The genome of Geobacter bemidjiensis, exemplar for the subsurface clade of Geobacter species that predominate in Fe(III)-reducing subsurface environments.
© Aklujkar et al; licensee BioMed Central Ltd. 2010
Received: 5 April 2010
Accepted: 9 September 2010
Published: 9 September 2010
Geobacter species in a phylogenetic cluster known as subsurface clade 1 are often the predominant microorganisms in subsurface environments in which Fe(III) reduction is the primary electron-accepting process. Geobacter bemidjiensis, a member of this clade, was isolated from hydrocarbon-contaminated subsurface sediments in Bemidji, Minnesota, and is closely related to Geobacter species found to be abundant at other subsurface sites. This study examines whether there are significant differences in the metabolism and physiology of G. bemidjiensis compared to non-subsurface Geobacter species.
Annotation of the genome sequence of G. bemidjiensis indicates several differences in metabolism compared to previously sequenced non-subsurface Geobacteraceae, which will be useful for in silico metabolic modeling of subsurface bioremediation processes involving Geobacter species. Pathways can now be predicted for the use of various carbon sources such as propionate by G. bemidjiensis. Additional metabolic capabilities such as carbon dioxide fixation and growth on glucose were predicted from the genome annotation. The presence of different dicarboxylic acid transporters and two oxaloacetate decarboxylases in G. bemidjiensis may explain its ability to grow by disproportionation of fumarate. Although benzoate is the only aromatic compound that G. bemidjiensis is known or predicted to utilize as an electron donor and carbon source, the genome suggests that this species may be able to detoxify other aromatic pollutants without degrading them. Furthermore, G. bemidjiensis is auxotrophic for 4-aminobenzoate, which makes it the first Geobacter species identified as having a vitamin requirement. Several features of the genome indicated that G. bemidjiensis has enhanced abilities to respire, detoxify and avoid oxygen.
Overall, the genome sequence of G. bemidjiensis offers surprising insights into the metabolism and physiology of Geobacteraceae in subsurface environments, compared to non-subsurface Geobacter species, such as the ability to disproportionate fumarate, more efficient oxidation of propionate, enhanced responses to oxygen stress, and dependence on the environment for a vitamin requirement. Therefore, an understanding of the activity of Geobacter species in the subsurface is more likely to benefit from studies of subsurface isolates such as G. bemidjiensis than from the non-subsurface model species studied so far.
Geobacter bemidjiensis is a member of the Geobacteraceae, a family of Fe(III)-respiring Deltaproteobacteria 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]. It was isolated from subsurface sediments in Bemidji, Minnesota, near a site where aromatic hydrocarbons were being degraded naturally . G. bemidjiensis is a member of the phylogenetic cluster designated subsurface clade 1, which predominates in a diversity of subsurface environments in which dissimilatory Fe(III) reduction is an important process . Environmental proteomic studies have demonstrated that Geobacter species closely related to G. bemidjiensis were metabolically active during the in situ bioremediation of uranium-contaminated groundwater .
Preliminary studies have suggested that genome-scale metabolic modeling of Geobacter species [6, 7] may aid in predicting the response of subsurface Geobacter species to subsurface bioremediation strategies [8, 9]. However, it is not known whether the metabolic potential of subsurface Geobacter species is essentially the same as that of non-subsurface Geobacter species, or significantly different. Therefore, comparative analysis of the genome of a representative of the subsurface clade 1 Geobacter species with the curated genomes of two non-subsurface Geobacter species, Geobacter sulfurreducens and Geobacter metallireducens[10, 11], was carried out to improve predictive modeling of the responses of Geobacteraceae to efforts to stimulate bioremediation of organic and metal contaminants in the subsurface.
Results and Discussion
Contents of the G. bemidjiensis genome
The automated annotation process identified 4040 protein-coding genes and 76 ribonucleic acid (RNA) genes in the genome of 4615150 bp. During manual curation, 56 genes were discarded, 40 genes were reannotated as pseudogenes, and another 79 protein-coding genes, 28 pseudogenes, and 778 non-protein-coding features were identified. Protein sequence alignments demonstrated that 27 pseudogenes were frameshifted within runs of five or more identical bases, where DNA replication is most error-prone, and seven of these are polymorphisms where the minor alleles contain no frameshift, indicating a subpopulation of cells that can produce functional proteins from these genes (Additional file 1: Table S1). Of the 4023 intact protein-coding genes in the G. bemidjiensis genome, 148 hypothetical proteins (3.6%) have no match in any other genome, including that of Geobacter sp. M21; a further 87 conserved hypothetical proteins were found only in these two closely related genomes.
Metabolism of pyruvate
An appropriate place to begin to compare metabolism between the subsurface exemplar G. bemidjiensis and the representative non-subsurface species G. sulfurreducens and G. metallireducens is with the central metabolic reactions that interconvert pyruvate and acetyl-CoA. Like other Geobacteraceae, G. bemidjiensis possesses two sets of genes encoding pyruvate dehydrogenase complexes (Gbem_2257 and Gbem_2251-Gbem_2250; Gbem_0459-Gbem_0461), which irreversibly convert pyruvate to acetyl-CoA. The ability of G. sulfurreducens to reverse this reaction and derive biomass from acetyl-CoA has been attributed to a homodimeric pyruvate:ferredoxin/flavodoxin oxidoreductase , for which homologs exist in all Geobacteraceae including G. bemidjiensis (Gbem_0209). G. bemidjiensis has an additional pyruvate:ferredoxin oxidoreductase (Gbem_4034), more closely related to those of sulfate-reducing bacteria. In addition to gluconeogenesis and oxidative decarboxylation to acetyl-CoA, a third fate of pyruvate in G. bemidjiensis may be oxidative decarboxylation to acetate by a putative quinone-reducing pyruvate decarboxylase (Gbem_0287) that is 32% identical to the E. coli enzyme . Thus, metabolism of pyruvate may be more complex in G. bemidjiensis than in non-subsurface Geobacter species.
Metabolism of propionate
Fatty acid metabolism
The G. bemidjiensis genome encodes many enzymes of acyl-CoA metabolism (Additional file 2: Table S2). This multiplicity of genes suggests that in addition to its known short-chain organic acid electron donors (butyrate, isobutyrate and valerate), G. bemidjiensis may also be able to utilize longer fatty acids as sources of carbon and electrons. Indeed, G. bemidjiensis and the other genomes of subsurface Geobacter species possess a very-long-chain fatty acyl-CoA dehydrogenase (fadE Gbem_2128) 50% identical to that of Bacillus subtilis, which is absent from G. sulfurreducens and G. metallireducens. Thus, the metabolism of fatty acids by subsurface Geobacter species may be better understood by examining G. bemidjiensis rather than non-subsurface relatives.
Growth of G. bemidjiensis by disproportionation of fumarate
Either the sodium/fumarate symporter DctA (Gbem_3225) that is 61% identical to the Salmonella typhimurium transporter  or the proton/fumarate symporter complex DctPQM (Gbem_2659-Gbem_2661) with 40% to 63% sequence identity to characterized homologs in Rhodobacter capsulatus may be a prerequisite for disproportionation of fumarate. Both transporters, which are absent from G. sulfurreducens, allow import of fumarate without the concomitant export of succinate required by the dicarboxylate exchange transporter DcuB (Gbem_2921, orthologous to GSU2751). Notably, the dcuB and dctA genes are each located 3' of a pair of genes encoding a periplasmic substrate-binding sensor histidine kinase and response regulator that are highly similar (Figure 2c); a third pair is located 5' of a phosphate-selective porin (Gbem_4037, orthologous to Gmet_1042). The three sensor kinases (Gbem_2923; Gbem_3228; Gbem_4039) are 47% to 56% identical and the three response regulators (Gbem_2922; Gbem_3227; Gbem_4038) are 65% to 69% identical, suggesting that G. bemidjiensis has developed parallel signalling pathways possibly linked to dicarboxylate transport.
In D. vulgaris, oxidation of fumarate proceeds through the decarboxylating malate oxidoreductase reaction (B. Giles and J. Wall, personal communication), but it would be surprising if this were the predominant pathway in G. bemidjiensis (Figure 2d). When G. sulfurreducens respires fumarate, the activity of its two malate oxidoreductases (NADP-dependent maeB GSU1700 and NAD-dependent mleA GSU2308) must be kept much lower than that of malate dehydrogenase, which converts malate to oxaloacetate, because the equal exchange of succinate for fumarate by DcuB requires that any malate that is decarboxylated to pyruvate must be replaced by carboxylation of pyruvate to oxaloacetate at the expense of one ATP, which is prohibitive . G. bemidjiensis possesses three maeB genes, all closely related to GSU1700, and no mleA gene. It is possible that one or more of these isozymes are upregulated during disproportionation of fumarate. However, complete oxidation of fumarate through acetyl-CoA using the malate oxidoreductases rather than malate dehydrogenase would result in NADH and NADPH being produced in a ratio of 3:2 rather than 4:1, requiring rerouting of reducing equivalents to meet energy demand. A more reasonable hypothesis is that conversion of fumarate to pyruvate is accomplished through decarboxylation of oxaloacetate by two parallel pathways (Figure 2d; gene diagrams in Figure 2e).
One oxaloacetate decarboxylase complex transfers the carboxyl group to propionyl-CoA as detailed above, forming (S)-methylmalonyl-CoA. In contrast with this same enzyme complex's predicted cyclic involvement in oxidation of propionate to pyruvate (see above, Figure 1), its catalysis of this step in the fumarate oxidation pathway requires that (S)-methylmalonyl-CoA be recycled to propionyl-CoA by methylmalonyl-CoA decarboxylase (Gbem_0684) (Figure 2d). The other oxaloacetate decarboxylase (oadA Gbem_1454) is 60% identical to the catalytic subunit of the sodium-translocating oxaloacetate decarboxylase of Klebsiella pneumoniae. No homologs of the other two subunits were found in G. bemidjiensis, indicating that decarboxylation of oxaloacetate is not coupled to a sodium pump.
The malate oxidoreductase and oxaloacetate decarboxylase pathways also account for the ability of G. bemidjiensis to grow with malate and succinate as sources of carbon and electrons. Thus, the genome annotation of G. bemidjiensis offers insight into its unique capability to metabolize dicarboxylic acids without excreting acetate, which could not be predicted correctly from studies of either the non-subsurface Geobacter genomes or non-Geobacter species.
Possible carbon dioxide fixation via citrate lyase
A reverse TCA cycle requires enzymes capable of carrying out two carboxylations: conversion of succinyl-CoA to 2-oxoglutarate and conversion of 2-oxoglutarate to isocitrate (Figure 3b). The first of these conversions has been inferred from a carbon flux analysis study of G. metallireducens and may be attributed to 2-oxoglutarate:ferredoxin oxidoreductase (Gbem_2896-Gbem_2899). The second conversion may be catalyzed by isocitrate dehydrogenase (Gbem_2901), which is 71% identical to the Chlorobium limicola enzyme that is known to be reversible . These genes form a cluster that includes malate dehydrogenase (Gbem_2900), suggesting that flux of oxaloacetate through the reverse TCA cycle may be coordinated.
The presence of homologous gene clusters in Pelobacter propionicus and Desulfuromonas acetoxidans suggests that citrate lyase was present in the common ancestor of the Geobacteraceae, and was lost by most species of the genus Geobacter. Interestingly, the 2'-(5''-triphosphoribosyl)-3'-dephospho-CoA synthase gene of the cluster, which is duplicated in G. bemidjiensis (citG-1 Gbem_3856, citG-2 Gbem_0190), is present in G. sulfurreducens (GSU0806), along with the gene for the enzyme that transfers 2'-(5''-triphosphoribosyl)-3'-dephospho-CoA to the acyl carrier protein of citrate lyase (citX Gbem_3857 = GSU0807), but the genes encoding structural components of citrate lyase are absent, suggesting that 2'-(5''-triphosphoribosyl)-3'-dephospho-CoA may have a second function unrelated to citrate lyase.
Both succinyl:acetate CoA transferase isozymes of G. sulfurreducens are doubly present in G. bemidjiensis (ato-3 Gbem_0795 is a duplicate of ato-1 Gbem_0468, and ato-4 Gbem_3897 is a duplicate of ato-2 Gbem_2843). One possible explanation for this is that the duplicates have distinct functions in the oxidative and reductive TCA cycles (Figure 3b). Another possibility is that each of the duplicate citrate synthases (Gbem_1652, Gbem_3905) utilizes the acetyl-CoA produced by a different pair of isozymes. Although these details remain to be worked out, the TCA cycle on the whole appears to be more complex in the subsurface isolate G. bemidjiensis than in the non-subsurface Geobacter species examined to date.
Carbon monoxide dehydrogenases and associated hydrogenase
Glucose as electron donor
Several unique genes discovered in the G. bemidjiensis genome suggested that G. bemidjiensis should be able to utilize glucose and galactose as carbon sources. These genes encode a glucose/galactose transporter (gluP Gbem_3671) 55% identical to that of Brucella abortus, a putative glucose 6-kinase (Gbem_2002) 33% identical to that of E. coli, a galactose 1-kinase (Gbem_4019) 35% identical to that of E. coli, and a uridine 5'-diphosphate (UDP)-glucose:galactose-1-phosphate uridylyltransferase (Gbem_4017) 32% identical to that of Thermotoga maritima. Most Geobacteraceae, which do not utilize glucose and galactose, possess only a putative glucose 6-kinase (Gbem_1326) similar to those of Streptomyces lividans and Streptomyces coelicolor[32, 33], a UDP-glucose/galactose 4-epimerase (Gbem_3215) and a different putative galactose-1-phosphate uridylyltransferase. G. bemidjiensis was able to grow with glucose, but not galactose, as electron donor and carbon source, using Fe(III) oxides as the terminal electron acceptor (D. Holmes, unpublished). This discovery illustrates the need for subsurface metabolic models to be based on subsurface genomes such as that of G. bemidjiensis, rather than approximations based on genomes of non-subsurface species.
Carbohydrate osmoprotectants and cell wall components
Like G. sulfurreducens and G. metallireducens, G. bemidjiensis is predicted to make trehalose from glucose storage polymers by the sequential action of maltooligosyltrehalose synthase (Gbem_0134) and maltooligosyltrehalose trehalohydrolase (Gbem_0132) . G. bemidjiensis lacks homologs of the enzymes predicted to make trehalose from glucose-6-phosphate in G. sulfurreducens, but may be able to isomerize maltose to trehalose by means of a maltose-active trehalose synthase (Gbem_0136) that is 33% identical to that of T. thermophilus. The presence of a fructose/mannose 6-kinase (mak Gbem_0370), 39% identical to that of E. coli and a mannitol dehydrogenase (Gbem_0401) with 47% identity to that of Apium graveolens suggests that G. bemidjiensis may synthesize and break down D-mannitol as an additional osmoprotectant.
The lipopolysaccharide of G. sulfurreducens contains no O-antigen . In contrast, the genome of G. bemidjiensis reveals many pathways for the production of various sugars that may be components of the cell wall (Additional file 3: Table S3). Thus, not only central metabolism of carbon but many specialized branch pathways appear to differ between subsurface and non-subsurface Geobacter species.
Biosynthesis of chorismate and folate in G. bemidjiensis
The G. bemidjiensis genome encodes no homolog of the putative 4-aminodeoxychorismate synthase/lyase of G. metallireducens and G. sulfurreducens (Gmet_3010 = GSU0523), and an attempt to grow G. bemidjiensis without vitamin supplementation confirmed that it is auxotrophic solely for the 4-aminobenzoate (PABA) moiety of folate (Figure 5b). This is the first report of a Geobacter species with any vitamin requirement, and suggests that the metabolic activity of subsurface Geobacter species may be stimulated by adding PABA.
Degradation of benzoate and other aromatic pollutants
Degradation of 3-hydroxypimelyl-CoA in G. bemidjiensis (Figure 6) is predicted to involve a non-decarboxylating glutaryl-CoA dehydrogenase (Gbem_1452) 44% identical to that of Desulfococcus multivorans, rather than a homolog of the decarboxylating glutaryl-CoA dehydrogenase of G. metallireducens. Subsequent decarboxylation of glutaconyl-CoA may take place through the product of the adjacent gene (gcdA Gbem_1453), which is 52% identical to the catalytic subunit of sodium-translocating glutaconyl-CoA decarboxylase of Acidaminococcus fermentans. No homologs of the other three subunits were found in the G. bemidjiensis genome, indicating that decarboxylation of glutaconyl-CoA is not coupled to a sodium pump. Although the GcdA protein of A. fermentans on its own is capable of decarboxylating glutaconyl-CoA with free biotin as a cofactor , it is notable that the oxaloacetate decarboxylase encoded by the gene adjacent to gcdA in G. bemidjiensis (oadA Gbem_1454) contains two biotin attachment domains, whereas its sodium pump-associated homologs contain only one. The possibility that the two decarboxylases cooperate as a complex deserves investigation.
Although the genome of G. bemidjiensis corroborates the observation that it cannot degrade as many aromatic compounds as G. metallireducens[39, 42, 46], it also suggests that G. bemidjiensis can detoxify some aromatic pollutants without degrading them. A homolog of the broad-specificity aldo-keto reductase YvgN of B. subtilis is present (Gbem_3980, 45% sequence identity), suggesting that G. bemidjiensis may convert chloro- and nitro- derivatives of benzaldehyde to the corresponding benzol derivatives. Although YvgN was previously described as a methylglyoxal reductase , detoxification of methylglyoxal, a byproduct of carbohydrate and lipid metabolism, may be of minor importance in G. bemidjiensis, as a methylglyoxal synthase was not found in G. bemidjiensis, but only in G. lovleyi (Glov_0611).
The fact that G. bemidjiensis is auxotrophic for PABA (Figure 5b) indicates that it has grown accustomed to an environment in which PABA is readily available, possibly in the form of 4-azobenzoate. G. bemidjiensis may convert azoaromatic compounds to arylamines by means of an azoreductase (azoR Gbem_2529) 30% identical to that of E. coli, which is not present in G. metallireducens. Furthermore, an arylamine N-acetyltransferase (Gbem_0306) not found in other Geobacteraceae may act in detoxification of aromatic compounds by G. bemidjiensis, and a putative amidohydrolase in the benzoate degradation gene cluster (Gbem_1458 = Gmet_2056) may also be involved in metabolism of aromatic compounds. Further studies of subsurface Geobacter species such as G. bemidjiensis are necessary to characterize their abilities to transform aromatic compounds.
The presence of an alkylmercury lyase (Gbem_0319) 36% identical to that of S. lividans) suggests that G. bemidjiensis possesses broad-spectrum resistance to mercury in various organic forms. A homolog of this enzyme was not found in any other Geobacter species, but genes that may encode mercuric reductases (e.g. Gbem_0457, Gbem_0640) exhibit vertical inheritance in the family, indicating that Geobacteraceae may generally have the ability to detoxify inorganic Hg(II) ions, whereas subsurface Geobacter species such as G. bemidjiensis may have acquired additional resistance to organomercuric compounds.
Expansion of transport systems for phosphate and molybdate
The ABC transport system for molybdate, consisting of a molybdate-binding protein (ModA), membrane protein (ModB) and ATP-binding protein (ModC), has also expanded in G. bemidjiensis (Figure 7): the modB 1 C 1 genes are located apart from the modA 1 gene, while the modA 2 B 2 C 2 genes remain an intact operon. The regulatory gene modE is located on the 5' side of the tungstate transporter genes tupABC (which are phylogenetically distinct from those of G. sulfurreducens and G. metallireducens; data not shown) in contrast with its location on the 5' side of modABC in G. sulfurreducens. The possibility that these expansions and rearrangements are a response by subsurface Geobacter species to molybdate limitation deserves to be investigated.
Oxygen respiration, oxygen detoxification, and possible anaerotaxis in G. bemidjiensis
Eight hemerythrin family proteins (Gbem_1252, Gbem_2241, Gbem_2255, Gbem_2262, Gbem_2701, Gbem_2773, Gbem_3870, Gbem_4009) were predicted from the genome of G. bemidjiensis, suggesting that it may have expanded its ability to sequester molecular oxygen and deliver it to respiratory or detoxifying enzymes, in contrast to G. metallireducens with two hemerythrin homologs; G. sulfurreducens has six hemerythrin homologs. To detoxify reactive oxygen species, G. bemidjiensis possesses a desulfoferrodoxin (Gbem_3292) 60% identical to that of Desulfoarculus baarsii and a rubredoxin:oxygen/nitric oxide oxidoreductase (Gbem_0186) 31% identical to that of D. gigas, in addition to the superoxide dismutase (Gbem_2204), peroxiredoxins (Gbem_0154, Gbem_0221, Gbem_1338, Gbem_2956, Gbem_4010) and two rubrerythrins (Gbem_2313, Gbem_3325) also present in G. sulfurreducens and G. metallireducens. Phylogenetic analysis (not shown) indicates that although the characterized cytochrome c peroxidase of G. sulfurreducens has an excellent homolog in G. bemidjiensis (Gbem_0020), this is actually an ortholog of MacA, implicated in Fe(III) reduction . As in G. metallireducens, there is no catalase in G. bemidjiensis, meaning that no oxygen is produced from detoxification of hydrogen peroxide; detoxification by rubrerythrins produces only water. All Geobacteraceae encode at least one iron-sulfur-oxygen hybrid cluster protein, thought to detoxify an unidentified reactive compound in response to nitric oxide stress , as well as hydrogen peroxide stress ; G. bemidjiensis and Geobacter sp. M21 alone have three hybrid cluster protein genes (Gbem_1033, Gbem_1168, Gbem_1239), evidently derived by expansion of a single ancestral gene. G. bemidjiensis also has a quinol-oxidizing nitric oxide reductase (norZ Gbem_3901) 40% identical to that of Cupriavidus necator, with a distant homolog in G. metallireducens. Overall, the genome annotation indicates that G. bemidjiensis has evolved to cope with many kinds of reactive oxygen species, a finding that should improve models of Geobacter metabolism in the subsurface.
The outer surface: c-type cytochromes, pili, and flagella
Sigma factors and signalling proteins
The G. bemidjiensis genome was examined for features of gene regulation conserved between it and its non-subsurface relatives. Of the six sigma factors of RNA polymerase in G. sulfurreducens, G. bemidjiensis has orthologs of five: RpoD (Gbem_3694), RpoS (Gbem_2683), RpoN (Gbem_0869), RpoH (Gbem_0573), and FliA (Gbem_3764). No homolog of the putative stress response sigma factor RpoE was found. There are also two additional sigma factors (Gbem_1696 and Gbem_3169) unrelated to the unique sigma factor of G. metallireducens.
The G. bemidjiensis genome encodes 127 putative sensor histidine kinases containing HATPase_c domains (Additional file 5: Table S5), including 8 chemotaxis-type kinases (cheA genes), of which 47 genes (37%) have full-length homologs in G. sulfurreducens and/or G. metallireducens. There are 163 proteins with response receiver (REC) domains (Additional file 5: Table S5), including 19 that may belong to chemotaxis-type signalling pathways; of these, 82 genes (50%) have full-length homologs in G. sulfurreducens and/or G. metallireducens. Thus, G. bemidjiensis has a different and much larger repertoire of phosphorylation-dependent signalling proteins than either G. sulfurreducens or G. metallireducens. The G. bemidjiensis genome encodes 21 GGDEF domain proteins that may synthesize the intracellular messenger cyclic diguanylate (Additional file 5: Table S5), a similar number to G. sulfurreducens and G. metallireducens, but only 10 of these are conserved. These differences in the repertoire of predicted signalling proteins among subsurface and non-subsurface Geobacter species are remarkable, especially considering that some ancestral genes encoding signalling proteins appear to have undergone duplication or triplication in G. bemidjiensis (Additional file 5: Table S5).
Non-protein-coding features of the G. bemidjiensis genome
Riboswitches that have been identified in the non-subsurface species G. sulfurreducens and G. metallireducens[10, 67] were found in the subsurface G. bemidjiensis genome also. In addition, several families of multicopy nucleotide sequences were noted in G. bemidjiensis (Additional file 6: Table S6; Additional files 7, 8, 9, 10, 11, 12, 13, 14 and 15: Figures S1-S9), most of which have no counterparts in the G. sulfurreducens or G. metallireducens genomes. Some of these families are based on palindromic sequences, and others consist of direct repeats of 6 to 42 nucleotides that occupy intergenic regions throughout the genome. Multicopy sequences (other than rRNA and tRNA genes) are found in 12% of regions between protein-coding genes in G. bemidjiensis, and multiple sequences are present in 31% of such intergenic regions, indicating that insertion is not random. One nucleotide sequence family was found inserted into protein-coding genes (on both strands and in all reading frames, without causing frameshifts) as well as between genes, as previously observed for a different family in G. metallireducens. The implications of so many more multicopy sequences being present in the genome of a subsurface Geobacter species than in those of its non-subsurface relatives remain to be elucidated.
The complete genome sequence of G. bemidjiensis reveals many differences from the previously published genomes of non-subsurface Geobacter species. Enzymes that account for the metabolic versatility of G. bemidjiensis were identified, and further metabolic, physiological and genomic peculiarities were discovered, including a more efficient pathway for oxidation of propionate, a pathway of fumarate disproportionation without excretion of acetate, a reductive TCA cycle, utilization of glucose, a defective folate biosynthesis pathway, and enhanced abilities to respond to oxygen stress. This information is of utmost value for an understanding of the activity of Geobacteraceae in subsurface environments undergoing bioremediation accompanied by reduction of Fe(III).
Sequence analysis and annotation
The genome of G. bemidjiensis Bem(T)  was sequenced at the Joint Genome Institute (JGI) using a combination of 3 kb, 6 kb and 35 kb DNA libraries. Inserts were sequenced from both ends using the standard Sanger method. All three libraries provided 11-fold coverage of the genome. The Phred/Phrap/Consed software package http://www.phrap.com was used for sequence assembly and quality assessment [68–70]. After the shotgun stage, 65888 reads were assembled with parallel Phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher (Han, 2006) or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI). Gaps between contigs were closed by editing in Consed, by custom primer walks, or by PCR amplification (Roche Applied Science, Indianapolis, IN). A total of 2059 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The completed genome sequence of G. bemidjiensis Bem(T) contains 67990 reads, achieving an average of 11-fold sequence coverage per base with an error rate less than 1 in 100,000.
The protein-coding genes were predicted using Prodigal V1.0 . A BLASTP search of the translations vs. Genbank's non-redundant database (NR of Nov. 2007) at 1e-05 evalue was conducted. Matches to the Geobacter genus were excluded and the alignment of the N-terminus of each gene model vs. the best NR match was used to pick a preferred gene model. The gene/protein set was searched using BLASTP, hmmer, RPS-BLAST and Interpro. BLASTP searches were done vs. Swiss-Prot/TrEMBL, NR, and KEGG databases with a cutoff evalue of 1e-05. Hmmer searches were done vs. Pfam and TIGRfam databases using the trusted cutoff. RPS-BLAST searches against PRIAM used its 1e-30 high-confidence cutoff and searches against COGS used a 1e-10 cutoff. Interpro was run using its default cutoffs. Automated product assignment was made using the following hierarchy of data sources: PRIAM, TIGRFam, Pfam, Interpro profiles, Swiss-Prot/TrEMBL, KEGG, and COG group. tRNAs were annotated using tRNAscan-SE (v1.23). rRNAs were annotated using RNAmmer v 1.1 . The srpRNA was located using the SRPscan website. The rnpB and ssrA genes were located using the Rfam database and Infernal.
The automated genome annotation of G. bemidjiensis and the manually curated genome annotations of G. sulfurreducens and G. metallireducens were queried reciprocally with the protein BLAST algorithm  as implemented by OrthoMCL  using the default inflation parameter value (1.5), to identify mutual best hits as potential orthologs. The functional annotations of G. bemidjiensis genes were emended for consistency with their counterparts in G. sulfurreducens and G. metallireducens. The coordinates of numerous genes 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 G. bemidjiensis genes that were not matched to genes in G. sulfurreducens or G. metallireducens were checked by BLAST searches of NR and the Swiss-Prot database. Functional annotations in all three genomes were updated to match the experimental characterization of highly similar full-length homologs, with extensive reference to the EcoSal online textbook http://www.ecosal.org and the MetaCyc database . 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 non-protein-coding features had not been missed. All intergenic regions of 30 bp or larger were also checked, which led to the annotation of numerous conserved nucleotide sequences.
Phylogenetic analysis of selected proteins was performed. In each case, the protein sequence of interest was included, along with its relatives, as identified by BLAST , and the set of sequences was aligned by TCoffee . ProtTest  was used to select a model of molecular evolution and MrBayes  was used to create a Bayesian estimation of the phylogeny.
To monitor the disproportionation of fumarate, G. bemidjiensis was cultured under strictly anaerobic conditions at 30°C in an atmosphere of N2 and CO2 (80%:20%), as previously described for G. sulfurreducens, in rubber-stoppered 156 ml bottles containing NBAF medium  from which sodium acetate was omitted. Samples of 1 ml were removed aseptically using anoxic syringes to monitor growth, then diluted 50-fold, passed through a 0.22 μm filter to remove cells, and stored at 4°C until high-pressure liquid chromatography analysis was performed as described previously . The 4-aminobenzoate requirement of G. bemidjiensis was tested in rubber-stoppered 26 ml glass tubes containing 10 ml of NBAF medium  from which the vitamin solution and resazurin were omitted, with 4-aminobenzoate (Sigma Aldrich) added to individual tubes to a final concentration of 100 μg/L.
List of Abbreviations used
flavin adenine dinucleotide
nicotinamide adenine dinucleotide (reduced)
nicotinamide adenine dinucleotide 2'-phosphate (reduced)
We thank Mounir Izallalen for helpful discussions and P. Brown, T. Woodard, K. Nevin, T. Brettin, C. Detter, and C. Kuske 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). The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.
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