Evolution from a respiratory ancestor to fill syntrophic and fermentative niches: comparative genomics of six Geobacteraceae species
© Butler et al; licensee BioMed Central Ltd. 2009
Received: 28 October 2008
Accepted: 11 March 2009
Published: 11 March 2009
The anaerobic degradation of organic matter in natural environments, and the biotechnical use of anaerobes in energy production and remediation of subsurface environments, both require the cooperative activity of a diversity of microorganisms in different metabolic niches. The Geobacteraceae family contains members with three important anaerobic metabolisms: fermentation, syntrophic degradation of fermentation intermediates, and anaerobic respiration.
In order to learn more about the evolution of anaerobic microbial communities, the genome sequences of six Geobacteraceae species were analyzed. The results indicate that the last common Geobacteraceae ancestor contained sufficient genes for anaerobic respiration, completely oxidizing organic compounds with the reduction of external electron acceptors, features that are still retained in modern Geobacter and Desulfuromonas species. Evolution of specialization for fermentative growth arose twice, via distinct lateral gene transfer events, in Pelobacter carbinolicus and Pelobacter propionicus. Furthermore, P. carbinolicus gained hydrogenase genes and genes for ferredoxin reduction that appear to permit syntrophic growth via hydrogen production. The gain of new physiological capabilities in the Pelobacter species were accompanied by the loss of several key genes necessary for the complete oxidation of organic compounds and the genes for the c-type cytochromes required for extracellular electron transfer.
The results suggest that Pelobacter species evolved parallel strategies to enhance their ability to compete in environments in which electron acceptors for anaerobic respiration were limiting. More generally, these results demonstrate how relatively few gene changes can dramatically transform metabolic capabilities and expand the range of environments in which microorganisms can compete.
The global carbon cycle and the production of a number of biofuels depends on the cooperative interaction of a physiological diversity of anaerobic microorganisms. These include microorganisms that ferment complex substrates to simpler molecules; respiratory microorganisms that convert fermentation products to carbon dioxide and/or methane; and syntrophic organisms that anaerobically oxidize some fermentation products and other substrates in reactions that are only thermodynamically feasible when respiratory organisms consume the syntrophs' products. The family Geobacteraceae has representatives of all three metabolic groups, providing an opportunity to evaluate how microorganisms might evolve to fill these various niches in anaerobic ecosystems.
Within the Geobacteraceae, Geobacter and Desulfuromonas are specialists in the complete oxidation of organic compounds to carbon dioxide coupled to the reduction of insoluble, extracellular electron acceptors . In aquatic sediments and submerged soils they influence the cycling of carbon and metals by oxidizing acetate, the central fermentation intermediate in anaerobic environments, with the reduction of iron and manganese oxides [1, 2]. In hydrocarbon-contaminated subsurface environments Geobacter species can play an important role in the bioremediation of aromatic hydrocarbons by oxidizing the contaminants with the reduction of Fe(III) oxides naturally present in the subsurface [3, 4]. This is a process which can be artificially stimulated [5, 6]. Another bioremediation application involving Geobacter species is to add acetate to uranium-contaminated subsurface environments. This stimulates the growth of Geobacter species which reduce soluble, mobile U(VI) to insoluble U(IV)  and thus immobilize the uranium in situ [4, 8]. The ability of Geobacter and Desulfuromonas species to oxidize organic compounds with electron transfer to graphite electrodes provides a convenient method for harvesting electricity from aquatic sediments and organic wastes to power electronic devices [9–11].
In contrast, the primary niche of Pelobacter species is methanogenic environments, functioning either as fermentative microorganisms or living in syntrophic associations with methanogens by partially oxidizing organic compounds to hydrogen and acetate which the methanogens consume [12, 13]. Pelobacter species cannot oxidize acetate or other organic compounds completely to carbon dioxide and are ineffective in electron transfer to metals  or electrodes . Therefore, not only the natural environments, but also the technological applications of Geobacter/Desulfuromonas versus Pelobacter species are quite different.
Here we report on the sequenced genomes of six members of the Geobacteraceae family: Geobacter sulfurreducens , Geobacter metallireducens, Geobacter uraniireducens, Pelobacter propionicus, Pelobacter carbinolicus, and Desulfuromonas acetoxidans. We use comparative genomics in an effort to better understand the enzymes involved in these unusual metabolisms, and to provide insight in to the selective pressures that may have contributed to their evolution.
Results and discussion
Identification of protein families in the six genomes
General features of the Geobacteraceae genomes
GC Content (%)
30.7 kb and 202.4 kb
The pattern of conservation of the proteins families across all species (the phyletic pattern) was determined (see Additional files 2 and 3). By far the most common pattern was conservation across all species, 5,345 proteins were members of clusters that included at least one protein from each genome (see Additional file 3). The second most common pattern was conservation in the Geobacter species and P. propionicus only (1,158 proteins). Proteins unique to the respiratory or fermentative species were relatively rare. Only 370 proteins were found in the respiratory species but not the fermentative species (see Additional file 4), and only 260 proteins were found in the fermentative species but not the respiratory species (see Additional file 5).
Thus, based on phylogeny, there are at least three possibilities for how modern Geobacteraceae evolved to fill multiple metabolic niches. The ancestor may have been adapted to a fermentative/syntrophic lifestyle with no reliance on external electron acceptors. In this scenario, the Geobacter and Desulfuromonas species would have independently evolved strategies for the complete oxidation of organic compounds coupled to Fe(III) reduction. Alternatively, the ancestor may have been a respiratory microorganism, and the Pelobacter species independently evolved strategies for fermentation and syntrophy and lost the ability to transfer electrons out of the cell. Another option was that the ancestor may have had both types of metabolism, and the various branches of the family lost certain abilities. We investigated these possibilities by analyzing the conservation and evolutionary history of genes for energy metabolism.
Conservation of pathways for acetate oxidation
The Pelobacter species contained orthologs to many of the TCA cycle enzymes (see Additional file 7), but there were two notable exceptions. NADPH metabolism appears to catalyzed by different enzymes in the Pelobacter species. A monomeric-type isocitrate dehydrogenase (GSU1465) was found in all of the respiratory species (see Additional file 8), but was not conserved in either Pelobacter species (see Additional file 7). In acetate oxidizers, this reaction is the primary source of NADPH in the cell . Instead, the Pelobacter species contained non-orthologous isocitrate dehydrogenase genes (Ppro_0452 and Pcar_1038 in cluster 3107) of the more common, homodimeric type . In addition, both Pelobacter species lacked the dehydrogenase (GSU0509–GSU0510) believed to transfer the electrons from NADPH into the electron transport chain . This enzyme was conserved in all four of the respiratory species (see Additional file 7). These changes suggest that the NADPH produced by the Pelobacter species may not be used primarily as a source of electrons for energy metabolism, as it is for the respiratory species. Furthermore, P. carbinolicus contained a non-orthologous fumarase enzyme (Pcar_0324) predicted to be of the class II, Fe(III)-free type , rather than the class I, Fe(III) type found in the other Geobacteraceae (GSU0994).
Conservation of pathways for extracellular electron transfer
The final part of the electron transport chain in the respiratory species is transfer of electrons from the inner membrane to cytochromes for transport to the cell surface. The differences between the respiratory and fermentative species are substantial in this part of the pathway. The genomes of all four of the respiratory species contained at least one copy of a putative cytochrome bc complex (GSU2932–GSU2934), which is predicted to transfer electrons from membrane-bound quinones to periplasmic cytochromes  (Figure 2, see Additional file 7). Neither of the Pelobacter species had any orthologs to this complex. C-type cytochromes span the periplasm and outer membrane in the respiratory species . All of the Geobacter and Desulfuromonas genomes contained more than 100 ORFs that have the heme-binding motif  characteristic of c-type cytochromes (see Additional file 1). Both Pelobacter genomes had far fewer cytochrome genes, ca. 40 per genome (see Additional file 1). Many of the proteins found only in the respiratory species were multi-heme, c-type cytochromes (see Additional file 4), whereas none of the proteins specific to the fermentative species were multi-heme (see Additional file 5). In addition, both Pelobacter species lacked most of the specific cytochromes required for electron transfer in G. sulfurreducens in vivo. These include those conserved in all of the respiratory species: MacA, a c-type cytochrome associated with the inner membrane  and OmcB, an outer-membrane cytochrome .
Lateral transfer of fermentation genes
Thus, while the Pelobacter species contained many vertically-inherited genes for respiratory metabolism, several key enzymes are different or missing in both species. To investigate evolution of the fermentative and syntrophic metabolism, Pelobacter-specific genes were analyzed, and genes originating from lateral gene transfer were identified using a combination of phylogenetic and BLAST-based analysis (see Additional file 9). Both Pelobacter species catabolize butanediol and acetoin . The butanediol dehydrogenase (Bdh, Pcar_0330) and acetoin dehydrogenase (AcoABCL, Pcar_0343–Pcar0346), which catalyze the initial steps in the metabolism of these compounds, have been characterized in P. carbinolicus . P. propionicus has genes with high sequence similarity to these, but the operon structure of the putative acetoin dehydrogenase included a duplication of the A and B subunits (Bdh, Ppro_1043 and AcoABCABL, Ppro_1024–Ppro1029, see Additional file 1). Phylogenetic analysis showed that the most closely related enzymes are not from Geobacter or Desulfuromonas, nor any other delta-Proteobacteria species (see Additional file 10). Instead the Pelobacter genes were most closely related to the butanediol catabolic genes from Pseudomonas  and Gram-positive species, indicating that these genes originated from lateral gene transfer (see Additional file 10).
The subsequent steps in the catabolism of the acetyl-CoA and acetaldehyde from acetoin are markedly different in the two Pelobacter species. The pathway in P. propionicus is cyclic and requires membrane-bound electron transport enzymes (Figure 3) . Several of the key enzymes were predicted to have been acquired by lateral gene transfer. Pyruvate:ferredoxin oxidoreductase is required to convert acetyl-CoA to pyruvate (Figure 3), and there were two heterotetrameric pyruvate:ferredoxin oxidoreductases from lateral gene transfer. One (Ppro_0322–Ppro_0325, see Additional file 1) is most closely related to the enzymes from Clostridia tetani and Thermotoga species, and the other (Ppro_0469–Ppro_0472, see Additional file 1) is most similar to the enzyme in Syntrophobacter and several archaeal species.
Pyruvate is then converted to succinyl-CoA to regenerate NAD+ without the need for a cooperating hydrogen-oxidizing species (Figure 3). P. propionicus has orthologs of four TCA cycle enzymes and the NADH dehydrogenase from the respiratory Geobacteraceae that could carry out these reactions (Figure 3, see Additional file 7). Then, propionate is generated via a methylmalonyl-CoA mutase and transcarboxylase (Figure 3). Both of these enzyme were predicted to have been acquired from lateral gene transfer. The mutase (Ppro_1284–Ppro_1285, see Additional file 1) is related to enzymes from Chlorflexus and Geobacillus species, and the transcarboxylase (Ppro_0033–Ppro_0034, see Additional file 1) is related to that crystallized from Propionibacterium freudenreichii .
Thus, it appears that P. propionicus uses a mosaic of vertically inherited and laterally acquired genes for fermentation. Interestingly, the pathway uses a complete, vertically inherited, respiratory electron transport chain – the succinate dehydrogenase operates in reverse as a fumarate reductase, accepting reducing equivalents from the NADH dehydrogenase . This is equivalent to that used by Geobacter species growing by fumarate respiration [18, 36].
Differences between Pelobacter species associated with syntrophy
Some Pelobacter species live in syntrophic associations with methanogens by partially oxidizing organic compounds to hydrogen and acetate which the methanogens consume [12, 13]. Both Pelobacter species lacked orthologs to any of the Geobacter-type  hydrogenases (GSU0121–GSU0123, GSU0782–GSU0785, GSU2417–GSU2420 or GSU2718–GSU2722, see Additional file 1). Furthermore, the hydrogenases in the two Pelobacter species are dissimilar. This may reflect the fact that P. carbinolicus is capable of growing syntrophically with a hydrogen-consuming partner, most typically a methanogen . Reducing equivalents can be disposed with the production of hydrogen, as long as the methanogen maintains hydrogen concentrations low enough for hydrogen production to be thermodynamically favorable. P. propionicus does not grow in this manner . P. carbinolicus has two highly-similar, four-subunit hydrogenases (Pcar_1602–Pcar_1605 and Pcar_1633–Pcar_1636) related to the cytoplasmic, hydrogen-producing HndABCD hydrogenase in Desulfovibrio fructosovorans . P. propionicus, encodes two highly-similar, 12-subunit enzymes (Ppro_0587–Ppro_0598 and Ppro_3521–Ppro_3532) that are related to the membrane-bound, hydrogen-producing, NiFe hydrogenase in Pyrococcus furiosus .
Approximately -45 kJ/mol is predicted to be available to P. carbinolicus and its methanogenic partner when they grow by ethanol oxidation under syntrophic conditions . This is less than what is predicted to be required for generation of an ATP by substrate-level phosphorylation, and it is assumed that some of the ATP from acetyl-CoA conversion must be reinvested to drive the overall reaction, possibly by reverse electron transport . It has been suggested that a Rnf-type ion-translocating electron transfer complex may play a role in reverse electron transport in syntrophs , by generating low potential ferredoxin from NADH using the membrane potential . P. carbinolicus contains an Rnf gene cluster (Pcar_0260–Pcar_0265, Figure 4) that is similar to the enzymes from Rhodobacter and Clostridium species, both predicted to be involved in reduction of ferredoxin by NADH . P. propionicus, which does not grow syntrophically, contains no Rnf-like genes.
Both Pelobacter genomes contain unusual numbers and types of ATP synthases, which have been suggested to be important for maintenance of the membrane potential . Both contain the ATP synthase that is conserved in all of the Geobacteraceae, but these genes appear to have recently been duplicated in both organisms (Pcar_0015–Pcar_0016/Pcar_3130–Pcar_3136, Pcar_0944–Pcar_0952, and Ppro_0599–Ppro_0607, Ppro_1500–Ppro_1508, Figures 3 and 4). In addition, both Pelobacter species also have a third ATP synthase encoded in operons organized like that of Methanosarcina barkeri , though they were predicted to have been acquired from different sources. The genes in P. propionicus (Ppro_844–Ppro851) are most closely related to enzymes from species of gamma Proteobacteria: Hahella, Legionella, and Methylococcus. The P. carbinolicus genes (Pcar_2989–Pcar2997) are only distantly related to the Geobacter and Desulfuromonas operons, but because they are similar to genes from a variety of species including D. acetoxidans, it is difficult to be confident about the lateral transfer source.
These results provide insights into the evolution of Geobacteraceae species into different environmental niches and biotechnological applications. The results suggest that the last common ancestor of the Geobacteraceae was an acetate-oxidizing, respiratory species capable of extracellular electron transfer, and that specialization for fermentative/syntrophic growth evolved at least twice, allowing some Geobacteraceae to fill additional niches.
The primacy of the respiratory mode is evident from the conservation of genes for all steps in this process including acetate uptake, central metabolism, and electron transfer across the cell membranes in both of the clades of the family. The fermentative/syntrophic Pelobacter species also contain many of these genes. However, they have lost several key enzymes that leave the pathways incomplete, including several necessary for the oxidation of acetate and most of the cytochromes predicted to provide the electrical connection between the inner membrane and the outside of the cell. Instead, the Pelobacter species have appropriated genes via lateral gene transfer for fermentative/syntrophic growth. It is clear that this has happened on two separate occasions. Although both P. carbinolicus and P. propionicus have closely related dehydrogenase genes for the initial metabolism of their unique substrates, acetoin and 2,3-butanediol, the genes for the further fermentation of these substrates are unrelated in the two organisms, reflecting the separate evolution of distinct metabolic pathways. The fact that P. carbinolicus also fills a syntrophic niche, participating in interspecies hydrogen transfer with hydrogen-consuming methanogens, whereas P. propionicus does not, may be explained by the genes associated with reverse electron transfer that only P. carbinolicus has appropriated. The fact that both Pelobacter species have phylogenetically distinct hydrogenase genes that are different from each other as well as those of the Geobacter species, may also reflect the difference in syntrophic capabilities of these species.
The selective pressure to specialize in syntrophic/fermentative growth may have initially been found at the interface of redox boundaries in sedimentary environments. As respiratory Geobacteraceae deplete the supply of the terminal electron acceptor Fe(III) oxide, their capacity for growth is greatly diminished and organisms with other respiratory processes, such as sulfate reduction or methane production, become predominant . Some Geobacter species can oxidize acetate to carbon dioxide and hydrogen when they lack external electron acceptors , but the slow rate of this metabolism and the requirement for very low hydrogen partial pressures means that they are not competitive with acetate-utilizing sulfate reducers or methanogens. Acquiring the ability to ferment novel substrates and/or to grow syntrophically could have facilitated expansion into Fe(III) oxide depleted environments. Under such conditions investing energy in the production of respiratory enzymes such as the c-type cytochromes Geobacteraceae require to grow under Fe(III)-reducing conditions would be maladaptive.
These findings also provide insight into the types of metabolic changes that might take place as these organisms are being adapted for modern biotechnical applications. In applications such as the in situ bioremediation of uranium-contaminated groundwater and the conversion of organic compounds to electricity Geobacter species must deal with a scarcity of electron acceptor because electron donor is generally provided well in excess of electron acceptor availability. During in situ bioremediation Fe(III) oxides are rapidly depleted near the source of subsurface acetate amendments, limiting the growth and effectiveness of Geobacter-catalyzed U(VI) reduction . Geobacter species form thick biofilms on the electrodes of microbial fuel cells, forcing many of the cells to metabolize acetate at a significant distance from this artificial electron acceptor [45, 46]. Preliminary results suggest that, like the Pelobacter species described here, Geobacteraceae that predominate during in situ uranium reduction or on the anodes of microbial fuel cells have fewer c-type cytochromes (DRL, unpublished data). Enhanced ability to release excess electrons as hydrogen, in a manner similar to that of P. carbinolicus, could also be beneficial under conditions in which electron acceptor availability is limiting. Thus, these relatively few changes appear to have allowed a respiratory ancestor to radiate out into fermentative and syntrophic niches in addition to their respiratory roles in anaerobic environments. This information serves as a guide to the history of these organisms and provides information that could aid in optimizing their biotechnological applications.
Genome sequencing and annotation
With the exception of G. sulfurreducens , sequence data for the Geobacteraceae 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/ (Table 1).
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 .
All the ORFs from the six Geobacteraceae genomes and the outgroup species Anaeromyxobacter dehalogenans 2CP-C (NC_007760) 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 (see Additional file 6). 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.
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. 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 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.
We thank Peter Brown for his assistance. 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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