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Anaerobic degradation of xenobiotic isophthalate by the fermenting bacterium Syntrophorhabdus aromaticivorans.

ABSTRACT: Syntrophorhabdus aromaticivorans is a syntrophically fermenting bacterium that can degrade isophthalate (3-carboxybenzoate). It is a xenobiotic compound which has accumulated in the environment for more than 50 years due to its global industrial usage and can cause negative effects on the environment. Isophthalate degradation by the strictly anaerobic S. aromaticivorans was investigated to advance our understanding of the degradation of xenobiotics introduced into nature, and to identify enzymes that might have ecological significance for bioremediation. Differential proteome analysis of isophthalate- vs benzoate-grown cells revealed over 400 differentially expressed proteins of which only four were unique to isophthalate-grown cells. The isophthalate-induced proteins include a phenylacetate:CoA ligase, a UbiD-like decarboxylase, a UbiX-like flavin prenyltransferase, and a hypothetical protein. These proteins are encoded by genes forming a single gene cluster that putatively codes for anaerobic conversion of isophthalate to benzoyl-CoA. Subsequently, benzoyl-CoA is metabolized by the enzymes of the anaerobic benzoate degradation pathway that were identified in the proteomic analysis. In vitro enzyme assays with cell-free extracts of isophthalate-grown cells indicated that isophthalate is activated to isophthalyl-CoA by an ATP-dependent isophthalate:CoA ligase (IPCL), and subsequently decarboxylated to benzoyl-CoA by a UbiD family isophthalyl-CoA decarboxylase (IPCD) that requires a prenylated flavin mononucleotide (prFMN) cofactor supplied by UbiX to effect decarboxylation. Phylogenetic analysis revealed that IPCD is a novel member of the functionally diverse UbiD family (de)carboxylases. Homologs of the IPCD encoding genes are found in several other bacteria, such as aromatic compound-degrading denitrifiers, marine sulfate-reducers, and methanogenic communities in a terephthalate-degrading reactor. These results suggest that metabolic strategies adapted for degradation of isophthalate and other phthalate are conserved between microorganisms that are involved in the anaerobic degradation of environmentally relevant aromatic compounds.

SUBMITTER: Junghare M

PROVIDER: S-EPMC6474229 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe sulfate-reducing bacterium Desulfatiglans anilini can grow with phenol as sole source of carbon and energy under strictly anaerobic, sulfate-reducing conditions. In the nitrate-reducing bacterium Thauera aromatica, the enzymes involved in phenol degradation have been well elucidated, whereas the anaerobic phenol degradation pathway by D. anilini was not studied in detail yet.ResultsThe pathway of anaerobic phenol degradation by the sulfate-reducing bacterium Desulfatiglans anilini was studied by identification of genes coding for phenylphosphate synthase (encoded by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) in the genome of D. anilini, by analysis of the transcription and translation of pps-ppc genes, and by measurement of phenylphosphate synthase activity in cell-free extracts of phenol-grown cells. The majority of genes involved in phenol degradation were found to be organized in one gene cluster. The gene cluster contained genes ppsα (phenylphosphate synthase alpha subunit), ppsβ (phenylphosphate synthase beta subunit), ppcβ (phenylphosphate carboxylase beta subunit), as well as 4-hydroxybenzoyl-CoA ligase and 4-hydroxylbenzoyl-CoA reductase-encoding genes. The genes ppsγ (phenylphosphate synthase gamma subunit), ppcα (phenylphosphate carboxylase alpha subunit) and ppcδ (phenylphosphate carboxylase delta subunit) were located elsewhere in the genome of D. anilini, and no obvious homologue of ppcγ (phenylphosphate carboxylase gamma subunit) was found in the genome. Induction of genes pps and ppc during growth on phenol was confirmed by reverse transcription polymerase chain reaction. Total proteome analysis revealed that the abundance of enzymes encoded by the gene cluster under study was much higher in phenol-grown cells than that in benzoate-grown cells. In in-vitro enzyme assays with cell-free extracts of phenol-grown cells, phenylphosphate was formed from phenol in the presence of ATP, Mg2+, Mn2+, K+ as co-factors.ConclusionsThe genes coding for enzymes involved in the anaerobic phenol degradation pathway were identified in the sulfate-reducing bacterium D. anilini. The results indicate that the first steps of anaerobic phenol degradation in D. anilini are phosphorylation of phenol to phenylphosphate by phenylphosphate synthase and carboxylation of phenylphosphate by phenylphosphate carboxylase.

Project description:Increasing the resistance of plant-fermenting bacteria to lignocellulosic inhibitors is useful to understand microbial adaptation and to develop candidate strains for consolidated bioprocessing. Here, we study and improve inhibitor resistance in Clostridium phytofermentans (also called Lachnoclostridium phytofermentans), a model anaerobe that ferments lignocellulosic biomass. We survey the resistance of this bacterium to a panel of biomass inhibitors and then evolve strains that grow in increasing concentrations of the lignin phenolic, ferulic acid, by automated, long-term growth selection in an anaerobic GM3 automat. Ultimately, strains resist multiple inhibitors and grow robustly at the solubility limit of ferulate while retaining the ability to ferment cellulose. We analyze genome-wide transcription patterns during ferulate stress and genomic variants that arose along the ferulate growth selection, revealing how cells adapt to inhibitors through changes in gene dosage and regulation, membrane fatty acid structure, and the surface layer. Collectively, this study demonstrates an automated framework for in vivo directed evolution of anaerobes and gives insight into the genetic mechanisms by which bacteria survive exposure to chemical inhibitors.IMPORTANCE Fermentation of plant biomass is a key part of carbon cycling in diverse ecosystems. Further, industrial biomass fermentation may provide a renewable alternative to fossil fuels. Plants are primarily composed of lignocellulose, a matrix of polysaccharides and polyphenolic lignin. Thus, when microorganisms degrade lignocellulose to access sugars, they also release phenolic and acidic inhibitors. Here, we study how the plant-fermenting bacterium Clostridium phytofermentans resists plant inhibitors using the lignin phenolic, ferulic acid. We examine how the cell responds to abrupt ferulate stress by measuring changes in gene expression. We evolve increasingly resistant strains by automated, long-term cultivation at progressively higher ferulate concentrations and sequence their genomes to identify mutations associated with acquired ferulate resistance. Our study develops an inhibitor-resistant bacterium that ferments cellulose and provides insights into genomic evolution to resist chemical inhibitors.

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Anaerobic degradation of xenobiotic isophthalate by the fermenting bacterium Syntrophorhabdus aromaticivorans.

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