Genetic characterization of a single bifunctional enzyme for fumarate reduction and succinate oxidation in Geobacter sulfurreducens and engineering of fumarate reduction in Geobacter metallireducens

The mechanism of fumarate reduction in Geobacter sulfurreducens was investigated. The genome contained genes encoding a heterotrimeric fumarate reductase, FrdCAB, with homology to the fumarate reductase of Wolinella succinogenes and the succinate dehydrogenase of Bacillus subtilis. Mutation of the putative catalytic subunit of the enzyme resulted in a strain that lacked fumarate reductase activity and was unable to grow with fumarate as the terminal electron acceptor. The mutant strain also lacked succinate dehydrogenase activity and did not grow with acetate as the electron donor and Fe(III) as the electron acceptor. The mutant strain could grow with acetate as the electron donor and Fe(III) as the electron acceptor if fumarate was provided to alleviate the need for succinate dehydrogenase activity in the tricarboxylic acid cycle. The growth rate of the mutant strain under these conditions was faster and the cell yields were higher than for wild type grown under conditions requiring succinate dehydrogenase activity, suggesting that the succinate dehydrogenase reaction consumes energy. An orthologous frdCAB operon was present in Geobacter metallireducens, which cannot grow with fumarate as the terminal electron acceptor. When a putative dicarboxylic acid transporter from G. sulfurreducens was expressed in G. metallireducens, growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms, G. sulfurreducens and possibly G. metallireducens use the same enzyme for both fumarate reduction and succinate oxidation in vivo.     

OmcB,a c-type polyheme cytochrome,involved in Fe(III) reduction in Geobacter sulfurreducens

Microorganisms in the family Geobacteraceae are the predominant Fe(III)-reducing microorganisms in a variety of subsurface environments in which Fe(III) reduction is an important process, but little is known about the mechanisms for electron transport to Fe(III) in these organisms. The Geobacter sulfurreducens genome was found to contain a 10-kb chromosomal duplication consisting of two tandem three-gene clusters. The last genes of the two clusters, designated omcB and omcC, encode putative outer membrane polyheme c-type cytochromes which are 79% identical. The role of the omcB and omcC genes in Fe(III) reduction in G. sulfurreducens was investigated. OmcB and OmcC were both expressed during growth with acetate as the electron donor and either fumarate or Fe(III) as the electron acceptor. OmcB was ca. twofold more abundant under both conditions. Disrupting omcB or omcC by gene replacement had no impact on growth with fumarate. However, the OmcB-deficient mutant was greatly impaired in its ability to reduce Fe(III) both in cell suspensions and under growth conditions. In contrast, the ability of the OmcC-deficient mutant to reduce Fe(III) was similar to that of the wild type. When omcB was reintroduced into the OmcB-deficient mutant, the capacity for Fe(III) reduction was restored in proportion to the level of OmcB production. These results indicate that OmcB, but not OmcC, has a major role in electron transport to Fe(III) and suggest that electron transport to the outer membrane is an important feature in Fe(III) reduction in this organism.     

Isolation of Geobacter species from diverse sedimentary environments.

In an attempt to better understand the microorganisms responsible for Fe(III) reduction in sedimentary environments, Fe(III)-reducing microorganisms were enriched for and isolated from freshwater aquatic sediments, a pristine deep aquifer, and a petroleum-contaminated shallow aquifer. Enrichments were initiated with acetate or toluene as the electron donor and Fe(III) as the electron acceptor. Isolations were made with acetate or benzoate. Five new strains which could obtain energy for growth by dissimilatory Fe(III) reduction were isolated. All five isolates are gram-negative strict anaerobes which grow with acetate as the electron donor and Fe(III) as the electron acceptor. Analysis of the 16S rRNA sequence of the isolated organisms demonstrated that they all belonged to the genus Geobacter in the delta subdivision of the Proteobacteria. Unlike the type strain, Geobacter metallireducens, three of the five isolates could use H2 as an electron donor for Fe(III) reduction. The deep subsurface isolate is the first Fe(III) reducer shown to completely oxidize lactate to carbon dioxide, while one of the freshwater sediment isolates is only the second Fe(III) reducer known that can oxidize toluene. The isolation of these organisms demonstrates that Geobacter species are widely distributed in a diversity of sedimentary environments in which Fe(III) reduction is an important process.     

Characterization of metabolism in the Fe(III)-reducing organism Geobacter sulfurreducens by constraint-based modeling

Geobacter sulfurreducens is a well-studied representative of the Geobacteraceae, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate G. sulfurreducens central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why Geobacter species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of G. sulfurreducens could synthesize amino acids more efficiently than that of Escherichia coli due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of G. sulfurreducens central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.     

Growth advantage in stationary-phase (GASP) phenotype in long-term survival strains of Geobacter sulfurreducens

Geobacter sulfurreducens exists in the subsurface and has been identified in sites contaminated with radioactive metals, consistent with its ability to reduce metals under anaerobic conditions. The natural state of organisms in the environment is one that lacks access to high concentrations of nutrients, namely electron donors and terminal electron acceptors (TEAs). Most studies have investigated G. sulfurreducens under high-nutrient conditions or have enriched for it in environmental systems via acetate amendments. We replicated the starvation state through long-term batch culture of G. sulfurreducens, where both electron donor and TEA were scarce. The growth curve revealed lag, log, stationary, death, and survival phases using acetate as electron donor and either fumarate or iron(III) citrate as TEA. In survival phase, G. sulfurreducens persisted at a constant cell count for as long as 23 months without replenishment of growth medium. Geobacter sulfurreducens demonstrated an ability to acquire a growth advantage in stationary-phase phenotype (GASP), with strains derived from subpopulations from death- or survival phase being able to out-compete mid-log-phase populations when co-cultured. The molecular basis for GASP was not because of any detectable mutation in the rpoS gene (GSU1525) nor because of a mutation in a putative homolog to Escherichia coli lrp, GSU3370.     

MacA, a diheme c-type cytochrome involved in Fe(III) reduction by Geobacter sulfurreducens

A 36-kDa diheme c-type cytochrome abundant in Fe(III)-respiring Geobacter sulfurreducens, designated MacA, was more highly expressed during growth with Fe(III) as the electron acceptor than with fumarate. Although MacA has homology to proteins with in vitro peroxidase activity, deletion of macA had no impact on response to oxidative stress. However, the capacity for Fe(III) reduction was greatly diminished, indicating that MacA, which is predicted to be localized in the periplasm, is a key intermediate in electron transfer to Fe(III).     

Possible nonconductive role of Geobacter sulfurreducens pilus nanowires in biofilm formation

Geobacter sulfurreducens required expression of electrically conductive pili to form biofilms on Fe(III) oxide surfaces, but pili were also essential for biofilm development on plain glass when fumarate was the sole electron acceptor. Furthermore, pili were needed for cell aggregation in agglutination studies. These results suggest that the pili of G. sulfurreducens also have a structural role in biofilm formation.     

Preferential reduction of FeIII over fumarate by Geobacter sulfurreducens

The presence of Fe(III), but not that of Fe(II), resulted in ca. 20-fold-lower levels of mRNA for fumarate reductase, inhibiting fumarate reduction and favoring utilization of fumarate as an electron donor in chemostat cultures of Geobacter sulfurreducens, despite the fact that growth yield with fumarate was 3-fold higher than with Fe(III).     

Fluorescent properties of c-type cytochromes reveal their potential role as an extracytoplasmic electron sink in Geobacter sulfurreducens

A novel fluorescence technique for monitoring the redox status of c-type cytochromes in Geobacter sulfurreducens was developed in order to evaluate the capacity of these extracytoplasmic cytochromes to store electrons during periods in which an external electron acceptor is not available. When intact cells in which the cytochromes were in a reduced state were excited at a wavelength of 350 nm, they fluoresced with maxima at 402 and 437 nm. Oxidation of the cytochromes resulted in a loss of fluorescence. This method was much more sensitive than the traditional approach of detecting c-type cytochromes via visible light absorbance. Furthermore, fluorescence of reduced cytochromes in individual cells could be detected via fluorescence microscopy, and the cytochromes in a G. sulfurreducens biofilm, remotely excited with an optical fibre, could be detected at distances as far as 5 cm. Fluorescence analysis of cytochrome oxidation and reduction of the external electron acceptor, anthraquinone-2,6-disulfonate, suggested that the extracytoplasmic cytochromes of G. sulfurreducens could store approximately 10(7) electrons per cell. Independent analysis of the haem content of the cells determined from analysis of incorporation of (55)Fe into cytochromes provided a similar estimate of cytochrome electron-storage capacity. This electron-storage capacity could, in the absence of an external electron acceptor, permit continued electron transfer across the inner membrane sufficient to supply the maintenance energy requirements for G. sulfurreducens for up to 8 min or enough proton motive force to power flagella motors for G. sulfurreducens motility. The fluorescence approach described here provides a sensitive method for evaluating the redox status of Geobacter species in culture and/or its environments. Furthermore, these results suggest that the periplasmic and outer-membrane cytochromes of Geobacter species act as capacitors, allowing continued electron transport, and thus viability and motility, for Geobacter species as they move between heterogeneously dispersed Fe(III) oxides during growth in the subsurface.     

The RpoS sigma factor in the dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens

Geobacter sulfurreducens RpoS sigma factor was shown to contribute to survival in stationary phase and upon oxygen exposure. Furthermore, a mutation in rpoS decreased the rate of reduction of insoluble Fe(III) but not of soluble forms of iron. This study suggests that RpoS plays a role in regulating metabolism of Geobacter under suboptimal conditions in subsurface environments.     

Graphite electrode as a sole electron donor for reductive dechlorination of tetrachlorethene by Geobacter lovleyi

The possibility that graphite electrodes can serve as the direct electron donor for microbially catalyzed reductive dechlorination was investigated with Geobacter lovleyi. In an initial evaluation of whether G. lovleyi could interact electronically with graphite electrodes, cells were provided with acetate as the electron donor and an electrode as the sole electron acceptor. Current was produced at levels that were ca. 10-fold lower than those previously reported for Geobacter sulfurreducens under similar conditions, and G. lovleyi anode biofilms were correspondingly thinner. When an electrode poised at -300 mV (versus a standard hydrogen electrode) was provided as the electron donor, G. lovleyi effectively reduced fumarate to succinate. The stoichiometry of electrons consumed to succinate produced was 2:1, the ratio expected if the electrode served as the sole electron donor for fumarate reduction. G. lovleyi effectively reduced tetrachloroethene (PCE) to cis-dichloroethene with a poised electrode as the sole electron donor at rates comparable to those obtained when acetate serves as the electron donor. Cells were less abundant on the electrodes when the electrodes served as an electron donor than when they served as an electron acceptor. PCE was not reduced in controls without cells or when the current supply to cells was interrupted. These results demonstrate that G. lovleyi can use a poised electrode as a direct electron donor for reductive dechlorination of PCE. The ability to colocalize dechlorinating microorganisms with electrodes has several potential advantages for bioremediation of subsurface chlorinated contaminants, especially in source zones where electron donor delivery is challenging and often limits dechlorination.     

Interactions between the Fe(III)-reducing bacterium Geobacter sulfurreducens and arsenate, and capture of the metalloid by biogenic Fe(II)

Previous work has shown that microbial communities in As-mobilizing sediments from West Bengal were dominated by Geobacter species. Thus, the potential of Geobacter sulfurreducens to mobilize arsenic via direct enzymatic reduction and indirect mechanisms linked to Fe(III) reduction was analyzed. G. sulfurreducens was unable to conserve energy for growth via the dissimilatory reduction of As(V), although it was able to grow in medium containing fumarate as the terminal electron acceptor in the presence of 500 muM As(V). There was also no evidence of As(III) in culture supernatants, suggesting that resistance to 500 muM As(V) was not mediated by a classical arsenic resistance operon, which would rely on the intracellular reduction of As(V) and the efflux of As(III). When the cells were grown using soluble Fe(III) as an electron acceptor in the presence of As(V), the Fe(II)-bearing mineral vivianite was formed. This was accompanied by the removal of As, predominantly as As(V), from solution. Biogenic siderite (ferrous carbonate) was also able to remove As from solution. When the organism was grown using insoluble ferrihydrite as an electron acceptor, Fe(III) reduction resulted in the formation of magnetite, again accompanied by the nearly quantitative sorption of As(V). These results demonstrate that G. sulfurreducens, a model Fe(III)-reducing bacterium, did not reduce As(V) enzymatically, despite the apparent genetic potential to mediate this transformation. However, the reduction of Fe(III) led to the formation of Fe(II)-bearing phases that are able to capture arsenic species and could act as sinks for arsenic in sediments.     

Geobacter lovleyi sp. nov. strain SZ, a novel metal-reducing and tetrachloroethene-dechlorinating bacterium

A bacterial isolate, designated strain SZ, was obtained from noncontaminated creek sediment microcosms based on its ability to derive energy from acetate oxidation coupled to tetrachloroethene (PCE)-to-cis-1,2-dichloroethene (cis-DCE) dechlorination (i.e., chlororespiration). Hydrogen and pyruvate served as alternate electron donors for strain SZ, and the range of electron acceptors included (reduced products are given in brackets) PCE and trichloroethene [cis-DCE], nitrate [ammonium], fumarate [succinate], Fe(III) [Fe(II)], malate [succinate], Mn(IV) [Mn(II)], U(VI) [U(IV)], and elemental sulfur [sulfide]. PCE and soluble Fe(III) (as ferric citrate) were reduced at rates of 56.5 and 164 nmol min(-1) mg of protein(-1), respectively, with acetate as the electron donor. Alternate electron acceptors, such as U(VI) and nitrate, did not inhibit PCE dechlorination and were consumed concomitantly. With PCE, Fe(III) (as ferric citrate), and nitrate as electron acceptors, H(2) was consumed to threshold concentrations of 0.08 +/- 0.03 nM, 0.16 +/- 0.07 nM, and 0.5 +/- 0.06 nM, respectively, and acetate was consumed to 3.0 +/- 2.1 nM, 1.2 +/- 0.5 nM, and 3.6 +/- 0.25 nM, respectively. Apparently, electron acceptor-specific acetate consumption threshold concentrations exist, suggesting that similar to the hydrogen threshold model, the measurement of acetate threshold concentrations offers an additional diagnostic tool to delineate terminal electron-accepting processes in anaerobic subsurface environments. Genetic and phenotypic analyses classify strain SZ as the type strain of the new species, Geobacter lovleyi sp. nov., with Geobacter (formerly Trichlorobacter) thiogenes as the closest relative. Furthermore, the analysis of 16S rRNA gene sequences recovered from PCE-dechlorinating consortia and chloroethene-contaminated subsurface environments suggests that Geobacter lovleyi belongs to a distinct, dechlorinating clade within the metal-reducing Geobacter group. Substrate versatility, consumption of electron donors to low threshold concentrations, and simultaneous reduction of electron acceptors suggest that strain SZ-type organisms have desirable characteristics for bioremediation applications.