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Role of ornibactin biosynthesis in the virulence of Burkholderia cepacia: characterization of pvdA, the gene encoding L-ornithine N(5)-oxygenase.

ABSTRACT: Burkholderia cepacia is a frequent cause of respiratory infections in cystic fibrosis patients. B. cepacia has been shown to produce at least four siderophores which may play a role in the virulence of this organism. To characterize genes involved in the synthesis of siderophores, Tn5-OT182 mutants were isolated in strain K56-2, which produces two siderophores, salicylic acid (SA) and ornibactins. Two mutants were characterized that did not produce zones on Chrome Azurol S agar in a commonly used assay to detect siderophore activity. These mutants were determined to produce sevenfold more SA than K56-2 yet did not produce detectable amounts of ornibactins. These mutants, designated I117 and T10, had a transposon insertion in genes with significant homology to pyoverdine biosynthesis genes of Pseudomonas aeruginosa. I117 contained an insertion in a pvdA homolog, the gene for the enzyme L-ornithine N(5)-oxygenase, which catalyzes the hydroxylation of L-ornithine. Ornibactin synthesis in this mutant was partially restored when the precursor L-N(5)-OH-Orn was added to the culture medium. T10 contained an insertion in a pvdD homolog, which is a peptide synthetase involved in pyoverdine synthesis. beta-Galactosidase activity was iron regulated in both I117 and T10, suggesting that the transposon was inserted downstream of an iron-regulated promoter. Tn5-OT182 contains a lacZ gene that is expressed when inserted downstream of an active promoter. Both I117 and T10 were deficient in uptake of iron complexed to either ornibactins or SA, suggesting that transposon insertions in ornibactin biosynthesis genes also affected other components of the iron transport mechanism. The B. cepacia pvdA homolog was approximately 47% identical and 59% similar to L-ornithine N(5)-oxygenase from P. aeruginosa. Three clones were identified from a K56-2 cosmid library that partially restored ornibactin production, SA production, and SA uptake to parental levels but did not affect the rate of (59)Fe-ornibactin uptake in I117. A chromosomal pvdA deletion mutant was constructed that had a phenotype similar to that of I117 except that it did not hyperproduce SA. The pvdA mutants were less virulent than the parent strain in chronic and acute models of respiratory infection. A functional pvdA gene appears to be required for effective colonization and persistence in B. cepacia lung infections.

SUBMITTER: Sokol PA

PROVIDER: S-EPMC96763 | biostudies-literature | 1999 Sep

REPOSITORIES: biostudies-literature

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Role of ornibactin biosynthesis in the virulence of Burkholderia cepacia: characterization of pvdA, the gene encoding L-ornithine N(5)-oxygenase.

Sokol P A PA Darling P P Woods D E DE Mahenthiralingam E E Kooi C C

Infection and immunity 19990901 9

Burkholderia cepacia is a frequent cause of respiratory infections in cystic fibrosis patients. B. cepacia has been shown to produce at least four siderophores which may play a role in the virulence of this organism. To characterize genes involved in the synthesis of siderophores, Tn5-OT182 mutants were isolated in strain K56-2, which produces two siderophores, salicylic acid (SA) and ornibactins. Two mutants were characterized that did not produce zones on Chrome Azurol S agar in a commonly use ...[more]

PMID: 10456885

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Project description:The Burkholderia cepacia complex (Bcc) displays a wealth of metabolic diversity with great biotechnological potential, but the utilization of these bacteria is limited by their opportunistic pathogenicity to humans. The third replicon of the Bcc, megaplasmid pC3 (0.5 to 1.4 Mb, previously chromosome 3), is important for various phenotypes, including virulence, antifungal, and proteolytic activities and the utilization of certain substrates. Approximately half of plasmid pC3 is well conserved throughout sequenced Bcc members, while the other half is not. To better locate the regions responsible for the key phenotypes, pC3 mutant derivatives of Burkholderia cenocepacia H111 carrying large deletions (up to 0.58 Mb) were constructed with the aid of the FLP-FRT (FRT, flippase recognition target) recombination system from Saccharomyces cerevisiae The conserved region was shown to confer near-full virulence in both Caenorhabditis elegans and Galleria mellonella infection models. Antifungal activity was unexpectedly independent of the part of pC3 bearing a previously identified antifungal gene cluster, while proteolytic activity was dependent on the nonconserved part of pC3, which encodes the ZmpA protease. To investigate to what degree pC3-encoded functions are dependent on chromosomally encoded functions, we transferred pC3 from Burkholderia cenocepacia K56-2 and Burkholderia lata 383 into other pC3-cured Bcc members. We found that although pC3 is highly important for virulence, it was the genetic background of the recipient that determined the pathogenicity level of the hybrid strain. Furthermore, we found that important phenotypes, such as antifungal activity, proteolytic activity, and some substrate utilization capabilities, can be transferred between Bcc members using pC3.IMPORTANCE The Burkholderia cepacia complex (Bcc) is a group of closely related bacteria with great biotechnological potential. Some strains produce potent antifungal compounds and can promote plant growth or degrade environmental pollutants. However, their agricultural potential is limited by their opportunistic pathogenicity, particularly for cystic fibrosis patients. Despite much study, their virulence remains poorly understood. The third replicon, pC3, which is present in all Bcc isolates and is important for pathogenicity, stress resistance, and the production of antifungal compounds, has recently been reclassified from a chromosome to a megaplasmid. In this study, we identified regions on pC3 important for virulence and antifungal activity and investigated the role of the chromosomal background for the function of pC3 by exchanging the megaplasmid between different Bcc members. Our results may open a new avenue for the construction of antifungal but nonpathogenic Burkholderia hybrids. Such strains may have great potential as biocontrol strains for protecting fungus-borne diseases of plant crops.

Dataset Information

Role of ornibactin biosynthesis in the virulence of Burkholderia cepacia: characterization of pvdA, the gene encoding L-ornithine N(5)-oxygenase.

Publications

Role of ornibactin biosynthesis in the virulence of Burkholderia cepacia: characterization of pvdA, the gene encoding L-ornithine N(5)-oxygenase.

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