IDENTIFICATION OF ALCR, AN ARAC-TYPE REGULATOR OF ALCALIGIN SIDEROPHORE SYNTHESIS IN BORDETELLA-BRONCHISEPTICA AND BORDETELLA-PERTUSSIS

A Fur titration assay was used to isolate DNA fragments bearing putati ve Fur binding sites (FBS) from a partial Bordetella bronchiseptica ge nomic DNA library. A recombinant plasmid bearing a 3.5-kb DNA insert w as further studied. Successive deletions in the cloned fragment enable d us to map a putative FBS at about 2 kb from one end. Sequence analys is revealed the presence of an FBS upstream from a new gene encoding a n AraC-type transcriptional regulator. The deduced protein displays si milarity to PchR, an activator of pyochelin siderophore and ferripyoch elin receptor synthesis in Pseudomonas aeruginosa. Homologous genes in Bordetella pertussis and Bordetella parapertussis were PCR amplified, and sequence comparisons indicated a very high conservation in the th ree species. The B. pertussis and B. bronchiseptica chromosomal genes were inactivated by allelic exchange. Under low-iron growth conditions , the mutants did not secrete the alcaligin siderophore and lacked Alc C, an alcaligin biosynthetic enzyme. Alcaligin production was restored after transformation with a plasmid bearing the wild-type gene. On th e basis of its role in regulation of alcaligin biosynthesis, the new g ene was designated alcR. Additional sequence determination showed that alcR is located about 2 kb downstream from the alcABC operon and is t ranscribed in the same orientation. Two tightly linked open reading fr ames, alcD and alcE, were identified between alcC and alcR. AlcE is a putative iron-sulfur protein; AlcD shows no homology with the proteins in the database. The production of major virulence factors and coloni zation in the mouse respiratory infection model are AlcR independent.