs); Gmet_H4## (for putative global regulatory elements consisting of four tandem heptanucleotide repeats); and Gmet_C### (for the spacers of clustered regularly interspaced short palindromic repeats – CRISPR). Newly added features in the G. sulfurreducens genome were assigned unique numbers with decimal points (GSU####.#) in accordance with earlier corrections.
Phylogenetic analysis of selected proteins was performed on alignments generated using T-COFFEE , manually corrected in Mesquite . Phylogenetic trees were constructed by the neighbour-joining method using Phylip software , with 500 bootstrap replications.
clustered regularly interspaced short palindromic repeats
histone-like DNA-binding proteins
integration host factors
methyl-accepting chemotaxis protein
nicotinamide adenine dinucleotide (reduced)
nicotinamide adenine dinucleotide phosphate (reduced)
We thank Maddalena Coppi, Jessica Butler, Ned Young, Mounir Izallalen and Radhakrishnan Mahadevan for helpful discussions. We also thank Jose F. Barbe and Marko Puljic for technical assistance. This research was supported by the Office of Science (Biological and Environmental Research), U.S. Department of Energy (Grant No. DE-FC02-02ER63446).
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