Isolation and Characterization ofBurkholderia cenocepaciaMutants Deficient in Pyochelin Production: Pyochelin Biosynthesis Is Sensitive to Sulfur Availability

Abstract

The opportunistic pathogen Burkholderia cenocepacia produces the yellow-green fluorescent siderophore, pyochelin. To isolate mutants which do not produce this siderophore, we mutagenized B. cenocepacia with the transposon mini-Tn5Tp. Two nonfluorescent mutants were identified which were unable to produce pyochelin. In both mutants, the transposon had integrated into a gene encoding an orthologue of CysW, a component of the sulfate/thiosulfate transporter. The cysW gene was located within a putative operon encoding other components of the transporter and a polypeptide exhibiting high homology to the LysR-type regulators CysB and Cbl. Sulfate uptake assays confirmed that both mutants were defective in sulfate transport. Growth in the presence of cysteine, but not methionine, restored the ability of the mutants to produce pyochelin, suggesting that the failure to produce the siderophore was the result of a depleted intracellular pool of cysteine, a biosynthetic precursor of pyochelin. Consistent with this, the wild-type strain did not produce pyochelin when grown in the presence of lower concentrations of sulfate that still supported efficient growth. We also showed that whereas methionine and certain organosulfonates can serve as sole sulfur sources for this bacterium, they do not facilitate pyochelin biosynthesis. These observations suggest that, under conditions of sulfur depletion, cysteine cannot be spared for production of pyochelin even under iron starvation conditions.

Role of phages in the pathogenesis of Burkholderia, or ‘Where are the toxin genes in Burkholderia phages?’

“…Although PAPS-reductase has not been demonstrated to be a virulence factor, it is reasonable that it could be a fitness factor under conditions of sulfur limitation. In Burkholderia, it has been shown that sulfur limitation preferentially affects biosynthesis of the siderophore pyochelin over that of cysteine synthesis [41]. Siderophores are known Burkholderia pathogenicity factors and pyochelin is one of a number of endogenous and exogenous siderophores that Burkholderia utilizes, contributing to success in the iron-limiting lung environment [42].…”

Pore-forming pyocin S5 utilizes the FptA ferripyochelin receptor to kill Pseudomonas aeruginosa

“…The opportunistic pathogen B. cenocepacia also produces the siderophore pyochelin and an FptA receptor highly similar (63 % identity, 76 % similarity) to the ferripyochelin receptor from P. aeruginosa (Farmer & Thomas, 2004;Mislin et al, 2006). Fig.…”

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

“…Tn5 derivatives have been shown to be useful for genetic analysis of members of the genus Burkholderia (Dennis & Zylstra, 1998;Sokol et al, 1999;Farmer & Thomas, 2004;Agnoli et al, 2006). To increase the versatility of these transposons, we inserted a promoterless lac cassette into the unique NotI site of mini-Tn5Cm and mini-Tn5Tp, thereby allowing lacZ to serve as a reporter of transcription originating in genomic DNA flanking the O end of the integrated transposon.…”

“…Isolation of B. cenocepacia mutants in which production of both ornibactin and pyochelin is decreased or abolished B. cenocepacia strain 715j produces the siderophores ornibactin and pyochelin (Darling et al, 1998;Visser et al, 2004;Farmer & Thomas, 2004;Agnoli et al, 2006). To test the effectiveness of mini-Tn5CmlacZYA, it was used to generate mutants of 715j that were deficient in production of both siderophores (Orb 2 Pch 2 phenotype).…”

“…Overnight cultures of bacteria were inoculated at 1 : 100 dilution into 3 ml M9-glucose (for analysis of ornibactin production) or 30 ml M9-glucose containing 0.5 % CAA (for analysis of pyochelin production) containing appropriate antibiotics, and the bacteria were grown with aeration at 37 uC for 36-48 h. The analysis of pyochelin (and salicylic acid) production was carried out by TLC, as previously described (Farmer & Thomas, 2004). Under these conditions, the two naturally occurring stereoisomers of pyochelin (pyochelin I and pyochelin II) migrate as two very closely spaced yellow-green fluorescent bands midway between the origin and the solvent front, while salicylate runs as a blue fluorescent band very close to the solvent front (Rinehart et al, 1995;Farmer & Thomas, 2004;Schlegel et al, 2004). Ornibactin production was assessed by the IEF-CAS overlay method (Koedam et al, 1994;Agnoli et al, 2006).…”