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-Smal 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.
Kang
H Y
HY
Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27858-4354, USA.
Brickman
T J
TJ
Beaumont
F C
FC
Armstrong
S K
SK
D000595:Amino Acid Sequence; D001483:Base Sequence; D016950:Bordetella bronchiseptica / Q000235:genetics* / Q000378:metabolism; D001886:Bordetella pertussis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D003227:Conjugation, Genetic; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial; D006877:Hydroxamic Acids; D007501:Iron / Q000494:pharmacology*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016376:Oligonucleotides, Antisense; D009876:Operon; D015183:Restriction Mapping; D017262:Siderophores / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial; D006877:Hydroxamic Acids; D016376:Oligonucleotides, Antisense; D017262:Siderophores; C092056:alcaligin; D007501:Iron