Project description:The ability of Geobacter species to readily donate electrons to extracellular electron acceptors makes the study of their physiology not only important for the understanding of environmental processes, but also for industrial applications such as bioelectronics and electrosynthesis. Studies in G. sulfurreducens have shown that outer surface components, such as c-type cytochromes and conductive type IV pili play an important role in direct electron transfer to extracellular electron acceptors such as Fe(III) oxides and electrodes. However, many of these thoroughly studied outer surface components, including c-type cytochromes, are not well conserved among Geobacter species. In order to better understand which components are involved in extracellular electron transfer in Geobacter species other than G. sulfurreducens, studies were conducted with its close relative G. metallireducens. Whole-genome microarray analysis revealed that 23 of the 91 putative c-type cytochromes encoded in the G. metallireducens genome were upregulated at least 2-fold in cells grown with Fe(III) oxide compared to cells in which Fe(III) citrate was provided as the terminal electron acceptor. Protein identification with liquid-chromatography/mass spectrometry detected 6 c-type cytochromes that were more abundant in the outer surface cell fraction of cells that were grown with Fe(III) oxide as the terminal electron acceptor compared to cells grown on Fe(III) citrate. 22 genes encoding c-type cytochromes were chosen for gene deletion. Deletion of 6 genes encoding for c-type cytochromes, a gene encoding for a lipopolysaccharide biosynthesis-associated protein, and a gene encoding for a NHL- repeat containing protein inhibited growth when Fe(III) oxide was provided as the electron acceptor. This study suggests that there are different roads for extracellular electron transfer in Geobacteraceae since homologous c-type cytochromes have different functions from one species to the other, and novel components not previously found to be essential for extracellular electron transfer were identified. An eight-chip study using total RNA recovered from four separate cultures of Geobacter metallireducens GS-15 grown with acetate (10mM)-Fe(III) oxide (100 mmol l-1) (experimental condition) or with acetate (10 mM)-Fe(III) citrate (55mM) (control condition) during exponential growth. Each chip measures the expression level of 3,627 genes from Geobacter metallireducens GS-15 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

Project description:The conductive pili of Geobacter sulfurreducens are essential for optimal extracellular electron transfer to Fe(III) and 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 for PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, slow rates of Fe(III) reduction were detected after extended (> 30 days) incubation 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, proteomic, and gene deletion studies indicated that this adaptation was associated with greater production 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 three days, the wild-type strain out-competed the adapted strain. A possible explanation for this is that the necessity to produce additional PgcA, to replace the PgcA continually removed, put the adapted strain at a competitive disadvantage, similar to the apparent selection against electron-shuttling producing Fe(III) reducers in most 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 cannot be achieved without PilA-pili. An eight-chip study using total RNA recovered from four separate cultures of Geobacter sulfurreducens JS-1 (experimental condition) or Geobacter sulfurreducens KN400 (control condition) grown with acetate (10mM)-Fe(III) oxide (100 mmol l-1) exponential growth. Each chip measures the expression level of 3,328 genes from Geobacter sulfurreducens KN400 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

Project description:Anaerobic activation of benzene is expected to represent a novel biochemistry of environmental significance but research into the mechanisms has been stymied by a lack of a genetically tractable pure culture which unequivocally does not use molecular oxygen to activate benzene. Geobacter metallireducens grew in a medium in which benzene was the sole electron donor and Fe(III) was the sole electron acceptor with a stoichiometry of benzene loss and Fe(III) reduction consistent with benzene oxidation to carbon dioxide coupled with Fe(III) reduction. Phenol labeled with 18O was produced when the medium was labeled with H218O, as expected for a true anaerobic conversion of benzene to phenol. Gene expression patterns indicated that benzene was metabolized through a phenol intermediate rather than benzoate or toluene. Deletion of ppcB, which encodes a subunit of the phenylphosphate carboxylase, an enzyme required for phenol metabolism, inhibited metabolism of benzene. Deleting genes specific for benzoate or toluene metabolism did not. Comparison of gene expression patterns in cells grown on benzene versus cells grown on phenol revealed genes specifically expressed in benzene-grown cells. Deletion of one of these, Gmet_3376, inhibited anaerobic benzene oxidation, but not the metabolism of phenol, benzoate, or toluene. The availability of a genetically tractable pure culture that can anaerobically convert benzene to phenol with oxygen derived from water should significantly accelerate elucidation of the mechanisms by which benzene can be activated in the absence of molecular oxygen. Total RNA from three separate cultures of G. metallireducens grown with 250 ÂµM benzene three separate cultures of G. metallireducens grown with 500 ÂµM phenol three separate cultures of G. metallireducens grown with 1 mM benzoate three separate cultures of G. metallireducens grown with 500 ÂµM toluene three separate cultures of G. metallireducens grown with 10 mM acetate were used to study [1] Anaerobic oxidation of benzene by G. metallireducens (Benzene vs. acetate, Benzene vs. benzoate, Benzene vs. phenol, Benzene vs. toluene) [2] Anaerobic oxidation of benzoate by G. metallireducens (Benzoate vs. acetate) [3] Anaerobic oxidation of phenol by G. metallireducens (Phenol vs. acetate) [4] Anaerobic oxidation of toluene by G. metallireducens (Toluene vs. acetate) Each chip measures the expression level of 3,627 genes from G. metallireducens DSM 7210 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

Project description:Anaerobic benzene oxidation coupled to the reduction of Fe(III) was studied in Ferroglobus placidus in order to learn more about how such a stable molecule could be metabolized under strict anaerobic conditions. F. placidus conserved energy to support growth at 85°C in a medium with benzene provided as the sole electron donor and Fe(III) as the sole electron acceptor. The stoichiometry of benzene loss and Fe(III) reduction, as well as the conversion of [14C]-benzene to [14C]-carbon dioxide, was consistent with complete oxidation of benzene to carbon dioxide with electron transfer to Fe(III). Benzoate, but not phenol or toluene, accumulated at low levels during benzene metabolism and [14C]-benzoate was produced from [14C]-benzene. Analysis of gene transcript levels revealed increased expression of genes encoding enzymes for anaerobic benzoate degradation during growth on benzene versus growth on acetate, but genes involved in phenol degradation were not up-regulated during growth on benzene. A gene for a putative carboxylase that was more highly expressed in benzene- versus benzoate-grown cells was identified. These results suggest that benzene is carboxylated to benzoate and that phenol is not an important intermediate in the benzene metabolism of F. placidus. This is the first demonstration of a microorganism in pure culture that can grow on benzene under strict anaerobic conditions and for which there is strong evidence for degradation of benzene via clearly defined anaerobic metabolic pathways. Thus, F. placidus provides a much needed pure culture model for further studies on the anaerobic activation of benzene in microorganisms. A five-chip study using total RNA recovered from three separate cultures of Ferroglobus placidus DSM 10642 grown with 0.5 mM phenol (experimental condition) and two separate cultures of Ferroglobus placidus DSM 10642 grown on 1 mM benzoate (control condition). Each chip measures the expression level of 2,613 genes from Ferroglobus placidus DSM 10642 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

Project description:Anaerobic benzene oxidation coupled to the reduction of Fe(III) was studied in Ferroglobus placidus in order to learn more about how such a stable molecule could be metabolized under strict anaerobic conditions. F. placidus conserved energy to support growth at 85°C in a medium with benzene provided as the sole electron donor and Fe(III) as the sole electron acceptor. The stoichiometry of benzene loss and Fe(III) reduction, as well as the conversion of [14C]-benzene to [14C]-carbon dioxide, was consistent with complete oxidation of benzene to carbon dioxide with electron transfer to Fe(III). Benzoate, but not phenol or toluene, accumulated at low levels during benzene metabolism and [14C]-benzoate was produced from [14C]-benzene. Analysis of gene transcript levels revealed increased expression of genes encoding enzymes for anaerobic benzoate degradation during growth on benzene versus growth on acetate, but genes involved in phenol degradation were not up-regulated during growth on benzene. A gene for a putative carboxylase that was more highly expressed in benzene- versus benzoate-grown cells was identified. These results suggest that benzene is carboxylated to benzoate and that phenol is not an important intermediate in the benzene metabolism of F. placidus. This is the first demonstration of a microorganism in pure culture that can grow on benzene under strict anaerobic conditions and for which there is strong evidence for degradation of benzene via clearly defined anaerobic metabolic pathways. Thus, F. placidus provides a much needed pure culture model for further studies on the anaerobic activation of benzene in microorganisms. A six-chip study using total RNA recovered from three separate cultures of Ferroglobus placidus DSM 10642 grown with 1 mM benzene (experimental condition) and three separate cultures of Ferroglobus placidus DSM 10642 grown on 0.5 mM phenol (control condition). Each chip measures the expression level of 2,613 genes from Ferroglobus placidus DSM 10642 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

Project description:Anaerobic benzene oxidation coupled to the reduction of Fe(III) was studied in Ferroglobus placidus in order to learn more about how such a stable molecule could be metabolized under strict anaerobic conditions. F. placidus conserved energy to support growth at 85°C in a medium with benzene provided as the sole electron donor and Fe(III) as the sole electron acceptor. The stoichiometry of benzene loss and Fe(III) reduction, as well as the conversion of [14C]-benzene to [14C]-carbon dioxide, was consistent with complete oxidation of benzene to carbon dioxide with electron transfer to Fe(III). Benzoate, but not phenol or toluene, accumulated at low levels during benzene metabolism and [14C]-benzoate was produced from [14C]-benzene. Analysis of gene transcript levels revealed increased expression of genes encoding enzymes for anaerobic benzoate degradation during growth on benzene versus growth on acetate, but genes involved in phenol degradation were not up-regulated during growth on benzene. A gene for a putative carboxylase that was more highly expressed in benzene- versus benzoate-grown cells was identified. These results suggest that benzene is carboxylated to benzoate and that phenol is not an important intermediate in the benzene metabolism of F. placidus. This is the first demonstration of a microorganism in pure culture that can grow on benzene under strict anaerobic conditions and for which there is strong evidence for degradation of benzene via clearly defined anaerobic metabolic pathways. Thus, F. placidus provides a much needed pure culture model for further studies on the anaerobic activation of benzene in microorganisms. A six-chip study using total RNA recovered from three separate cultures of Ferroglobus placidus DSM 10642 grown with 1 mM benzene (experimental condition) and three separate cultures of Ferroglobus placidus DSM 10642 grown on 10 mM acetate (control condition). Each chip measures the expression level of 2,613 genes from Ferroglobus placidus DSM 10642 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

Project description:Anaerobic benzene oxidation coupled to the reduction of Fe(III) was studied in Ferroglobus placidus in order to learn more about how such a stable molecule could be metabolized under strict anaerobic conditions. F. placidus conserved energy to support growth at 85°C in a medium with benzene provided as the sole electron donor and Fe(III) as the sole electron acceptor. The stoichiometry of benzene loss and Fe(III) reduction, as well as the conversion of [14C]-benzene to [14C]-carbon dioxide, was consistent with complete oxidation of benzene to carbon dioxide with electron transfer to Fe(III). Benzoate, but not phenol or toluene, accumulated at low levels during benzene metabolism and [14C]-benzoate was produced from [14C]-benzene. Analysis of gene transcript levels revealed increased expression of genes encoding enzymes for anaerobic benzoate degradation during growth on benzene versus growth on acetate, but genes involved in phenol degradation were not up-regulated during growth on benzene. A gene for a putative carboxylase that was more highly expressed in benzene- versus benzoate-grown cells was identified. These results suggest that benzene is carboxylated to benzoate and that phenol is not an important intermediate in the benzene metabolism of F. placidus. This is the first demonstration of a microorganism in pure culture that can grow on benzene under strict anaerobic conditions and for which there is strong evidence for degradation of benzene via clearly defined anaerobic metabolic pathways. Thus, F. placidus provides a much needed pure culture model for further studies on the anaerobic activation of benzene in microorganisms. A five-chip study using total RNA recovered from three separate cultures of Ferroglobus placidus DSM 10642 grown with 1 mM benzene (experimental condition) and two separate cultures of Ferroglobus placidus DSM 10642 grown on 1 mM benzoate (control condition). Each chip measures the expression level of 2,613 genes from Ferroglobus placidus DSM 10642 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

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FCV-CNRS Aerobic-Anaerobic A. ferrooxidans

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