Project description:Geobacter sulfurreducens is a dissimilatory metal-reducing bacterium capable of forming thick electron-conducting biofilms on solid electrodes in the absence of alternative electron acceptors. The remarkable ability of such biofilms to transfer electrons, liberated from soluble organic electron donors, over long distances has attracted scientific interest as to the mechanism for this process, and technological interest for application to microbial fuel and electrolysis cells and sensors. Here, we employ comparative proteomics to identify key metabolic pathways involved in G. sulfurreducens respiration by planktonic cells versus electron-conducting biofilms, in an effort to elucidate long-range electron transfer mechanisms.
Project description: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 ?M) 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 (18)O during growth in H2(18)O, 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.
Project description:BackgroundGeobacter metallireducens was the first organism that can be grown in pure culture to completely oxidize organic compounds with Fe(III) oxide serving as electron acceptor. Geobacter species, including G. sulfurreducens and G. metallireducens, are used for bioremediation and electricity generation from waste organic matter and renewable biomass. The constraint-based modeling approach enables the development of genome-scale in silico models that can predict the behavior of complex biological systems and their responses to the environments. Such a modeling approach was applied to provide physiological and ecological insights on the metabolism of G. metallireducens.ResultsThe genome-scale metabolic model of G. metallireducens was constructed to include 747 genes and 697 reactions. Compared to the G. sulfurreducens model, the G. metallireducens metabolic model contains 118 unique reactions that reflect many of G. metallireducens' specific metabolic capabilities. Detailed examination of the G. metallireducens model suggests that its central metabolism contains several energy-inefficient reactions that are not present in the G. sulfurreducens model. Experimental biomass yield of G. metallireducens growing on pyruvate was lower than the predicted optimal biomass yield. Microarray data of G. metallireducens growing with benzoate and acetate indicated that genes encoding these energy-inefficient reactions were up-regulated by benzoate. These results suggested that the energy-inefficient reactions were likely turned off during G. metallireducens growth with acetate for optimal biomass yield, but were up-regulated during growth with complex electron donors such as benzoate for rapid energy generation. Furthermore, several computational modeling approaches were applied to accelerate G. metallireducens research. For example, growth of G. metallireducens with different electron donors and electron acceptors were studied using the genome-scale metabolic model, which provided a fast and cost-effective way to understand the metabolism of G. metallireducens.ConclusionWe have developed a genome-scale metabolic model for G. metallireducens that features both metabolic similarities and differences to the published model for its close relative, G. sulfurreducens. Together these metabolic models provide an important resource for improving strategies on bioremediation and bioenergy generation.
Project description:Geobacter metallireducens serves as the model for Geobacter species that anaerobically oxidize aromatic contaminants with the reduction of Fe(III) oxides in contaminated sediments. Analysis of the complete G. metallireducens genome sequence revealed a 307 kb region, designated the aromatics island, not found in closely related species that do not degrade aromatics. This region encoded enzymes for the degradation of benzoate and other aromatic compounds with the exception of the genes for the conversion of toluene to benzyol-CoA which were in a different region of the genome. Predicted aromatic degradation pathways were similar to those described in more well-studied organisms except that no genes encoding a benzoyl-CoA reductase were present. A genome-wide comparison of gene transcript levels during growth on benzoate versus growth on acetate demonstrated that the majority of the most significant increases in transcript levels were among genes within the aromatics island. Of particular interest were highly expressed genes that encode redox proteins of unknown function, one of which had a homolog outside the aromatics island that was also highly expressed. There was also an apparent shift in the acetyl-CoA oxidation pathway to the use of a putative ATP-yielding succinyl-CoA synthase during growth on benzoate. These results provide new insights into the pathways for the degradation of aromatic compounds in G. metallireducens, indicate genes whose role in benzoate metabolism need to be evaluated further, and suggest target genes whose expression may be monitored in order to better assess the degradation of aromatic compounds in contaminated environments. Keywords: Metabolism
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.