IDENTIFICATION AND CHARACTERIZATION OF ALCR, A GENE ENCODING AN ARAC-LIKE REGULATOR OF ALCALIGIN SIDEROPHORE BIOSYNTHESIS AND TRANSPORT IN BORDETELLA-PERTUSSIS AND BORDETELLA-BRONCHISEPTICA

A Bordetella bronchiseptica iron transport mutant,vas isolated followi ng an enrichment procedure based on streptonigrin resistance, The muta nt displayed a growth defect on iron-restricted medium containing ferr ic alcaligin as the sole iron source. In addition to the apparent inab ility to acquire iron from tile siderophore, the mutant failed to prod uce alcaligin as well as two known iron-regulated proteins, one of whi ch is the AlcC alcaligin biosynthesis protein, A 1.6-kb KpnI-PstI Bord etella pertussis DNA fragment mapping downstream of the alcaligin bios ynthesis genes alcABC restored both siderophore biosynthesis and expre ssion of the iron-regulated proteins to the mutant. Nucleotide sequenc ing of this complementing 1,6-kb region identified an open reading fra me predicted to encode a protein with strong similarity to members of the AraC family of transcriptional regulators, for which we propose th e gene designation alcR. Primer extension analysis localized an iron-r egulated transcription initiation site upstream of the alcR open readi ng frame and adjacent to sequences homologous to the consensus Fur rep ressor binding site, The AlcR protein was produced by using an Escheri chia coli expression system and visualized in electrophoretic gels, In -frame alcR deletion mutants of B. pertussis and B. bronchiseptica wer e constructed, and the defined mutants exhibited the alcR mutant pheno type, characterized by the inability to produce and transport alcaligi n and express the two-iron repressed proteins, The cloned alcR gene pr ovided in trans restored these siderophore system activities to the mu tants, Together, these results indicate that AlcR is involved in the r egulation of Bordetella alcaligin biosynthesis and transport genes and is required for their full expression.