bvg Repression of alcaligin synthesis in Bordetella bronchiseptica is associated with phylogenetic lineage.

Recent studies have shown that Bordetella bronchiseptica utilizes a siderophore-mediated transport system for acquisition of iron from the host iron-binding proteins lactoferrin and transferrin. We recently identified the B. bronchiseptica siderophore as alcaligin, which is also produced by B. pertussis. Alcaligin production by B. bronchiseptica is repressed by exogenous iron, a phenotype of other microbes that produce siderophores. In this study, we report that alcaligin production by B. bronchiseptica RB50 and GP1SN was repressed by the Bordetella global virulence regulator, bvg, in addition to being Fe repressed. Modulation of bvg locus expression with 50 mM MgSO4 or inactivation of bvg by deletion allowed strain RB50 to produce alcaligin. In modulated organisms, siderophore production remained Fe repressed. These observations contrasted with our previous data indicating that alcaligin production by B. bronchiseptica MBORD846 and B. pertussis was repressed by Fe but bvg independent. Despite bvg repression of alcaligin production, strain RB50 was still able to acquire Fe from purified alcaligin, suggesting that expression of the bacterial alcaligin receptor was not repressed by bvg. We tested 114 B. bronchiseptica strains and found that bvg repression of alcaligin production was strongly associated with Bordetella phylogenetic lineage and with host species from which the organisms were isolated.     

The ornithine decarboxylase gene odc is required for alcaligin siderophore biosynthesis in Bordetella spp.: putrescine is a precursor of alcaligin.

Chromosomal insertions defining Bordetella bronchiseptica siderophore phenotypic complementation group III mutants BRM3 and BRM5 were found to reside approximately 200 to 300 bp apart by restriction mapping of cloned genomic regions associated with the insertion markers. DNA hybridization analysis using B. bronchiseptica genomic DNA sequences flanking the cloned BRM3 insertion marker identified homologous Bordetella pertussis UT25 cosmids that complemented the siderophore biosynthesis defect of the group III B. bronchiseptica mutants. Subcloning and complementation analysis localized the complementing activity to a 2.8-kb B. pertussis genomic DNA region. Nucleotide sequencing identified an open reading frame predicted to encode a polypeptide exhibiting strong similarity at the primary amino acid level with several pyridoxal phosphate-dependent amino acid decarboxylases. Alcaligin production was fully restored to group III mutants by supplementation of iron-depleted culture media with putrescine (1,4-diaminobutane), consistent with defects in an ornithine decarboxylase activity required for alcaligin siderophore biosynthesis. Concordantly, the alcaligin biosynthesis defect of BRM3 was functionally complemented by the heterologous Escherichia coli speC gene encoding an ornithine decarboxylase activity. Enzyme assays confirmed that group III B. bronchiseptica siderophore-deficient mutants lack an ornithine decarboxylase activity required for the biosynthesis of alcaligin. Siderophore production by an analogous mutant of B. pertussis constructed by allelic exchange was undetectable. We propose the designation odc for the gene defined by these mutations that abrogate alcaligin siderophore production. Putrescine is an essential precursor of alcaligin in Bordetella spp.     

Iron and pH‐responsive FtrABCD ferrous iron utilization system of Bordetella species

A putative operon encoding an uncharacterized ferrous iron transport (FtrABCD) system was previously identified in cDNA microarray studies. In growth studies using buffered medium at pH values ranging from pH 6.0 to 7.6, Bordetella pertussis and Bordetella bronchiseptica FtrABCD system mutants showed dramatic reductions in growth yields under iron‐restricted conditions at pH 6.0, but had no growth defects at pH 7.6. Supplementation of culture medium with 2 mM ascorbate reductant was inhibitory to alcaligin siderophore‐dependent growth at pH 7.6, but had a neglible effect on FtrABCD system‐dependent iron assimilation at pH 6.0 consistent with its predicted specificity for ferrous iron. Unlike Bordetella siderophore‐dependent and haem iron transport systems, and in agreement with its hypothesized role in transport of inorganic iron from periplasm to cytoplasm, FtrABCD system function did not require the TonB energy transduction complex. Gene fusion analysis revealed that ftrABCD promoter activity was maximal under iron‐restricted growth conditions at acidic pH. The pH of human airway surface fluids ranges from pH 5.5 to 7.9, and the FtrABCD system may supply ferrous iron necessary for Bordetella growth in acidic host microenvironments in which siderophores are ineffective for iron retrieval.     

Identification of alcaligin as the siderophore produced by Bordetella pertussis and B. bronchiseptica.

The siderophores produced by iron-starved Bordetella pertussis and B. bronchiseptica were purified and were found to be identical. Using mass spectrometry and proton nuclear magnetic resonance, we determined that the siderophore produced by these organisms was identical to alcaligin, a siderophore produced by Alcaligenes denitrificans.     

Purification, spectroscopic analysis and biological activity of the macrocyclic dihydroxamate siderophore alcaligin produced by Bordetella pertussis and Bordetella bronchiseptica

Hydroxamate siderophores of virulent Bordetella pertussis and Bordetella bronchiseptica strains were purified using a simple large-scale isolation procedure, and identified by various spectroscopic techniques as the macrocyclic dihydroxamate siderophore trivially known as alcaligin, 1,8(S),11,18(S)-tetrahydroxy-1,6,11,16-tetraazacycloeicosane-2,5,12,15-tetrone, which was previously isolated from the taxonomically-related bacterial species Alcaligenes denitrificans subsp. xylosoxydans. Alcaligin purified from iron-depleted cultures of B. pertussis and B. bronchiseptica exhibited specific growth-promoting activity under iron-restricted conditions for Bordetella indicator strains, and were active in [⁵⁵Fe]ferric alcaligin transport assays. Evidence suggests that several C ₂-symmetric conformations of alcaligin exist simultaneously in both methanolic and aqueous solution.     

Differential expression of Bordetella pertussis iron transport system genes during infection

Temporal expression patterns of the Bordetella pertussis alcaligin, enterobactin and haem iron acquisition systems were examined using alcA-, bfeA- and bhuR-tnpR recombinase fusion strains in a mouse respiratory infection model. The iron systems were differentially expressed in vivo, showing early induction of the alcaligin and enterobactin siderophore systems, and delayed induction of the haem system in a manner consistent with predicted changes in host iron source availability during infection. Previous mixed infection competition studies established the importance of alcaligin and haem utilization for B. pertussis in vivo growth and survival. In this study, the contribution of the enterobactin system to the fitness of B. pertussis was confirmed using wild-type and enterobactin receptor mutant strains in similar competition infection experiments. As a correlate to the in vivo expression studies of B. pertussis iron systems in mice, sera from uninfected and B. pertussis-infected human donors were screened for antibody reactivity with Bordetella iron-repressible cell envelope proteins. Pertussis patient sera recognized multiple iron-repressible proteins including the known outer membrane receptors for alcaligin, enterobactin and haem, supporting the hypothesis that B. pertussis is iron-starved and responds to the presence of diverse iron sources during natural infection.     

Alcaligin siderophore production by Bordetella bronchiseptica strain RB50 is not repressed by the BvgAS virulence control system

A previous study found that alcaligin siderophore production by Bordetella bronchiseptica strain RB50 is Bvg repressed. In contrast, we report that alcaligin production by RB50 does not require Bvg phenotypic phase modulation and that isogenic Bvg(Con) and Bvg(-) phase-locked mutants both produce alcaligin in response to iron starvation.     

Siderophore-mediated iron uptake in Alcaligenes eutrophus CH34 and identification of aleB encoding the ferric iron-alcaligin E receptor.

Siderophore production in response to iron limitation was observed in Alcaligenes eutrophus CH34, and the corresponding siderophore was named alcaligin E. Alcaligin E was characterized as a phenolate-type siderophore containing neither catecholate nor hydroxamate groups. Alcaligin E promoted the growth of siderophore-deficient A. eutrophus mutants under iron-restricted conditions and promoted 59Fe uptake by iron-limited cells. However, the growth of the Sid- mutant AE1152, which was obtained from CH34 by Tn5-Tc mutagenesis, was completely inhibited by the addition of alcaligin E. AE1152 also showed strongly reduced 59Fe uptake in the presence of alcaligin E. This indicates that a gene, designated aleB, which is involved in transport of ferric iron-alcaligin E across the membrane is inactivated. The aleB gene was cloned, and its putative amino acid sequence showed strong similarity to those of ferric iron-siderophore receptor proteins. Both wild-type strain CH34 and aleB mutant AE1152 were able to use the same heterologous siderophores, indicating that AleB is involved only in ferric iron-alcaligin E uptake. Interestingly, no utilization of pyochelin, which is also a phenolate-type siderophore, was observed for A. eutrophus CH34. Genetic studies of different Sid- mutants, obtained after transposon mutagenesis, showed that the genes involved in alcaligin E and ferric iron-alcaligin E receptor biosynthesis are clustered in a 20-kb region on the A. eutrophus CH34 chromosome in the proximity of the cys-232 locus.