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Differential membrane proteome analysis reveals novel proteins involved in the degradation of aromatic compounds in Geobacter metallireducens.

ABSTRACT: Aromatic compounds comprise a large class of natural and man-made compounds, many of which are of considerable concern for the environment and human health. In aromatic compound-degrading anaerobic bacteria the central intermediate of aromatic catabolism, benzoyl coenzyme A, is attacked by dearomatizing benzoyl-CoA reductases (BCRs). An ATP-dependent BCR has been characterized in facultative anaerobes. In contrast, a previous analysis of the soluble proteome from the obligately anaerobic model organism Geobacter metallireducens identified genes putatively coding for a completely different dearomatizing BCR. The corresponding BamBCDEFGHI complex is predicted to comprise soluble molybdenum or tungsten, selenocysteine, and FeS cluster-containing components. To elucidate key processes involved in the degradation of aromatic compounds in obligately anaerobic bacteria, differential membrane protein abundance levels from G. metallireducens grown on benzoate and acetate were determined by the MS-based spectral counting approach. A total of 931 proteins were identified by combining one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with liquid chromatography-tandem mass spectrometry. Several membrane-associated proteins involved in the degradation of aromatic compounds were newly identified including proteins with similarities to modules of NiFe/heme b-containing and energy-converting hydrogenases, cytochrome bd oxidases, dissimilatory nitrate reductases, and a tungstate ATP-binding cassette transporter system. The transcriptional regulation of differentially expressed genes was analyzed by quantitative reverse transcription-PCR; in addition benzoate-induced in vitro activities of hydrogenase and nitrate reductase were determined. The results obtained provide novel insights into the poorly understood degradation of aromatic compounds in obligately anaerobic bacteria.

SUBMITTER: Heintz D

PROVIDER: S-EPMC2742446 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Differential membrane proteome analysis reveals novel proteins involved in the degradation of aromatic compounds in Geobacter metallireducens.

Heintz Dimitri D Gallien Sébastien S Wischgoll Simon S Ullmann Anja Kerstin AK Schaeffer Christine C Kretzschmar Antje Karen AK van Dorsselaer Alain A Boll Matthias M

Molecular & cellular proteomics : MCP 20090603 9

Aromatic compounds comprise a large class of natural and man-made compounds, many of which are of considerable concern for the environment and human health. In aromatic compound-degrading anaerobic bacteria the central intermediate of aromatic catabolism, benzoyl coenzyme A, is attacked by dearomatizing benzoyl-CoA reductases (BCRs). An ATP-dependent BCR has been characterized in facultative anaerobes. In contrast, a previous analysis of the soluble proteome from the obligately anaerobic model o ...[more]

PMID: 19497847

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Project description:BackgroundGroundwater and subsurface environments contaminated with aromatic compounds can be remediated in situ by Geobacter species that couple oxidation of these compounds to reduction of Fe(III)-oxides. Geobacter metallireducens metabolizes many aromatic compounds, but the enzymes involved are not well known.ResultsThe complete G. metallireducens genome contained a 300 kb island predicted to encode enzymes for the degradation of phenol, p-cresol, 4-hydroxybenzaldehyde, 4-hydroxybenzoate, benzyl alcohol, benzaldehyde, and benzoate. Toluene degradation genes were encoded in a separate region. None of these genes was found in closely related species that cannot degrade aromatic compounds. Abundant transposons and phage-like genes in the island suggest mobility, but nucleotide composition and lack of synteny with other species do not suggest a recent transfer. The inferred degradation pathways are similar to those in species that anaerobically oxidize aromatic compounds with nitrate as an electron acceptor. In these pathways the aromatic compounds are converted to benzoyl-CoA and then to 3-hydroxypimelyl-CoA. However, in G. metallireducens there were no genes for the energetically-expensive dearomatizing enzyme. Whole-genome changes in transcript levels were identified in cells oxidizing benzoate. These supported the predicted pathway, identified induced fatty-acid oxidation genes, and identified an apparent shift in the TCA cycle to a putative ATP-yielding succinyl-CoA synthase. Paralogs to several genes in the pathway were also induced, as were several putative molybdo-proteins. Comparison of the aromatics degradation pathway genes to the genome of an isolate from a contaminated field site showed very similar content, and suggested this strain degrades many of the same compounds. This strain also lacked a classical dearomatizing enzyme, but contained two copies of an eight-gene cluster encoding redox proteins that was 30-fold induced during benzoate oxidation.ConclusionG. metallireducens appears to convert aromatic compounds to benzoyl-CoA, then to acetyl-CoA via fatty acid oxidation, and then to carbon dioxide via the TCA cycle. The enzyme responsible for dearomatizing the aromatic ring may be novel, and energetic investments at this step may be offset by a change in succinate metabolism. Analysis of a field isolate suggests that the pathways inferred for G. metallireducens may be applicable to modeling in situ bioremediation.

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.

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Differential membrane proteome analysis reveals novel proteins involved in the degradation of aromatic compounds in Geobacter metallireducens.

Publications

Differential membrane proteome analysis reveals novel proteins involved in the degradation of aromatic compounds in Geobacter metallireducens.

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