The pobA gene of Burkholderia cenocepacia encodes a group I Sfp-type phosphopantetheinyltransferase required for biosynthesis of the siderophores ornibactin and pyochelin.

The opportunistic pathogen Burkholderia cenocepacia produces the siderophores ornibactin and pyochelin under iron-restricted conditions. Biosynthesis of both siderophores requires the involvement of non-ribosomal peptide synthetases (NRPSs). Using a transposon containing the lacZ reporter gene, two B. cenocepacia mutants were isolated which were deficient in siderophore production. Mutant IW10 was shown to produce normal amounts of ornibactin but only trace amounts of pyochelin, whereas synthesis of both siderophores was abolished in AHA27. Growth of AHA27, but not IW10, was inhibited under iron-restricted conditions. In both mutants, the transposon had integrated into the pobA gene, which encodes a polypeptide exhibiting similarity to the Sfp-type phosphopantetheinyltransferases (PPTases). These enzymes are responsible for activation of NRPSs by the covalent attachment of the 4'-phosphopantetheine (P-pant) moiety of coenzyme A. Previously characterized PPTase genes from other bacteria were shown to efficiently complement both mutants for siderophore production when provided in trans. The B. cenocepacia pobA gene was also able to efficiently complement an Escherichia coli entD mutant for production of the siderophore enterobactin. Using mutant IW10, in which the lacZ gene carried by the transposon is inserted in the same orientation as pobA, it was shown that pobA is not appreciably iron-regulated. Finally, we confirmed that Sfp-type bacterial PPTases can be subdivided into two distinct groups, and we present the amino acid signature sequences which characterize each of these groups.

Regulation of sulfur assimilation pathways in Burkholderia cenocepacia: identification of transcription factors CysB and SsuR and their role in control of target genes.

Two genes encoding transcriptional regulators involved in sulfur assimilation pathways in Burkholderia cenocepacia strain 715j have been identified and characterized functionally. Knockout mutations in each of the B. cenocepacia genes were constructed and introduced into the genome of 715j by allelic replacement. Studies on the utilization of various sulfur sources by 715j and the obtained mutants demonstrated that one of the B. cenocepacia regulators, designated CysB, is preferentially involved in the control of sulfate transport and reduction, while the other, designated SsuR, is required for aliphatic sulfonate utilization. Using transcriptional promoter-lacZ fusions and DNA-binding experiments, we identified several target promoters for positive control by CysB and/or SsuR--sbpp (preceding the sbp cysT cysW cysA ssuR cluster), cysIp (preceding the cysI cysD1 cysN cysH cysG cluster), cysD2p (preceding a separate cluster, cysD2 cysNC), and ssuDp (located upstream of the ssuDCB operon)--and we demonstrated overlapping functions of CysB and SsuR at particular promoters. We also demonstrated that the cysB gene is negatively controlled by both CysB and SsuR but the ssuR gene itself is not significantly regulated as a separate transcription unit. The function of B. cenocepacia CysB (in vivo and in vitro) appeared to be independent of the presence of acetylserine, the indispensable coinducer of the CysB regulators of Escherichia coli and Salmonella. The phylogenetic relationships among members of the "CysB family" in the gamma and beta subphyla are presented.