Identification and characterization of iron-regulated Bordetella pertussis alcaligin siderophore biosynthesis genes

Abstract

Bordetella bronchiseptica mutants BRM1, BRM6, and BRM9 fail to produce the native dihydroxamate siderophore alcaligin. A 4.5-kb BamHI-SmaI Bordetella pertussis genomic DNA fragment carried multiple genes required to restore alcaligin production to these siderophore-deficient mutants. Phenotypic complementation analysis using subclones of the 4.5-kb genomic region demonstrated that the closely linked BRM1 and BRM9 mutations were genetically separable from the BRM6 mutation, and both insertions exerted strong polar effects on expression of the downstream gene defined by the BRM6 mutation, suggesting a polycistronic transcriptional organization of these alcaligin biosynthesis genes. Subcloning and complementation experiments localized the putative Bordetella promoter to a 0.7-kb BamHI-SphI subregion of the cloned genomic DNA fragment. Nucleotide sequencing, phenotypic analysis of mutants, and protein expression by the 4.5-kb DNA fragment in Escherichia coli suggested the presence of three alcaligin system genes, namely, alcA, alcB, and alcC. The deduced protein products of alcA, alcB, and alcC have significant primary amino acid sequence similarities with known microbial siderophore biosynthesis enzymes. Primer extension analysis mapped the transcriptional start site of the putative alcaligin biosynthesis operon containing alcABC to a promoter region overlapping a proposed Fur repressor-binding site and demonstrated iron regulation at the transcriptional level.Iron is a fundamental nutritional requirement for virtually all cells, and its assimilation is considered essential for invading pathogenic bacteria to establish infection in the iron-limiting environment of the host (13, 56). Additionally, iron serves as an environmental modulator of the production of certain virulence factors in a number of bacteria (14,24,31,32,46). Despite host iron sequestration, mediated primarily by the glycoprotein family of iron-binding transferrins, pathogens multiply successfully in vivo because they express efficient ironscavenging systems in response to decreased iron availability. These iron retrieval systems utilize two general strategies: one involving high-affinity iron-chelating soluble siderophores (30,40) and the other using siderophore-independent cell surface receptor mechanisms allowing iron uptake directly from host sources such as transferrin, lactoferrin, and heme compounds (7,35,38,54).Bordetella pertussis, the causative agent of human whooping cough or pertussis, and Bordetella bronchiseptica, the agent of swine atrophic rhinitis and kennel cough in dogs, are bacterial pathogens that infect the respiratory epithelial mucosae of their hosts. Early reports described the production of putative siderophores by both B. pertussis and B. bronchiseptica in response to iron deficiency (1, 23). Armstrong and Clements isolated and characterized B. bronchiseptica transposon-induced siderophore-deficient mutants; DNA hybridization studies using sequences flanking those transposon insertions confirmed the existence of homologs o...

Norepinephrine Mediates Acquisition of Transferrin-Iron in Bordetella bronchiseptica

“…When FeSO 4 was added to norepinephrine-containing cultures at a concentration calculated to exceed the iron-binding capacity of transferrin, cell association of 55 Fe decreased dramatically. This result suggests that expression of norepinephrine-mediated iron transport functions is repressible when iron is abundant (perhaps at the transcriptional level by the Fur repressor), similar to the regulation of other Bordetella TonB-dependent iron transport systems (3,10,27,55). Importantly, the tonB mutant strain exhibited low levels of bound iron in both the presence and absence of norepinephrine, further reinforcing the conclusion that the TonB system is required for norepinephrine-mediated iron acquisition.…”

“…The respiratory pathogens Bordetella bronchiseptica and Bordetella pertussis have multiple characterized mechanisms of iron acquisition, including transport of heme-iron (55), biosynthesis and utilization of the alcaligin siderophore (10,27), and uptake of catechol xenosiderophores, such as enterobactin (4,8). In addition, there are other putative outer membrane iron transporters encoded in the Bordetella genome for which the ligand is unknown (41).…”

Discovery of a Nonclassical Siderophore, Legiobactin, Produced by Strains ofLegionella pneumophila

“…For a number of reasons, we strongly believe that this CAS-reactive substance represents a bona fide siderophore. First, the CAS assay has reliably identified and characterized a range of siderophores encompassing the iron chelators of over 20 bacterial genera (2,3,5,12,18,20,21,27,33,36,37,38,47,48,51,58,66,68,74,80,82,85,98). Second, the presence of CAS-reactive material correlated with enhanced aerobic growth in an iron-depleted defined medium (68).…”

Reduced Virulence of aBordetella bronchisepticaSiderophore Mutant in Neonatal Swine

“…Previously, it was shown that in response to iron limitation B. bronchiseptica synthesizes and excretes the siderophore alcaligin, a low-molecular-weight, dihydroxamate Fe-chelating compound (37,39). Alcaligin is encoded by the alcABCDER operon (10,23,24,29,45). The genes alcA, alcB, and alcC show significant similarity to the Escherichia coli aerobactin biosynthesis genes iucD, iucB, and iucC, respectively (23,24,29).…”

“…Alcaligin is encoded by the alcABCDER operon (10,23,24,29,45). The genes alcA, alcB, and alcC show significant similarity to the Escherichia coli aerobactin biosynthesis genes iucD, iucB, and iucC, respectively (23,24,29). AlcE is predicted to be an iron-sulfur protein, while alcD has no homologs in the public databases which would suggest a function (45).…”