IDENTIFICATION AND CHARACTERIZATION OF IRON-REGULATED BORDETELLA-PERTUSSIS ALCALIGIN SIDEROPHORE BIOSYNTHESIS GENES

Bordetella bronchiseptica mutants BRM1, BRM6, and BRM9 fail to produce the native dihydroxamate siderophore alcaligin. A 4,5-kb BamHI-SmaI B ordetella pertussis genomic DNA fragment carried multiple genes requir ed to restore alcaligin production to these siderophore-deficient muta nts. Phenotypic complementation analysis using subclones of the 4.5-kb genomic region demonstrated that the closely linked BRM1 and BRM9 mut ations were genetically separable from the BRM6 mutation, and both ins ertions exerted strong polar effects on expression of the downstream g ene defined by the BRM6 mutation, suggesting a polycistronic transcrip tional organization of these alcaligin biosynthesis genes. Subcloning and complementation experiments localized the putative Bordetella prom oter to a 0.7-kb BamHI-SphI subregion of the cloned genomic DNA fragme nt. Nucleotide sequencing, phenotypic analysis of mutants, and protein expression by the 4.5-kb DNA fragment in Escherichia coli suggested t he presence of three alcaligin system genes, namely, alcA, alcB, and a lcC. The deduced protein products of alcA, alcB, and alcC have signifi cant primary amino acid sequence similarities with known microbial sid erophore biosynthesis enzymes. Primer extension analysis mapped the tr anscriptional start site of the putative alcaligin biosynthesis operon containing alcABC to a promoter region overlapping a proposed Fur rep ressor-binding site and demonstrated iron regulation at the transcript ional level.