Development of a Genetic System for the Chemolithoautotrophic Bacterium Thiobacillus denitrificans

摘要: Thiobacillus denitrificans is a widespread, chemolithoautotrophic bacterium with an unusual and environmentally relevant metabolic repertoire, which includes its ability to couple denitrification sulfur compound oxidation; catalyze anaerobic, nitrate-dependent oxidation of Fe(II) U(IV); oxidize mineral electron donors. Recent analysis genome sequence also revealed the presence genes encoding two [NiFe]hydrogenases, whose role in metabolism unclear, as sequenced strain does not appear be able grow on hydrogen sole donor under denitrifying conditions. In this study, we report development genetic system for T. denitrificans, insertion mutations can introduced by homologous recombination complemented trans. The antibiotic sensitivity was characterized, procedure transformation foreign DNA electroporation established. Insertion were generated vitro transposition, mutated amplified PCR, amplicons into electroporation. IncP plasmid pRR10 found useful vector complementation. effectiveness demonstrated hynL gene, encodes large subunit [NiFe]hydrogenase. Interruption hynL::kan mutant resulted 75% decrease specific hydrogenase activity relative wild type, whereas complementation mutation that 50% greater than type. availability will facilitate our understanding genetics biochemistry underlying metabolism.