Kathryn Byerly – Biochemistry & Molecular Genetics

Poster Presentation:

Abstract:

Identification of genes in Rhodosprillum rubrum required for the reduction of volatile organic sulfur compounds in a novel Methionine Salvage Pathway

Background: Sulfur is required for cellular processes. Sulfur uptake by bacteria is done through assimilation of sulfate into cysteine, a precursor to necessary sulfur-containing compounds. Bacteria also rely on sulfur-salvage from organic sulfur compounds via methionine-salvage pathways (MSP). Various methionine and dimethylsulfinopropionate degradation pathways produce volatile organic sulfur compounds (VOSCs) like (2-methylthio)ethanol (MT-EtOH) and dimethyl sulfide (DMS), as does the Dihydroxyacetone Phosphate (DHAP) Shunt pathway. We recently discovered an anaerobic MSP in Rhodospirillum rubrum during which MT-EtOH or DMS are reduced to the methionine precursor, methanethiol, and ethylene or methane, respectively. The goal of this work was to characterize the genes and corresponding enzymes directly responsible for reduction of MT-EtOH and DMS in this novel MSP.
Methods: Chromosomal deletion via homologous recombination and plasmid-based complementation of R. rubrum genes putatively involved in MT-EtOH and DMS metabolism were performed. The requirement of each gene for MT-EtOH and DMS metabolism was assessed based on the ability of the knockouts strains to grow anaerobically in defined minimal media with sulfate, MT-EtOH, or DMS. For strains that showed a growth defect, specific requirements of the genes was verified by restoration of growth upon complementation of the genes via expression from a plasmid. Ethylene and methane production, analyzed via gas chromatography, were used to determine functionality of MT-EtOH and DMS metabolism in the knockouts and complementation.
Results: An operon of four genes was required by R. rubrum to grow on MT-EtOH and DMS as the sulfur-source in anerobic conditions, as well as for the production of ethylene and methane respectively in the novel MSP pathway. Growth was restored only when the entire operon of all four genes was reintroduced in complementation studies. Coordinately, ethylene and methane production were abolished in the knockout strain and restored upon complementation with all four genes.
Conclusion: This novel gene cluster corresponds to a putative enzyme complex that functions as a methylthio-alkane reductase. Phylogenetic comparisons demonstrate these gene products belong to the nitrogenase superfamily. Future studies will isolate and characterize the active methylthio-alkane reductase complex and the precise reaction it performs in VOSC metabolism for methane, ethylene, and methionine biosynthesis.