Evolution of electron transfer out of the cell: comparative genomics of six Geobacter genomes

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Genome-based Geobacter phylogeny. Bayesian inference of the phylogenetic tree of the six Geobacter species discussed, using another Geobacteraceae species, Pelobacter propionicus, as the outgroup. The tree was based on a concatenation of the proteins in the 697 families that had exactly one ortholog conserved in each of the seven genomes (listed in Additional file 4 of the supplementary material). Values at branch points are posterior probabilities.

Figure 2

Conservation of the energy metabolism pathways of Geobacter sulfurreducens. Shown are the pathways for acetate activation and oxidation via the TCA cycle in the cytoplasm; inner membrane oxidation of TCA cycle products coupled with electron/proton transport and ATP generation; and periplasmic and outer membrane cytochromes known to be required in vivo for transfer of electrons to an extracellular acceptor. The genes encoding the enzymes of these pathways and their full conservation pattern across all of the Geobacter genomes are listed in Additional file 6 of the supplementary material. The enzymes are colored black if there were orthologs for every subunit in all of the species and red if there were not. OmcB is shown in gray because there positional but not sequence-based orthologs (see text and figure 5).

Figure 3

Neighbor-joining phylogeny of the large subunit of the four-subunit hydrogenase. This enzyme is specific to the Geobacter species of the subsurface clade, there are no orthologs in other Geobacter species. NCBI identification numbers of homologs and bootstrap values from 1000 replicates are shown.

Figure 4

The gene cluster (GSU2937 through GSU2930) encoding the putative inner-membrane cytochrome bc complex that is conserved in all six Geobacter species. Genes encoding c-type cytochromes are shown in yellow, the Fe-S cluster protein encoding gene is shown in purple, and the cytochrome b gene is shown in green. All of these protein are orthologs across all of the Geobacter genomes (Table 5). The c-type cytochromes contain 2, 10, 12, and 5 heme-binding motifs each, respectively (see Additional file 9).

Figure 5

The region of the operon of omcB (dark blue) in all six Geobacter species genomes. In G. sulfurreducens the multi-heme cytochrome OmcB, which is required for electron transport to extracellular acceptors, is encoded in an operon with two other genes, orf1 (red) and orf2(gray) that is duplicated in the genome [54]. Shown here are regions of the genomes that encode the orthologs to these genes in all six Geobacter genomes, with orthologs colored identically. In some cases, there were multi-heme cytochromes encoded in the position of OmcB, but the sequence similarity was too low to confidently predict orthology, so these genes are colored light blue.