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 µM benzene three separate cultures of G. metallireducens grown with 500 µM phenol three separate cultures of G. metallireducens grown with 1 mM benzoate three separate cultures of G. metallireducens grown with 500 µM 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: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