Evolution of electron transfer out of the cell: comparative genomics of six Geobacter genomes
© Butler et al; licensee BioMed Central Ltd. 2010
Received: 22 June 2009
Accepted: 17 January 2010
Published: 17 January 2010
Geobacter species grow by transferring electrons out of the cell - either to Fe(III)-oxides or to man-made substances like energy-harvesting electrodes. Study of Geobacter sulfurreducens has shown that TCA cycle enzymes, inner-membrane respiratory enzymes, and periplasmic and outer-membrane cytochromes are required. Here we present comparative analysis of six Geobacter genomes, including species from the clade that predominates in the subsurface. Conservation of proteins across the genomes was determined to better understand the evolution of Geobacter species and to create a metabolic model applicable to subsurface environments.
The results showed that enzymes for acetate transport and oxidation, and for proton transport across the inner membrane were well conserved. An NADH dehydrogenase, the ATP synthase, and several TCA cycle enzymes were among the best conserved in the genomes. However, most of the cytochromes required for Fe(III)-reduction were not, including many of the outer-membrane cytochromes. While conservation of cytochromes was poor, an abundance and diversity of cytochromes were found in every genome, with duplications apparent in several species.
These results indicate there is a common pathway for acetate oxidation and energy generation across the family and in the last common ancestor. They also suggest that while cytochromes are important for extracellular electron transport, the path of electrons across the periplasm and outer membrane is variable. This combination of abundant cytochromes with weak sequence conservation suggests they may not be specific terminal reductases, but rather may be important in their heme-bearing capacity, as sinks for electrons between the inner-membrane electron transport chain and the extracellular acceptor.
Species of the Geobacter clade specialize in the oxidation of organic compounds to carbon dioxide coupled to the reduction of insoluble, extracellular electron acceptors . These species play an important role in pristine sediments and soils where they oxidize fermentation by-products like acetate and reduce naturally occurring insoluble Fe(III) and Mn(IV) oxides . In addition, they play important roles in three biotechnical applications: they are able to degrade hydrocarbon contaminants in soils, they are able to insolubilize uranium in contaminated aquifers, and finally, they are able to transfer electrons from a variety of substrates onto graphite electrodes, from which electricity can be harvested [2–4].
The mechanisms of electron transfer to Fe(III) and extracellular electron acceptors generally are not well understood . While soluble electron acceptors like oxygen and nitrate can diffuse into the cell, Geobacter species must transfer electrons onto an essentially insoluble, and therefore extracellular, electron acceptor. Geobacter sulfurreducens is currently the model organism for the Geobacteraceae family; the genome is sequenced  and there is a genetic system . G. sulfurreducens completely oxidizes the electron donor acetate to carbon dioxide via TCA cycle reactions . Electrons are then transferred into the inner membrane, presumably via NADH dehydrogenase(s) , and a succinate dehydrogenase . Electron transfer out of the inner membrane, through the periplasm and outer membrane to Fe(III) presumably requires c-type cytochromes. Several cytochromes have been shown to be required for growth by Fe(III) reduction, both in G. sulfurreducens [10–16] and in the other well-studied dissimilatory Fe(III) reducer, Shewanella oneidensis [17, 18]. However, a specific electron transport chain to extracellular Fe(III) has not been determined for any organism.
The genomes of several closely related Fe(III)-reducing organisms in the Geobacter family have recently been sequenced. This work compares the complete or 10×-coverage draft genome sequences of six species: G. sulfurreducens, Geobacter metallireducens, Geobacter uraniireducens, Geobacter bemidjiensis, Geobacter strain FRC-32 and Geobacter lovleyi. The six Geobacter genomes were compared and conservation of electron transport proteins was determined in order to identify electron transport genes that may be critical for the reduction of Fe(III) and other terminal electron acceptors, to better understand the evolution of the family, and to help provide foundational data for modeling of subsurface bioremediation.
Results and Discussion
Identification of the protein families in the six Geobacter genomes
Characteristics of genomes used in the comparative analysis
Geobacter sp. FRC-32
GC Content (%)
For each protein family, its phyletic pattern - the pattern of which species encode the proteins in that family - was determined (see Additional files 2 and 3). By far the most common pattern was conservation across all species, 35% of the proteins (7,774) were in families that included at least one ortholog from each genome (see Additional file 3). The second most common pattern was conservation in all of the species except G. lovleyi - 6% (1,246) of the proteins had this phyletic pattern (see Additional file 3).
The largest families of orthologous proteins (at least 10 members) excluding transposases
ATP-dependent protease La
elongation factor G
sensory box histidine kinase
CzcA family heavy metal efflux protein
glycosyl transferase, group 1
sodium/solute symporter family protein
group II intron, maturase
Fis family transcriptional regulator
iron-sulfur cluster-binding protein
electron transfer flavoprotein, alpha subunit
molybdenum cofactor biosynthesis protein A
cytochrome c family protein
Hybrid cluster protein
potassium transporter family protein
cold-shock domain-contain protein
high-molecular-weight cytochrome c
elongation factor Tu
methyl-accepting chemotaxis protein
sensor histidine kinase/response regulator
DNA-binding response regulator
nickel-dependent hydrogenase, large subunit
hypothetical protein GSU3410
cytochrome c family protein
Conservation of acetate and hydrogen metabolism
The genes for the eight reactions for acetate oxidation via the TCA cycle were conserved in all species (Figure 2). All of the subunits for acetyl-CoA transferase, citrate synthase, aconitase, isocitrate dehydrogenase, keto/oxoacid ferredoxin oxidoreductase, succinate dehydrogenase (complex II), fumarase, and malate dehydrogenase were conserved in all six of the species (see Additional file 6).
After acetate or hydrogen oxidation, the electrons are transferred into an inner-membrane bound electron transport chain, and protons are pumped out of the cytoplasm for ATP synthesis via an ATP synthase (Figure 2). G. sulfurreducens encodes two NADH dehydrogenases (complex I), one with 12 subunits and one with 14. This reaction is predicted to be the only one at which protons are pumped during Fe(III) or fumarate respiration . All six Geobacter species contained orthologs to one of these enzymes, the 14-subunit enzyme (GSU0338-GSU0351) (see Additional file 6). The 12-subunit enzyme was conserved in all species except G. loveyi (see Additional file 6). The putative NADPH dehydrogenase  was conserved in all six Geobacter species (Figure 2, Additional file 6).
Regardless of whether acetate or hydrogen is the electron donor, ATP is synthesized with an inner-membrane bound ATP synthase. G. sulfurreducens encoded one ATP synthase enzyme in two gene clusters (GSU0108-GSU0114 and GSU0333-GSU0334). All six Geobacter species contained orthologs to all subunits of this enzyme (Figure 2, Additional file 6).
The best conserved proteins in the Geobacter species
Proteins with orthologs in every genome and average bit score ratio ≥ 90%
Geobacter sulfurreducens gene
ATP synthase subunit B
translation initiation factor IF-1
ribosomal protein S21
translation elongation factor Tu
nitrogen regulatory protein P-II
iron-sulfur cluster-binding protein
transcription termination factor Rho
ribosomal protein S3
hypothetical protein GSU0966
nitrogen regulatory protein
ATP synthase subunit A
60 kDa chaperonin
radical SAM domain protein
acyl carrier protein
NADH dehydrogenase I, B subunit
NADH dehydrogenase I, A subunit
cell division protein FtsA
rod shape-determining protein MreB
30S ribosomal protein S11
ribosomal protein L14
heavy metal efflux pump
ribosomal protein L33
imidazoleglycerol phosphate synthase
ribosomal protein L11
nitrogenase iron protein
preprotein translocase SecY
NADH dehydrogenase I, D subunit
NADH dehydrogenase I, F subunit
carboxyl transferase domain protein
type IV pilus biogenesis protein PilB
heavy metal efflux pump
ribosomal protein L31
50S ribosomal protein L2
heterodisulfide reductase subunit
GTP-binding protein LepA
delta-aminolevulinic acid dehydratase
thiamine biosynthesis protein ThiC
ribosomal protein S19
NADH dehydrogenase I, E subunit
translation elongation factor G
hypothetical protein GSU3309
Identifying genes encoding cytochromes
After the electron donors have been oxidized and the electrons have been transferred into the inner membrane, they must then be transferred out of the cell to the extracellular electron acceptor like Fe(III) or electrodes. This pathway presumably requires periplasmic and outer-membrane c-type cytochromes, an abundance of which is the hallmark of Geobacter species [30, 32–34]. Several cytochromes have been shown to be required for optimal growth by Fe(III) reduction or on electrodes in G. sulfurreducens: PpcA, MacA, OmcB, OmcE, OmcF, OmcG, OmcH, OmcS, OmcT, OmcX (M. Izallalan, unpublished), and OmcZ (B.C. Kim, unpublished).
Searching all six Geobacter species genomes showed that at least 100 ORFs in each genome contained at least one occurrence of the motif for covalent heme binding (CXXCH), indicating that these may be cytochromes (see Additional file 8). This was more than was found in 16 other genomes including those of Shewanella, Desulfovibrio, Rhodoferax, and Anaeromyxobacter species known to be cytochrome rich (see Additional file 8). Since this definition of cytochrome is minimal, a more stringent definition was created using 26 sequence profiles described in the protein database Interpro as c-type cytochromes. These profiles were compared against all of the proteins in the six Geobacter genomes. Proteins were considered cytochromes if their sequence contained at least one profile match and at least one CXXCH motif (see Additional file 9).
Characteristics of cytochromes found in each genome
ORFs in genome
cytochromes (% genome)
cytochromes with >1 heme
cytochromes (% multiheme)
hemes per cytochrome (average)
G. strain FRC-32
Conservation of cytochromes
Characteristics of cytochrome families with members in every genome
Geobacter sulfurreducens gene
ppcA and ppcB
orf2 OmcBC operon
There was poor conservation across the species of many of the cytochromes that have been shown to be required in vivo in G. sulfurreducens for wild-type levels of Fe(III) or electrode reduction (Figure 2). Most of the cytochromes required in G. sulfurreducens for growth on extracellular acceptors were not conserved in all species, including OmcE, OmcF, OmcS, OmcT, OmcX, OmcZ, and MacA (Figure 2, Additional file 9).
Only one of the nine well-conserved cytochrome families contained a cytochrome, PpcA, known to be required for wild type levels of Fe(III) reduction . At least one homolog to PpcA was found in every genome, and there were multiple homologs in most of the genomes: five in G. sulfurreducens, five in G. metallireducens, four in G. uraniireducens, three in G. bemidjiensis, two in G. strain FRC-32, and one in G. lovleyi (see Additional file 2). In related sulfate- and sulfur-reducing δ-Proteobacteria species, the most abundant and best studied cytochromes are the of the tetra-heme c3 type [36, 37], while those of PpcA family are of the tri-heme c7 type .
There were three other cytochrome families that were well conserved in all six genomes: families of 3-heme, 9-heme, and 12-heme cytochromes (Table 5). None of these cytochromes have been studied.
Duplications of cytochrome genes
Twenty-eight of the 115 families that included cytochromes had more than one protein member per genome (see Additional file 9). In other words, they included paralogs, which may represent duplicated cytochrome genes. The largest cytochrome family had 12 members from 5 genomes (family 23, Additional file 9). Several families were made up of cytochromes from only a single genome, indicating recent duplication or triplication of the cytochrome since that species diverged (families 3111, 3250, 3413, and 3597).
Several cytochromes known to be required for wild type Fe(III) metabolism appeared to have been duplicated within single genomes. The OmcS family (64) had nine members, all 6-heme cytochromes, found in four of the Geobacter genomes (see Additional file 9). The G. bemidjiensis genome contained four OmcS proteins, G. sulfurreducens three, and one in both G. FRC-32 and G. uraniireducens (see Additional file 9). The OmcZ family (2307) contained four members from three genomes: G. sulfurreducens had two members (see Additional file 9). All six of the Geobacter genomes contained more than one PpcA-like protein (Table 5).
Lateral gene transfer
The data presented above indicates that cytochromes are abundant in each genome, but not very well conserved across the genomes. Cytochrome duplication and divergence appears to have played a role in these genotypes. In addition, to investigate whether cytochromes were less well conserved because they were acquired laterally rather than inherited vertically, genes originating from lateral gene transfer were identified using a combination of phylogenetic and BLAST-based analysis. A neighbor-joining phylogenetic tree was inferred for every protein from the six genomes and homologous sequences for each protein were selected from the non-redundant protein database. These trees were used to identify proteins for which the nearest relative was not from the Geobacteraceae. If the phylogeny was strongly supported (bootstrap ≥ 50) or if the phylogeny was weakly supported and the most similar sequence in the non-redundant protein was not a Geobacteraceae species, the protein was considered a lateral gene transfer candidate.
2,196 of the 21,434 proteins in these six genomes (9.8%) were predicted to have originated from recent transfer from a distantly related organism (see Additional file 1). Only 19 of the 472 predicted cytochromes (4.0%) were identified as lateral gene transfer candidates - 1 in G. bemidjiensis, 5 in G. lovleyi, 6 in G. metallireducens, 2 in G. sulfurreducens, and 3 in G. uraniireducens (see Additional file 9). None of the cytochromes shown to be required for wild type electron transport in G. sulfurreducens were predicted to have originated from lateral gene transfer (see Additional file 9). These data indicated that the abundance of cytochromes in these six species cannot be explained by frequent lateral gene transfer.
The results show that the genes for oxidizing acetate and transferring electrons to cytoplasmic carriers, and for inner membrane electron transport, are well conserved between the Geobacter genomes. These results indicate that the Geobacter species and their last common ancestor all oxidized acetate using the same TCA cycle pathway that produces NADH, NADPH, and reduced ferredoxin. These substances are then oxidized at the inner membrane, and ATP is generated via oxidative phosphorylation. The previously unidentified site of quinol oxidation in the inner membrane is suggested to be a cytochrome bc complex encoded in an unique gene cluster that is conserved in all six species. The pathways used by the better-studied species were also found to be conserved in the newly discovered species that predominate in subsurface environments undergoing bioremediation, suggesting that the current metabolic model for G. sulfurreducens provides a good foundation for broader modeling of microbial metabolism in contaminated subsurfaces during bioremediation. However, the role of the newly identified hydrogenase unique to these subsurface species merits further investigation.
In stark contrast to the conservation of the pathway for ATP generation from acetate is the lack of conservation of the enzymes that dispose of the electrons after ATP production. The six Geobacter genomes contain an average of 79 cytochrome genes each, with each cytochrome predicted to bind an average of more than 7 hemes. So an abundance of extracytoplasmic heme is clearly important in these species. However, only 14% of the cytochromes are conserved in all six of the genomes. More surprisingly, even the cytochromes that have been shown to be required in G. sulfurreducens for electron transport to Fe(III) or electrodes are not well conserved.
Cells of G. sulfurreducens have been shown to be capable of storing ca. 1.6 × 10-17 mol electrons in the iron of their cytochromes . This has lead to the proposal that cytochromes may act as electric capacitors, accepting and storing the electrons from energy metabolism for short time spans in the absence of an extracellular electron-accepting surface . The data presented here indicates that in these species there is a combination of strong pressure to maintain many cytochrome genes with weak pressure to maintain the sequence of most cytochrome genes. This lack of conservation of cytochrome genes suggests that in Geobacter species there may not be a single common pathway for electron transport outside the cell, and that cytochromes may be required for general Fe-bearing capacity, as sinks for electrons between the inner-membrane electron transport chain and the extracellular acceptor.
Genome sequencing and annotation
With the exception of G. sulfurreducens , sequence data for the genomes were produced by the US Department of Energy Joint Genome Institute http://www.jgi.doe.gov, using a whole-genome shotgun strategy for the Sanger-sequencing of 3-Kb, 8-Kb, and 40-Kb DNA libraries to 8-9X depth. Open reading frames and their translations and predicted function based on automated annotation were taken from NCBI http://www.ncbi.nlm.nih.gov/, and are listed in Additional file 1 of the supplementary material. The following motifs were used to annotate cytochromes (showing Interpro identification http://www.ebi.ac.uk/interpro/): IPR000298, IPR000763, IPR000883, IPR000883, IPR001128, IPR002016, IPR002016, IPR002321, IPR002322, IPR002585, IPR003317, IPR004203, IPR009056, IPR010176, IPR010177, IPR010255, IPR010960, IPR011031, IPR011048, IPR012282, IPR012292, SSF47175, SSF48613, SSF48695, SSF81342, SSF81648.
Clustering orthologs into protein families
All proteins in the genomes were clustered into families of orthologs and recent paralogs using OrthoMCL , which uses reciprocal best similarity pairs from all-vs-all BLAST  to identify orthologs and recent paralogs, which are then clustered together across all the genomes using the Markov clustering algorithm . A functional role was predicted for each cluster using the G. sulfurreducens in silico model annotation  and COG categorization . The level of sequence similarity among conserved proteins was estimated using bit score ratios between reciprocal orthologs .
All the ORFs from the six genomes and the outgroup species Pelobacter propionicus (NC_008609) were put into orthologous groups using Hal , with inflation parameters from 1.1-5.0 for the clustering algorithm. The proteins used for the phylogeny were those that were part of a cluster generated with any inflation value that had exactly one member from each genome, and are listed in Additional file 4 of the supplementary material. All of the proteins in the cluster were concatenated and the resulting sequences aligned by ClustalW . ProtTest  was used to select a model of molecular evolution and MrBayes  was used to create a Bayesian estimation of the phylogeny. The single gene phylogeny was inferred from a ClustalW alignment of homologs to the large subunit of the hydrogenase from the NCBI non-redundant database. Distances and branching order were determined by the neighbor-joining method with bootstrap values from 1000 replicates in Mega.
Lateral gene transfer
A phylogenetic tree was inferred using PhyloGenie  for every protein from the six genomes. Homologous sequences for each protein were selected by BLAST from the non-redundant protein database from NCBI http://www.ncbi.nlm.nih.gov/, alignments were created with ClustalW , and the phylogeny was inferred using neighbor-joining  and 100 bootstrapped replicates. If, for a given protein, a phylogenetic relationship with non-Geobacteraceae was strongly-supported (bootstrap ≤ 50) or if the relationship was weakly supported and the most similar sequence in the non-redundant protein database from NCBI was not a Geobacteraceae species, the protein was considered a candidate. If the next branch out contained a single sequence not from Geobacteraceae species, the query gene was defined as being from lateral transfer. If the next branch contained a single sequence from Geobacteraceae, it was not. If the sister group was a clade or was not strongly supported, the ancestral condition was inferred  and used to determine lateral transfer.
This research was supported by the Office of Science (BER), U. S. Department of Energy, Cooperative Agreement No. DE-FC02-02ER63446.
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