Maddalena V. Coppi

Development of a Genetic System for Geobacter sulfurreducens

ABSTRACT Members of the genus Geobacter are the dominant metal-reducing microorganisms in a variety of anaerobic subsurface environments and have been shown to be involved in the bioremediation of both organic and metal contaminants. To facilitate the study of the physiology of these organisms, a genetic system was developed forGeobacter sulfurreducens. The antibiotic sensitivity of this organism was characterized, and optimal conditions for plating it at high efficiency were established. A protocol for the introduction of foreign DNA into G. sulfurreducens by electroporation was also developed. Two classes of broad-host-range vectors, IncQ and pBBR1, were found to be capable of replication in G. sulfurreducens. In particular, the IncQ plasmid pCD342 was found to be a suitable expression vector for this organism. When the information and novel methods described above were utilized, thenifD gene of G. sulfurreducens was disrupted by the single-step gene replacement method. Insertional mutagenesis of this key gene in the nitrogen fixation pathway impaired the ability of G. sulfurreducens to grow in medium lacking a source of fixed nitrogen. Expression of thenifD gene in trans complemented this phenotype. This paper constitutes the first report of genetic manipulation of a member of the Geobacter genus.

Outer Membrane c-Type Cytochromes Required for Fe(III) and Mn(IV) Oxide Reduction in Geobacter sulfurreducens

ABSTRACT The potential role of outer membrane proteins in electron transfer to insoluble Fe(III) oxides by Geobacter sulfurreducens was investigated because this organism is closely related to the Fe(III) oxide-reducing organisms that are predominant in many Fe(III)-reducing environments. Two of the most abundant proteins that were easily sheared from the outer surfaces of intact cells were c-type cytochromes. One, designated OmcS, has a molecular mass of ca. 50 kDa and is predicted to be an outer membrane hexaheme c-type cytochrome. Transcripts for omcS could be detected during growth on Fe(III) oxide, but not on soluble Fe(III) citrate. The omcS mRNA consisted primarily of a monocistronic transcript, and to a lesser extent, a longer transcript that also contained the downstream gene omcT, which is predicted to encode a second hexaheme outer membrane cytochrome with 62.6% amino acid sequence identity to OmcS. The other abundant c-type cytochrome sheared from the outer surface of G. sulfurreducens, designated OmcE, has a molecular mass of ca. 30 kDa and is predicted to be an outer membrane tetraheme c-type cytochrome. When either omcS or omcE was deleted, G. sulfurreducens could no longer reduce Fe(III) oxide but could still reduce soluble electron acceptors, including Fe(III) citrate. The mutants could reduce Fe(III) in Fe(III) oxide medium only if the Fe(III) chelator, nitrilotriacetic acid, or the electron shuttle, anthraquinone 2,6-disulfonate, was added. Expressing omcS or omcE in trans restored the capacity for Fe(III) oxide reduction. OmcT was not detected among the sheared proteins, and genetic studies indicated that G. sulfurreducens could not reduce Fe(III) oxide when omcT was expressed but OmcS was absent. In contrast, Fe(III) oxide was reduced when omcS was expressed in the absence of OmcT. These results suggest that OmcS and OmcE are involved in electron transfer to Fe(III) oxides in G. sulfurreducens. They also emphasize the importance of evaluating mechanisms for Fe(III) reduction with environmentally relevant Fe(III) oxide, rather than the more commonly utilized Fe(III) citrate, because additional electron transfer components are required for Fe(III) oxide reduction that are not required for Fe(III) citrate reduction.

Biochemical and Genetic Characterization of PpcA, a Periplasmic c-Type Cytochrome in Geobacter Sulfurreducens

A 9.6 kDa periplasmic c -type cytochrome, designated PpcA, was purified from the Fe(III)-reducing bacterium Geobacter sulfurreducens and characterized. The purified protein is basic (pI 9.5), contains three haems and has an N-terminal amino acid sequence closely related to those of the previously described trihaem c (7) cytochromes of Geobacter metallireducens and Desulfuromonas acetoxidans. The gene encoding PpcA was identified from the G. sulfurreducens genome using the N-terminal sequence, and encodes a protein of 71 amino acids (molecular mass 9.58 kDa) with 49% identity to the c (7) cytochrome of D. acetoxidans. In order to determine the physiological role of PpcA, a knockout mutant was prepared with a single-step recombination method. Acetate-dependent Fe(III) reduction was significantly inhibited in both growing cultures and cell suspensions of the mutant. When ppcA was expressed in trans, the full capacity for Fe(III) reduction with acetate was restored. The transfer of electrons from acetate to anthraquinone 2,6-disulphonate (AQDS; a humic acid analogue) and to U(VI) was also compromised in the mutant, but acetate-dependent reduction of fumarate was not altered. The rates of reduction of Fe(III), AQDS, U(VI) and fumarate were also the same in the wild type and ppcA mutant when hydrogen was supplied as the electron donor. When taken together with previous studies on other electron transport proteins in G. sulfurreducens, these results suggest that PpcA serves as an intermediary electron carrier from acetate to terminal Fe(III) reductases in the outer membrane, and is also involved in the transfer of electrons from acetate to U(VI) and humics.

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.

Characterization of Metabolism in the Fe(III)-Reducing Organism Geobacter sulfurreducens by Constraint-Based Modeling

ABSTRACT 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.

Importance of c-Type Cytochromes for U(VI) Reduction by Geobacter Sulfurreducens

BackgroundIn order to study the mechanism of U(VI) reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI) with acetate serving as the electron donor was investigated.ResultsThe ability of several c-type cytochrome deficient mutants to reduce U(VI) was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50–60%) the ability of G. sulfurreducens to reduce U(VI). Involvement in U(VI) reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI) reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI) reduction. A subpopulation of both wild type and U(VI) reduction-impaired cells, 24–30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI) reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III) revealed no correlation between the impact of cytochrome deletion on U(VI) reduction and reduction of Fe(III) hydroxide and chelated Fe(III).ConclusionThis study indicates that c-type cytochromes are involved in U(VI) reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI) reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI) reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium reduction. Periplasmic uranium accumulation may reflect the ability of uranium to penetrate the outer membrane rather than the occurrence of enzymatic U(VI) reduction. Elimination of cytochromes rarely had a similar impact on both Fe(III) and U(VI) reduction, suggesting that there are differences in the routes of electron transfer to U(VI) and Fe(III). Further studies are required to clarify the pathways leading to U(VI) reduction in G. sulfurreducens.

Geobacter sulfurreducens Can Grow with Oxygen as a Terminal Electron Acceptor

ABSTRACT Geobacter sulfurreducens, previously classified as a strict anaerobe, tolerated exposure to atmospheric oxygen for at least 24 h and grew with oxygen as the sole electron acceptor at concentrations of 10% or less in the headspace. These results help explain how Geobacter species may survive in oxic subsurface environments, being poised to rapidly take advantage of the development of anoxic conditions.

Gene expression and deletion analysis of mechanisms for electron transfer from electrodes to Geobacter sulfurreducens

Geobacter sulfurreducens is one of the few microorganisms available in pure culture known to directly accept electrons from a negatively poised electrode. Microarray analysis was used to compare gene transcript abundance in biofilms of G. sulfurreducens using a graphite electrode as the sole electron donor for fumarate reduction compared with transcript abundance in biofilms growing on the same material, but not consuming current. Surprisingly, genes for putative cell-electrode connections, such as outer-surface cytochromes and pili, which are highly expressed in current-producing biofilms, were not highly expressed in current-consuming biofilms. Microarray analysis of G. sulfurreducens gene transcript abundance in current-consuming biofilms versus current-producing biofilms gave similar results. In both comparative studies current-consuming biofilms had greater transcript abundance for a gene (GSU3274) encoding a putative monoheme, c-type cytochrome. Deletion of genes for outer-surface proteins previously shown to be essential for optimal electron transfer to electrodes had no impact on electron transfer from electrodes. Deletion of GSU3274 completely inhibited electron transfer from electrodes, but had no impact on electron transfer to electrodes. These differences in gene expression patterns and the impact of gene deletions suggest that the mechanisms for electron transfer from electrodes to G. sulfurreducens differ significantly from the mechanisms for electron transfer to electrodes.

OmcF, a Putative c-Type Monoheme Outer Membrane Cytochrome Required for the Expression of Other Outer Membrane Cytochromes in Geobacter sulfurreducens

Outer membrane cytochromes are often proposed as likely agents for electron transfer to extracellular electron acceptors, such as Fe(III). The omcF gene in the dissimilatory Fe(III)-reducing microorganism Geobacter sulfurreducens is predicted to code for a small outer membrane monoheme c-type cytochrome. An OmcF-deficient strain was constructed, and its ability to reduce and grow on Fe(III) citrate was found to be impaired. Following a prolonged lag phase (150 h), the OmcF-deficient strain developed the ability to grow in Fe(III) citrate medium with doubling times and yields that were ca. 145% and 70% of those of the wild type, respectively. Comparison of the c-type cytochrome contents of outer membrane-enriched fractions prepared from wild-type and OmcF-deficient cultures confirmed the outer membrane association of OmcF and revealed multiple changes in the cytochrome content of the OmcF-deficient strain. These changes included loss of expression of two previously characterized outer membrane cytochromes, OmcB and OmcC, and overexpression of a third previously characterized outer membrane cytochrome, OmcS, during growth on Fe(III) citrate. The omcB and omcC transcripts could not be detected in the OmcF-deficient mutant by either reverse transcriptase PCR or Northern blot analyses. Expression of the omcF gene in trans restored both the capacity of the OmcF-deficient mutant to reduce Fe(III) and wild-type levels of omcB and omcC mRNA and protein. Thus, elimination of OmcF may impair Fe(III) reduction by influencing expression of OmcB, which has previously been demonstrated to play a critical role in Fe(III) reduction.

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).