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.

FIG. 1

Uptake of ⁵⁹Fe-siderophore complexes by B. cepacia K56-2, I117, and T10. (A) Uptake assays were initiated by the addition of ⁵⁹Fe-ornibactins; 1-ml samples were removed at intervals, and the amount of ⁵⁹Fe accumulated was determined. Cultures used in the assay were grown in the presence (open symbols) or absence (closed symbols) of ornibactins. (B) Uptake assays were initiated by the addition of ⁵⁹Fe-SA; 1-ml samples were removed at intervals, and the amount of ⁵⁹Fe accumulated was determined from a standard curve. Values represent the means ± the standard deviations of triplicate assays. These experiments were repeated at least three times with similar results.

FIG. 2

Computer-generated alignment of the deduced amino acid sequence of B. cepacia PvdA with P. aeruginosa PvdA (64) (accession number A49892), Ustilago maydis Sid1 (32) (accession number A47266), B. bronchiseptica AlcA (19) (accession number U32117), and E. coli IucD (22) (M18968) determined by using the programs PC/GENE CLUSTAL and Seqvu. Boxed shaded areas indicate amino acids identical in at least four of the five proteins. Unboxed shaded areas indicate regions of similarity. Gaps introduced to increase homology are indicated by dashes.

FIG. 3

Comparison of ⁵⁹Fe-siderophore uptake by I117 and K56pvdA::tp. (A) Uptake of ⁵⁹Fe-ornibactins. (B) Uptake of ⁵⁹Fe-SA as described in Fig. 1. Values represent the means ± the standard deviations of triplicate assays. These experiments were repeated at least three times with similar results.

FIG. 4

Siderophore production assays. (A) Comparison of SA production by K56-2, I117, and K56pvdA::tp. Values represent the means ± the standard deviations of triplicate assays. I117 significantly different than K56-2 and K56pvdA::tp (P < 0.001) by using analysis of variance (ANOVA). (B) Partial complementation of I117 CAS activity (ornibactin production) by cosmid clones pSBC-4, pSBC-8, and pSBC-13. Values represent the means ± the standard deviation of triplicate assays. CAS activity is per 10 μl of culture supernatant. K56-2, pSBC-4, pSBC-8, and pSBC-13 are significantly different than I117 or pScosBC1 (P < 0.001) by ANOVA.

FIG. 5

Comparison of the ability of cosmid clones to restore ⁵⁹Fe-ornibactin uptake (A) or ⁵⁹Fe-SA uptake (B) in I117. Assays were performed as described in Fig. 1. Values represent the means ± the standard deviations of triplicate assays. These experiments were repeated at least three times, with similar results.

FIG. 6

(Lanes 1 to 3) Resolution of the linearized forms of the three large replicons (3.5, 3.0, and 0.8 Mb) from K56-2 by PFGE. Lane 1, Saccharomyces cerevisiae YNN295 molecular weight marker; lanes 2 and 3, K56-2 genomic DNA. (Lanes 4 and 5) Southern hybridization of PFGE in panel A hybridized to [³²P]dCTP-labelled pvdA probe. The gel and autoradiogram were scanned with Hewlett-Packard ScanJet 4c and Hewlett-Packard Deskscan II software.

FIG. 7

Hematoxylin-and-eosin-stained sections of rat lungs infected with B. cepacia K56-2. (A) Mixed cellular infiltrate composed primarily of lymphocytes and mononuclear phagocytes. Also shown is a swollen lymphoid follicle (magnification, ×75). (B) Photomicrograph of higher-power magnification (×150) demonstrating the mononuclear nature of the infiltrate.