The Ornibactin Biosynthesis and Transport Genes of Burkholderia cenocepacia Are Regulated by an Extracytoplasmic Function σ Factor Which Is a Part of the Fur Regulon

Abstract

Burkholderia cenocepacia mutants that fail to produce the siderophore ornibactin were obtained following mutagenesis with mini-Tn5Tp. These mutants were shown to be growth restricted under conditions of iron depletion. In eight of the mutants, the transposon had integrated into one of two genes, orbI and orbJ, encoding nonribosomal peptide synthetases. In the other mutant, the transposon had inserted into an open reading frame, orbS, located upstream from orbI. The polypeptide product of orbS exhibits a high degree of similarity to the Pseudomonas aeruginosa extracytoplasmic function (ECF) factor PvdS but possesses an N-terminal extension of approximately 29 amino acids that is not present in PvdS. Three predicted OrbS-dependent promoters were identified within the ornibactin gene cluster, based on their similarity to PvdS-dependent promoters. The iron-regulated activity of these promoters was shown to require OrbS. Transcription of the orbS gene was found to be under the control of an iron-regulated 70 -dependent promoter. This promoter, but not the OrbS-dependent promoters, was shown to be a target for repression by the global regulator Fur. Our results demonstrate that production of ornibactin by B. cenocepacia in response to iron starvation requires transcription of an operon that is dependent on the Fur-regulated ECF factor gene orbS. A mechanism is also proposed for the biosynthesis of ornibactin.

Iron acquisition in the cystic fibrosis lung and potential for novel therapeutic strategies

“…There are three known species of this linear hydroxamate siderophore: ornibactin-C4, ornibactin-C6 and ornibactin-C8 produced by Bcc strains (Stephan et al, 1993), and ferrated ornibactin is transported across the outer membrane by a ferric-ornibactin receptor, OrbA. Similar to Pseudomonas siderophore biosynthesis, the ornibactin operon is Fur regulated (Agnoli et al, 2006). The second siderophore produced by some strains of the Bcc is pyochelin (Sokol, 1986), and the production of ornibactin and pyochelin correlates with morbidity and mortality in CF (Sokol, 1986;Visser et al, 2004).…”

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

“…Most clinical isolates of B. cenocepacia produce the siderophores ornibactin and pyochelin (Sokol, 1986;Visca et al, 1993;Meyer et al, 1995;Darling et al, 1998;Thomas, 2007), and their production has been correlated with morbidity and mortality in CF patients and/or shown to contribute to pathology in animal models of respiratory infection (Sokol, 1986;Sokol & Woods, 1988;Sokol et al, 1999Sokol et al, , 2000Visser et al, 2004;Uehlinger et al, 2009). Ornibactin contains an L-ornithine-D-hydroxyaspartate-L-serine-L-ornithine backbone that requires two non-ribosomal peptide synthetases (NRPSs), OrbI and OrbJ, for its assembly (Stephan et al, 1993;Agnoli et al, 2006). The yellow-green fluorescent siderophore pyochelin is biosynthesized from salicylate by the successive addition and cyclization of two molecules of cysteine (Ankenbauer et al, 1988;Reimmann et al, 1998;Quadri et al, 1999;Quadri, 2000;Crosa & Walsh, 2002).…”

“…This enzyme transfers the P-pant moiety from coenzyme A (CoASH) to an invariant serine residue in the conserved phosphopantetheinylation site [FFxLGG(D/H)S(L/I)] of the PCP/ArCP domain Marahiel et al, 1997;Quadri et al, 1998b). A total of four predicted PCP domains are present in the ornibactin biosynthetic machinery (three in OrbI and one in OrbJ), each of which binds one of the four amino acid building blocks, and three PCP/ArCP domains occur in the NRPSs involved in pyochelin biosynthesis (two in PchE and one in PchF) (Quadri, 2000;Agnoli et al, 2006).…”

“…For strains carrying lacZ fusions, X-Gal was included in solid medium at 40 mg ml 21 . CAS plates used to screen B. cenocepacia mutants for siderophore production were made as described by Agnoli et al (2006). For assaying enterobactin production by E. coli strains, CAS agar was made by adding 10 ml CAS reagent (Schwyn & Neilands, 1987) For measurement of the effect of iron availability on the bacterial growth rate, B. cenocepacia strains were grown to stationary phase at 37 uC (i.e.~20 h) in M9-CAA medium (25 mg ml 21 trimethoprim was included when pAHA14 derivatives were employed).…”

Volatile communication between fungi and bacteria

“…Our analysis revealed that all six strains encode biosynthesis clusters which resemble ornibactin (Fig. 4E), a siderophore synthesis cluster of Burkholderia cenocepacia (Agnoli et al, 2006). Siderophore production was confirmed for all the six Collimonas strains, albeit with different production efficiency (Fig.…”