Mapping of the Denitrification Pathway in Burkholderia thailandensis by Genome-Wide Mutant Profiling.

is a soil saprophyte that is closely related to the pathogen , the etiological agent of melioidosis in humans. The environmental niches and infection sites occupied by these bacteria are thought to contain only limited concentrations of oxygen, where they can generate energy via denitrification. However, knowledge of the underlying molecular basis of the denitrification pathway in these bacteria is scarce. In this study, we employed a transposon sequencing (Tn-Seq) approach to identify genes conferring a fitness benefit for anaerobic growth of Of the 180 determinants identified, several genes were shown to be required for growth under denitrifying conditions: the nitrate reductase operon , the gene encoding a previously unknown nitrite reductase, and the genes encoding a cytochrome , as well as three novel regulators that control denitrification. Our Tn-Seq data allowed us to reconstruct the entire denitrification pathway of and shed light on its regulation. Analyses of growth behaviors combined with measurements of denitrification metabolites of various mutants revealed that nitrate reduction provides sufficient energy for anaerobic growth, an important finding in light of the fact that some pathogenic species can use nitrate as a terminal electron acceptor but are unable to complete denitrification. Finally, we demonstrated that a nitrous oxide reductase mutant is not affected for anaerobic growth but is defective in biofilm formation and accumulates NO, which may play a role in the dispersal of biofilms. is a soil-dwelling saprophyte that is often used as surrogate of the closely related pathogen , the causative agent of melioidosis and a classified biowarfare agent. Both organisms are adapted to grow under oxygen-limited conditions in rice fields by generating energy through denitrification. Microoxic growth of is also considered essential for human infections. Here, we have used a Tn-Seq approach to identify the genes encoding the enzymes and regulators required for growth under denitrifying conditions. We show that a mutant that is defective in the conversion of NO to N, the last step in the denitrification process, is unaffected in microoxic growth but is severely impaired in biofilm formation, suggesting that NO may play a role in biofilm dispersal. Our study identified novel targets for the development of therapeutic agents to treat meliodiosis.