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Genome of Geobacter sulfurreducens: Metal Reduction in Subsurface Environments

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Science  12 Dec 2003:
Vol. 302, Issue 5652, pp. 1967-1969
DOI: 10.1126/science.1088727

Abstract

The complete genome sequence of Geobacter sulfurreducens, a δ-proteobacterium, reveals unsuspected capabilities, including evidence of aerobic metabolism, one-carbon and complex carbon metabolism, motility, and chemotactic behavior. These characteristics, coupled with the possession of many two-component sensors and many c-type cytochromes, reveal an ability to create alternative, redundant, electron transport networks and offer insights into the process of metal ion reduction in subsurface environments. As well as playing roles in the global cycling of metals and carbon, this organism clearly has the potential for use in bioremediation of radioactive metals and in the generation of electricity.

G. sulfurreducens, a member of the δ-Proteobacteria and of the family Geobacteraceae, is an important component of subsurface biota. Geobacter spp. generate energy as adenosine triphosphate by using metal ion–mediated electron transport to oxidize organic compounds to CO2. For instance, Fe(III) oxides are abundant in the subsurface environment and are commonly used as terminal electron acceptors. However, the considerable interest in using Geobacter spp. for bioremediation stems from their ability to precipitate soluble metals, such as uranium, as a product of electron transport. Preferential stimulation of native populations of Geobacter spp. to promote metal precipitation from groundwater is readily achieved in situ by the addition of acetate (13). Beyond the opportunities for bioremediation, interest in Geobacter spp. lies in biotechnological efforts to capture energy from the catabolism of organic waste with energy-harvesting electrodes (4). Not forgetting, of course, the critical roles that Geobacter spp. play in the global cycling of metals and carbon.

The G. sulfurreducens genome is a single circular chromosome of 3,814,139 base pairs (bp) with a total of 3466 predicted protein-encoding open reading frames [coding sequences (CDSs)] (Table 1 and fig. S1) (5). This genome offers a phylogenetic framework for evolutionary studies on metal ion reduction. Analysis of gene distribution patterns across lineages (phylogenetic profiling) revealed that G. sulfurreducens and the metal ion–reducing γ-Proteobacterium Shewanella oneidensis (6) shared only two genes not found in any other species: both encoding c-type cytochromes. Hence, the metal ion–reducing capabilities of these species are not simply related to their sharing an exclusive set of genes; the expansion of specific gene families and the presence of novel genes are also involved. Similar analysis revealed many global similarities in gene content across a wider range of taxa (table S1). One example is a cluster of 20 genes found in organisms including Desulfovibrio vulgaris, low-G+C firmicutes, and Archaea. Most of the genes with known functions in this cluster are involved in energy metabolism (such as those encoding heterodisulfide reductases). The presence of four additional conserved hypothetical CDSs in this cluster suggests that their functional roles may be related to energy metabolism as well.

Table 1.

General features of the G. sulfurreducens genome.

Size (bp) 3,814,139
G + C percentage 60.9
Number of predicted CDSs 3466
Average size of CDS (bp) 989
Percentage coding 90
Number of ribosomal RNA operons (16S-23S-5S) 2
Number of transfer RNAs 49
Number of structural RNAs 2
Number of CDSs similar to known protein 2011
Number of CDSs similar to proteins of unknown function 445
Number of conserved hypothetical proteins 384
Number of hypothetical proteins 633
Number of Rho-independent terminators 376

G. sulfurreducens encodes genes for glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway. With one notable exception, the TCA genes appear to be bacterial forms; however, both G. sulfurreducens and G. metallireducens (7) encode a form of citrate synthase previously reported only in eukaryotes (8) (fig. S2).

Central to the metabolism of G. sulfurreducens is the ability to anaerobically oxidize acetate (an abundant electron donor and carbon source in subsurface zones) completely to CO2 and water using a variety of electron acceptors including metal ions, elemental sulfur, and fumarate. The lack of identifiable transporters for sugar uptake highlights the central importance of acetate metabolism to this organism (fig. S3). Based on its predicted membrane transporter complement, amino acids and carboxylates appear to be the predominant organic substrates for G. sulfurreducens. However, G. sulfurreducens does possess a complex set of phosphotransferase enzymes that presumably serve solely regulatory purposes.

G. sulfurreducens encodes enzymes that might participate in the acetyl–coenzyme A (acetyl-CoA) pathway. This versatile pathway can use acetate and one-carbon (C1) compounds as substrates for energy generation, and can also be used to assimilate carbon by CO2 reduction (911). However, G. sulfurreducens is missing a key enzyme of this pathway: formyl tetrahydrofolate synthetase (FTS). Instead, it may use reverse electron transport (coupling energetically unfavorable redox reactions to the expenditure of a membrane ion gradient), analogously to some methanogens, hence circumventing the need for the missing FTS (12) (fig. S3).

Additional evidence of C1 metabolism is supplied by the presence of the anaerobic form of carbon monoxide dehydrogenase (CODH), which catalyzes the oxidation of carbon monoxide to CO2 and hydrogen. In addition to participation in the acetyl-CoA pathway, CODH can be used chemolithoautotrophically in reactions distinct from this pathway (12).

G. sulfurreducens appears to have a versatile approach to capturing energy and carbon, having three enzyme systems, each of which is capable of converting pyruvate to acetyl-CoA. These include pyruvate-ferredoxin oxidoreductase and pyruvate-formate lyase, used by anaerobes, and a putative pyruvate dehydrogenase complex found largely in aerobic organisms (13).

Alternate electron transport pathways in G. sulfurreducens take electrons generated from central metabolism in the cytoplasm and transfer them by direct contact to extracellular electron acceptors, such as Fe(III) oxides (2). In contrast, other metal ion reducers, including Shewanella spp., also use soluble electron shuttling compounds in addition to direct contact (14). There were an unprecedented number of putative c-type cytochromes found in G. sulfurreducens, with 111 CDSs containing at least one match to the c-type cytochrome motif that identifies heme groups (15) (table S2). Seventy-three c-type cytochromes contain two or more heme groups, including one that possesses 27. The abundance of cytochromes highlights the importance of electron transport to this organism and suggests that flexibility and redundancy in the electron transfer networks it can create are important for the reduction of diverse metal ions in natural environments.

Of the c-type cytochromes in G. sulfurreducens, many are more similar to those from S. oneidensis than from the metal ion–reducing δ-Proteobacterium D. vulgaris (16). For instance, 23 are best matches to CDSs in S. oneidensis, as compared with 10 in D. vulgaris. Grouping c-type cytochromes into families revealed that some are shared across all three genomes (tables S3 and S4), although 43 candidates were unique to G. sulfurreducens (table S6).

Other well-recognized electron transport components, including dehydrogenases, quinones, iron-sulfur proteins, and b-type cytochromes, are present. Overall, however, the G. sulfurreducens genome has a markedly different set of electron transport components as compared with those of other metal ion reducers (12) (figs. S4 and S5); for instance, 51% of the electron transport proteins in G. sulfurreducens have no homolog in S. oneidensis (table S5).

G. sulfurreducens can couple the oxidation of hydrogen to the reduction of Fe(III). Besides using environmental hydrogen sources to fuel this reaction, molecular hydrogen can also be formed as a byproduct of the organism's own metabolism (for example, from nitrogen fixation) and subsequently cycled back into the electron transport network to yield energy. G. sulfurreducens has at least three NiFe-hydrogenases (large- and small-subunit) (12) that could be involved, as well as two multisubunit nicotinamide adenine dinucleotide (NAD+)–reducing hydrogenases. The latter are similar to the NADH: quinone oxidoreductases (proton pumps that establish membrane ion gradients) and may also be involved in reverse electron transport or hydrogen cycling (12).

Many of the electron transport proteins were predicted to reside in the periplasm or outer membrane of G. sulfurreducens, making export through the plasma membrane important to energy metabolism. Supporting evidence included sequences encoding machinery for twin arginine transport and a type II secretian-dependent pathway for the translocation of proteins from the periplasmic space to the outer membrane. Several sequences representing components of the type II secretion system in G. sulfurreducens were divergent as compared to other type II secretion protein sequences, suggesting distinctions in its transport mechanisms.

Geobacter spp. have previously been characterized as strict anaerobes (17). However, there was considerable evidence in the genome of an oxidative capacity, which could be used to exploit or provide protection from oxic episodes. Homologs have been found for the high-oxygen-affinity cytochrome d–ubiquinol terminal oxidase and rubredoxin-oxygen oxidoreductase, as well as for the low-oxygen-affinity cytochrome c oxidase from the heme-copper oxidase superfamily (18, 19). The occurrence of homologs for catalase, superoxide dismutase, ruberythrin, and other peroxidases suggests an ability to scavenge oxygen radicals. G. sulfurreducens also possesses a CDS related to the oxygen-dependent form of the enzyme protoporphyrinogen oxidase, which catalyzes the penultimate step in the porphyrin biosynthetic pathway. This is unexpected, because most facultative or anaerobic bacteria capable of this reaction use a multienzyme complex linked to the respiratory chain. It is more usual for strict aerobes to use this single protein, which is dependent on oxygen as a terminal electron acceptor (20).

It is possible that G. sulfurreducens metabolizes complex carbon compounds through aerobic metabolism. A putative dioxygenase could break aromatic rings, and the presence of isoquinoline-oxidoreductase indicates complex heterocyclic ring catabolism (21). The presence of indolepyruvate oxidoreductases suggests that aryl pyruvates can be catabolized anaerobically. Hence, G. sulfurreducens seems to have a choice of carbon catabolism pathways it can use, permitting versatility under changing conditions.

The large number of regulatory genes found probably reflects the need to adapt to rapidly changing conditions (6). Four percent of the CDSs were two-component regulators (histidine kinases and response regulators); of these, 43% have PAS (PER-ARNT-SIM) domains or sensory modules that detect oxygen tension, redox state, or light or energy levels (22). Transcriptional regulators in the G. sulfurreducens genome included multiple members of DNA binding protein families that regulate metal-responsive genes, such as the Fur and ArsR families (23, 24).

Chemotactic behavior requires complex regulatory networks and a mechanism for motility. G. sulfurreducens possesses multiple copies of methyl-accepting chemotaxis proteins (MCPs), including at least two previously undescribed and a homolog of DifA, which is crucial to social gliding motility (25). Essential Che proteins are also found in multiple copies, including the sensor kinase CheA and response regulator CheY. Although G. sulfurreducens was previously thought to be nonmotile, our analysis has revealed CDSs for both flagella and pili production.

The G. sulfurreducens genome has not only provided remarkable new insights into its unique metabolic capabilities and strategies for environmental survival, but has also made us rethink Geobacter physiology. This species may be neither immobile nor a strict anaerobe. It also possesses extraordinary electron transport capability and sensory potential, highlighted by the unprecedented collection of newly reported c-type cytochromes, the range and depth of which will best be appreciated in the light of comparative studies. Genomic analysis continues to further our understanding of the role of Geobacter spp. in the environment, as well as the evolution of metal ion reduction and how these processes relate to bioremediation and energy generation.

Supporting Online Material

www.sciencemag.org/cgi/content/full/302/5652/1967/DC1

Materials and Methods

SOM Text

Figs. S1 to S5

Tables S1 to S6

References

References and Notes

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