Identification of Genes Involved in Biofilm Formation and Respiration via Mini-Himar Transposon Mutagenesis of Geobacter sulfurreducens

Electron transfer from cells to metals and electrodes by the Fe(III)-reducing anaerobe Geobacter sulfurreducens requires proper expression of redox proteins and attachment mechanisms to interface bacteria with surfaces and neighboring cells. We hypothesized that transposon mutagenesis would complement targeted knockout studies in Geobacter spp. and identify novel genes involved in this process. Escherichia coli mating strains and plasmids were used to develop a conjugation protocol and deliver mini-Himar transposons, creating a library of over 8,000 mutants that was anaerobically arrayed and screened for a range of phenotypes, including auxotrophy for amino acids, inability to reduce Fe(III) citrate, and attachment to surfaces. Following protocol validation, mutants with strong phenotypes were further characterized in a three-electrode system to simultaneously quantify attachment, biofilm development, and respiratory parameters, revealing mutants defective in Fe(III) reduction but unaffected in electron transfer to electrodes (such as an insertion in GSU1330, a putative metal export protein) or defective in electrode reduction but demonstrating wild-type biofilm formation (due to an insertion upstream of the NHL domain protein GSU2505). An insertion in a putative ATP-dependent transporter (GSU1501) eliminated electrode colonization but not Fe(III) citrate reduction. A more complex phenotype was demonstrated by a mutant containing an insertion in a transglutaminase domain protein (GSU3361), which suddenly ceased to respire when biofilms reached approximately 50% of the wild-type levels. As most insertions were not in cytochromes but rather in transporters, two-component signaling proteins, and proteins of unknown function, this collection illustrates how biofilm formation and electron transfer are separate but complementary phenotypes, controlled by multiple loci not commonly studied in Geobacter spp.Geobacter sulfurreducens is a member of the metal-reducing Geobacteraceae family and was originally isolated based on its ability to transfer electrons from internal oxidative reactions to extracellular electron acceptors such as insoluble Fe(III) or Mn(IV) oxides (5). G. sulfurreducens is also able to use an electrode as its sole electron acceptor for respiration, a phenotype which has many possible biotechnological applications (28, 29), and serves as a useful tool for direct measurement of electron transfer rates (2, 31). As G. sulfurreducens was the first Geobacteraceae genome sequence available (34) and the only member of this family with a robust genetic system (7), it serves as a model organism for extracellular electron transfer studies.The proteins facilitating electron transfer to insoluble Fe(III) oxides by individual Geobacter cells and how these cells interact in multicellular biofilms are not fully understood. Many genes implicated in Fe(III) and electrode reduction were identified based on proteomic and microarray analysis of cultures grown with fumarate versus Fe(III) citrate as a terminal electron acceptor (9, 15, 35). More recently, similar expression data from Fe(III) oxide and electrode-grown cultures have also become available (8, 12, 16). In most extracellular electron transfer studies, outer membrane proteins (such as c-type cytochromes) have been the focus (4, 23, 27, 32), leading to targeted knockout studies of at least 14 cytochromes to date.To reduce an insoluble electron acceptor, Geobacter spp. must achieve direct contact with the substrate (36). While contact with small Fe(III) oxide particles may be transient, growth on Fe(III)-coated surfaces or electron-accepting electrodes requires biofilm formation (31, 39). For example, when G. sulfurreducens produces an exponentially increasing rate of electron transfer at an electrode, this demonstrates that all newly divided cells remain embedded in the growing, conductive biofilm (2, 31). Thus, in addition to the need for an array of outer membrane cytochromes, there is also a need for control of both cell-cell contact and cell-surface contact.While a genetic system for G. sulfurreducens has been developed, conjugal transfer of a plasmid or a transposon has not been reported (7). The broad-host-range cloning vector pBBR1MCS-2 has previously been electroporated into G. sulfurreducens, but its mobilization capabilities were not utilized (7). Similarly, a number of suicide vectors have been identified for G. sulfurreducens, but none have been used to deliver transposons for mutagenesis. mariner-based transposon mutagenesis systems have been successful in a variety of Bacteria and Archaea, producing random insertions (20, 25, 40, 41, 43, 46, 48, 49). For example, genes involved in Shewanella oneidensis cytochrome maturation were discovered using the modified transposon mini-Himar RB1 (3).In this work, we describe a system for the conjugal transfer of the pBBR1MCS family of plasmids from Escherichia coli to G. sulfurreducens, which allowed transposon mutagenesis based on pMiniHimar RB1. Under strictly anaerobic conditions, a library of insertion mutants was constructed and screened to identify genes putatively involved in attachment and Fe(III) citrate reduction. Approximately 8,000 insertion mutants were isolated, with insertions distributed throughout the G. sulfurreducens chromosome. Subsequent characterization revealed mutants defective in metal reduction but unaffected in all aspects of electrode reduction, as well as mutants able to reduce metals but incapable of electrode reduction. These observations greatly expand the list of Geobacter mutants with defects in respiration or biofilm formation, and this library serves as a resource for further screening of extracellular electron transfer phenotypes.     

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.     

Efficient and precise control of gene expression in Geobacter sulfurreducens through new genetic elements and tools for pollutant conversion

Geobacter species, exhibiting exceptional extracellular electron transfer aptitude, hold great potential for applications in pollution remediation, bioenergy production, and natural elemental cycles. Nonetheless, a scarcity of well-characterized genetic elements and gene expression tools constrains the effective and precise fine-tuning of gene expression in Geobacter species, thereby limiting their applications. Here, we examined a suite of genetic elements and developed a new genetic editing tool in Geobacter sulfurreducens to enhance their pollutant conversion capacity. First, the performances of the widely used inducible promoters, constitutive promoters, and ribosomal binding sites (RBSs) elements in G. sulfurreducens were quantitatively evaluated. Also, six native promoters with superior expression levels than constitutive promoters were identified on the genome of G. sulfurreducens. Employing the characterized genetic elements, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system was constructed in G. sulfurreducens to achieve the repression of an essential gene-aroK and morphogenic genes-ftsZ and mreB. Finally, applying the engineered strain to the reduction of tungsten trioxide (WO₃), methyl orange (MO), and Cr(VI), We found that morphological elongation through ftsZ repression amplified the extracellular electron transfer proficiency of G. sulfurreducens and facilitated its contaminant transformation efficiency. These new systems provide rapid, versatile, and scalable tools poised to expedite advancements in Geobacter genomic engineering to favor environmental and other biotechnological applications.     

A Geobacter sulfurreducens Strain Expressing Pseudomonas aeruginosa Type IV Pili Localizes OmcS on Pili but Is Deficient in Fe(III) Oxide Reduction and Current Production

The conductive pili of Geobacter species play an important role in electron transfer to Fe(III) oxides, in long-range electron transport through current-producing biofilms, and in direct interspecies electron transfer. Although multiple lines of evidence have indicated that the pili of Geobacter sulfurreducens have a metal-like conductivity, independent of the presence of c-type cytochromes, this claim is still controversial. In order to further investigate this phenomenon, a strain of G. sulfurreducens, designated strain PA, was constructed in which the gene for the native PilA, the structural pilin protein, was replaced with the PilA gene of Pseudomonas aeruginosa PAO1. Strain PA expressed and properly assembled P. aeruginosa PilA subunits into pili and exhibited a profile of outer surface c-type cytochromes similar to that of a control strain expressing the G. sulfurreducens PilA. Surprisingly, the strain PA pili were decorated with the c-type cytochrome OmcS in a manner similar to the control strain. However, the strain PA pili were 14-fold less conductive than the pili of the control strain, and strain PA was severely impaired in Fe(III) oxide reduction and current production. These results demonstrate that the presence of OmcS on pili is not sufficient to confer conductivity to pili and suggest that there are unique structural features of the G. sulfurreducens PilA that are necessary for conductivity.     

A novel <Emphasis Type="Italic">Geobacteraceae</Emphasis>-specific outer membrane protein J (OmpJ) is essential for electron transport to Fe (III) and Mn (IV) oxides in <Emphasis Type="Italic">Geobacter sulfurreducens</Emphasis>

Background  

Metal reduction is thought to take place at or near the bacterial outer membrane and, thus, outer membrane proteins in the model dissimilatory metal-reducing organism Geobacter sulfurreducens are of interest to understand the mechanisms of Fe(III) reduction in the Geobacter species that are the predominant Fe(III) reducers in many environments. Previous studies have implicated periplasmic and outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer membrane protein of G. sulfurreducens, OmpJ, is not a cytochrome yet it is required for metal respiration.     

Identification of an uptake hydrogenase required for hydrogen-dependent reduction of Fe(III) and other electron acceptors by Geobacter sulfurreducens

Geobacter sulfurreducens, a representative of the family Geobacteraceae that predominates in Fe(III)-reducing subsurface environments, can grow by coupling the oxidation of hydrogen to the reduction of a variety of electron acceptors, including Fe(III), fumarate, and quinones. An examination of the G. sulfurreducens genome revealed two operons, hya and hyb, which appeared to encode periplasmically oriented respiratory uptake hydrogenases. In order to assess the roles of these two enzymes in hydrogen-dependent growth, Hya- and Hyb-deficient mutants were generated by gene replacement. Hyb was found to be required for hydrogen-dependent reduction of Fe(III), anthraquinone-2,6-disulfonate, and fumarate by resting cell suspensions and to be essential for growth with hydrogen and these three electron acceptors. Hya, in contrast, was not. These findings suggest that Hyb is an essential respiratory hydrogenase in G. sulfurreducens.     

Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation

Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.     

Anaerobic Benzene Oxidation by Geobacter Species

The abundance of Geobacter species in contaminated aquifers in which benzene is anaerobically degraded has led to the suggestion that some Geobacter species might be capable of anaerobic benzene degradation, but this has never been documented. A strain of Geobacter, designated strain Ben, was isolated from sediments from the Fe(III)-reducing zone of a petroleum-contaminated aquifer in which there was significant capacity for anaerobic benzene oxidation. Strain Ben grew in a medium with benzene as the sole electron donor and Fe(III) oxide as the sole electron acceptor. Furthermore, additional evaluation of Geobacter metallireducens demonstrated that it could also grow in benzene-Fe(III) medium. In both strain Ben and G. metallireducens the stoichiometry of benzene metabolism and Fe(III) reduction was consistent with the oxidation of benzene to carbon dioxide with Fe(III) serving as the sole electron acceptor. With benzene as the electron donor, and Fe(III) oxide (strain Ben) or Fe(III) citrate (G. metallireducens) as the electron acceptor, the cell yields of strain Ben and G. metallireducens were 3.2 × 10⁹ and 8.4 × 10⁹ cells/mmol of Fe(III) reduced, respectively. Strain Ben also oxidized benzene with anthraquinone-2,6-disulfonate (AQDS) as the sole electron acceptor with cell yields of 5.9 × 10⁹ cells/mmol of AQDS reduced. Strain Ben serves as model organism for the study of anaerobic benzene metabolism in petroleum-contaminated aquifers, and G. metallireducens is the first anaerobic benzene-degrading organism that can be genetically manipulated.     

Direct and Fe(II)-Mediated Reduction of Technetium by Fe(III)-Reducing Bacteria

The dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens reduced and precipitated Tc(VII) by two mechanisms. Washed cell suspensions coupled the oxidation of hydrogen to enzymatic reduction of Tc(VII) to Tc(IV), leading to the precipitation of TcO₂ at the periphery of the cell. An indirect, Fe(II)-mediated mechanism was also identified. Acetate, although not utilized efficiently as an electron donor for direct cell-mediated reduction of technetium, supported the reduction of Fe(III), and the Fe(II) formed was able to transfer electrons abiotically to Tc(VII). Tc(VII) reduction was comparatively inefficient via this indirect mechanism when soluble Fe(III) citrate was supplied to the cultures but was enhanced in the presence of solid Fe(III) oxide. The rate of Tc(VII) reduction was optimal, however, when Fe(III) oxide reduction was stimulated by the addition of the humic analog and electron shuttle anthaquinone-2,6-disulfonate, leading to the rapid formation of the Fe(II)-bearing mineral magnetite. Under these conditions, Tc(VII) was reduced and precipitated abiotically on the nanocrystals of biogenic magnetite as TcO₂ and was removed from solution to concentrations below the limit of detection by scintillation counting. Cultures of Fe(III)-reducing bacteria enriched from radionuclide-contaminated sediment using Fe(III) oxide as an electron acceptor in the presence of 25 μM Tc(VII) contained a single Geobacter sp. detected by 16S ribosomal DNA analysis and were also able to reduce and precipitate the radionuclide via biogenic magnetite. Fe(III) reduction was stimulated in aquifer material, resulting in the formation of Fe(II)-containing minerals that were able to reduce and precipitate Tc(VII). These results suggest that Fe(III)-reducing bacteria may play an important role in immobilizing technetium in sediments via direct and indirect mechanisms.     

Going Wireless: Fe(III) Oxide Reduction without Pili by Geobacter sulfurreducens Strain JS-1

Previous studies have suggested that the conductive pili of Geobacter sulfurreducens are essential for extracellular electron transfer to Fe(III) oxides and for optimal long-range electron transport through current-producing biofilms. The KN400 strain of G. sulfurreducens reduces poorly crystalline Fe(III) oxide more rapidly than the more extensively studied DL-1 strain. Deletion of the gene encoding PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, low rates of Fe(III) reduction were detected after extended incubation (>30 days) in the presence of Fe(III) oxide. After seven consecutive transfers, the PilA-deficient strain adapted to reduce Fe(III) oxide as fast as the wild type. Microarray, whole-genome resequencing, proteomic, and gene deletion studies indicated that this adaptation was associated with the production of larger amounts of the c-type cytochrome PgcA, which was released into the culture medium. It is proposed that the extracellular cytochrome acts as an electron shuttle, promoting electron transfer from the outer cell surface to Fe(III) oxides. The adapted PilA-deficient strain competed well with the wild-type strain when both were grown together on Fe(III) oxide. However, when 50% of the culture medium was replaced with fresh medium every 3 days, the wild-type strain outcompeted the adapted strain. A possible explanation for this is that the necessity to produce additional PgcA, to replace the PgcA being continually removed, put the adapted strain at a competitive disadvantage, similar to the apparent selection against electron shuttle-producing Fe(III) reducers in many anaerobic soils and sediments. Despite increased extracellular cytochrome production, the adapted PilA-deficient strain produced low levels of current, consistent with the concept that long-range electron transport through G. sulfurreducens biofilms is more effective via pili.     

Molecular evidence for the adaptive evolution of Geobacter sulfurreducens to perform dissimilatory iron reduction in natural environments

The electrically conductive pili (e-pili) of Geobacter species enable extracellular electron transfer to insoluble metallic minerals, electrodes and other microbial species, which confers biogeochemical significance and global prevalence on Geobacter in diverse anaerobic environments. E-pili are constructed by truncated PilA which is considered to have evolved from full-length pilin by gene fission under positive evolutionary selection. However, this hypothesis is based on phylogenetic analysis and has not yet been experimentally confirmed. Here, we reconstructed an ancestral strain of G. sulfurreducens (designated COMB) carrying full-length PilA by combining genes GSU1496 and GSU1497. The results demonstrated that strain COMB expressed and assembled the full-length fused PilA and exhibited an outer membrane c-type cytochrome profile similar to the wild-type strain. Surprisingly, the generated COMB-pili were also conductive, indicating the evolution of truncated PilA did not occur for conductivity. Moreover, strain COMB minimally reduced Fe(III) oxides but maintained its ability to respire electrodes, demonstrating the truncation of pilin enables iron respiration. This study provides the first experimental evidence that the truncation of pilin in Geobacter species confers adaption to Fe(III)-mineral-mediated selective pressures, and suggests an evolutionary event during which the separation of the GSU1497 gene helped Geobacter survive and thrive in natural environments.     

Effect of gonococcal lipooligosaccharide variation on human monocytic cytokine profile

Background

In 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.

Results

The 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).

Conclusion

This 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.     

Comparative proteomics of Geobacter sulfurreducens PCAT in response to acetate,formate and/or hydrogen as electron donor

Geobacter sulfurreducens is a model bacterium to study the degradation of organic compounds coupled to the reduction of Fe(III). The response of G. sulfurreducens to the electron donors acetate, formate, hydrogen and a mixture of all three with Fe(III) citrate as electron acceptor was studied using comparative physiological and proteomic approaches. Variations in the supplied electron donors resulted in differential abundance of proteins involved in the citric acid cycle (CAC), gluconeogenesis, electron transport, and hydrogenases and formate dehydrogenase. Our results provided new insights into the electron donor metabolism of G. sulfurreducens. Remarkably, formate was the preferred electron donor compared to acetate, hydrogen, or acetate plus hydrogen. When hydrogen was the electron donor, formate was formed, which was associated with a high abundance of formate dehydrogenase. Notably, abundant proteins of two CO₂ fixation pathways (acetyl-CoA pathway and the reversed oxidative CAC) corroborated chemolithoautotrophic growth of G. sulfurreducens with formate or hydrogen and CO₂, and provided novel insight into chemolithoautotrophic growth of G. sulfurreducens.     

A Periplasmic and Extracellular c-Type Cytochrome of Geobacter sulfurreducens Acts as a Ferric Iron Reductase and as an Electron Carrier to Other Acceptors or to Partner Bacteria

An extracellular electron carrier excreted into the growth medium by cells of Geobacter sulfurreducens was identified as a c-type cytochrome. The cytochrome was found to be distributed in about equal amounts in the membrane fraction, the periplasmic space, and the surrounding medium during all phases of growth with acetate plus fumarate. It was isolated from periplasmic preparations and purified to homogeneity by cation-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. The electrophoretically homogeneous cytochrome had a molecular mass of 9.57 ± 0.02 kDa and exhibited in its reduced state absorption maxima at wavelengths of 552, 522, and 419 nm. The midpoint redox potential determined by redox titration was −0.167 V. With respect to molecular mass, redox properties, and molecular features, this cytochrome exhibited its highest similarity to the cytochromes c of Desulfovibrio salexigens and Desulfuromonas acetoxidans. The G. sulfurreducens cytochrome c reduced ferrihydrite (Fe(OH)₃), Fe(III) nitrilotriacetic acid, Fe(III) citrate, and manganese dioxide at high rates. Elemental sulfur, anthraquinone disulfonate, and humic acids were reduced more slowly. G. sulfurreducens reduced the cytochrome with acetate as an electron donor and oxidized it with fumarate. Wolinella succinogenes was able to reduce externally provided cytochrome c of G. sulfurreducens with molecular hydrogen or formate as an electron donor and oxidized it with fumarate or nitrate as an electron acceptor. A coculture could be established in which G. sulfurreducens reduced the cytochrome with acetate, and the reduced cytochrome was reoxidized by W. succinogenes in the presence of nitrate. We conclude that this cytochrome can act as iron(III) reductase for electron transfer to insoluble iron hydroxides or to sulfur, manganese dioxide, or other oxidized compounds, and it can transfer electrons to partner bacteria.     

Ferrihydrite reduction by Geobacter species is stimulated by secondary bacteria

Geobacter species such as G. bremensis, G. pelophilus, and G. sulfurreducens are obligately anaerobic and grow in anoxic, non-reduced medium by fast reduction of soluble ferric citrate. In contrast, insoluble ferrihydrite was either only slowly or not reduced when supplied as electron acceptor in similar growth experiments. Ferrihydrite reduction was stimulated by addition of a reducing agent or by concomitant growth of secondary bacteria that were physiologically and phylogenetically as diverse as Escherichia coli, Lactococcus lactis, or Pseudomonas stutzeri. In control experiments with heat-inactivated Geobacter cells and viable secondary bacteria, no (E. coli, P. stutzeri) or only little (L. lactis) ferrihydrite was reduced. Redox indicator dyes showed that growing E. coli, P. stutzeri, or L. lactis cells lowered the redox potential of the medium in a similar way as a reducing agent did. The lowered redox potential was presumably the key factor that stimulated ferrihydrite reduction by all three Geobacter species. The observed differences in anoxic non-reduced medium with ferric citrate versus ferrihydrite as electron acceptor indicated that reduction of these electron acceptors involved different cellular components or different biochemical strategies. Furthermore, it appears that redox-sensitive components are involved, and/or that gene expression of components needed for ferrihydrite reduction is controlled by the redox state.Dedicated to Prof. Dr. Dr. h.c. mult. Hans Günter Schlegel on the occasion of his 80th birthday.     

Anaerobic Benzene Oxidation via Phenol in Geobacter metallireducens

Anaerobic activation of benzene is expected to represent a novel biochemistry of environmental significance. Therefore, benzene metabolism was investigated in Geobacter metallireducens, the only genetically tractable organism known to anaerobically degrade benzene. Trace amounts (<0.5 μM) of phenol accumulated in cultures of Geobacter metallireducens anaerobically oxidizing benzene to carbon dioxide with the reduction of Fe(III). Phenol was not detected in cell-free controls or in Fe(II)- and benzene-containing cultures of Geobacter sulfurreducens, a Geobacter species that cannot metabolize benzene. The phenol produced in G. metallireducens cultures was labeled with ¹⁸O during growth in H₂¹⁸O, as expected for anaerobic conversion of benzene to phenol. Analysis of whole-genome gene expression patterns indicated that genes for phenol metabolism were upregulated during growth on benzene but that genes for benzoate or toluene metabolism were not, further suggesting that phenol was an intermediate in benzene metabolism. Deletion of the genes for PpsA or PpcB, subunits of two enzymes specifically required for the metabolism of phenol, removed the capacity for benzene metabolism. These results demonstrate that benzene hydroxylation to phenol is an alternative to carboxylation for anaerobic benzene activation and suggest that this may be an important metabolic route for benzene removal in petroleum-contaminated groundwaters, in which Geobacter species are considered to play an important role in anaerobic benzene degradation.     

Lack of Electricity Production by Pelobacter carbinolicus Indicates that the Capacity for Fe(III) Oxide Reduction Does Not Necessarily Confer Electron Transfer Ability to Fuel Cell Anodes

The ability of Pelobacter carbinolicus to oxidize electron donors with electron transfer to the anodes of microbial fuel cells was evaluated because microorganisms closely related to Pelobacter species are generally abundant on the anodes of microbial fuel cells harvesting electricity from aquatic sediments. P. carbinolicus could not produce current in a microbial fuel cell with electron donors which support Fe(III) oxide reduction by this organism. Current was produced using a coculture of P. carbinolicus and Geobacter sulfurreducens with ethanol as the fuel. Ethanol consumption was associated with the transitory accumulation of acetate and hydrogen. G. sulfurreducens alone could not metabolize ethanol, suggesting that P. carbinolicus grew in the fuel cell by converting ethanol to hydrogen and acetate, which G. sulfurreducens oxidized with electron transfer to the anode. Up to 83% of the electrons available in ethanol were recovered as electricity and in the metabolic intermediate acetate. Hydrogen consumption by G. sulfurreducens was important for ethanol metabolism by P. carbinolicus. Confocal microscopy and analysis of 16S rRNA genes revealed that half of the cells growing on the anode surface were P. carbinolicus, but there was a nearly equal number of planktonic cells of P. carbinolicus. In contrast, G. sulfurreducens was primarily attached to the anode. P. carbinolicus represents the first Fe(III) oxide-reducing microorganism found to be unable to produce current in a microbial fuel cell, providing the first suggestion that the mechanisms for extracellular electron transfer to Fe(III) oxides and fuel cell anodes may be different.