Genetic characterization of wild-type and mutant fur genes of Bordetella avium

For most, if not all, organisms, iron (Fe) is an essential element. In resp onse to the nutritional requirement for Fe, bacteria evolved complex system s to acquire the element from the environment, The genes encoding these sys tems are often coordinately regulated in response to the Fe concentration. Recent investigations revealed that Bordetella avium, a respiratory pathoge n of birds, expressed a number of Fe-regulated genes (T. D. Connell, A. Dic kenson, A. J. Martone, K. T. Militello, M. J. Filiatraut, M. L. Hayman, and J. Pitula, Infect. Immun. 66:3597-3605, 1998). By using manganese selectio n on an engineered strain of B. avium that carried an Fe-regulated alkaline phosphatase reporter gene, a mutant was obtained that was affected in expr ession of Fe-regulated genes. To determine if Fe-dependent regulation in B. avium was mediated by a fur-like gene, a fragment of the B. avium chromoso me, corresponding to the fur locus of B, pertussis, was cloned by PCR. Sequ encing revealed that the fragment from B. avium encoded a polypeptide with 92% identity to the Fur protein of B. pertussis. In vivo experiments showed that the cloned gene complemented H1780, a fur mutant of Escherichia coil. Southern hybridizations and PCRs demonstrated that the manganese mutant ha d a deletion of 2 to 3 Mbp of nucleotide sequence in the region located imm ediately 5' of the fur open reading frame. A spontaneous PCR-derived mutant of the R. avium fur gene was isolated that encoded a Fur protein in which a histidine was substituted for an arginine at amino acid position 18 (R18H ). Genetic analysis showed that the R18H mutant gene when cloned into a low -copy-number vector did not complement the fur mutation in H1780, However, the R18H mutant gene was able to complement the fur mutation when cloned in to a high-copy-number vector. The cloned wild-type fur gene will be useful as a genetic tool to identify Fur-regulated genes in the B. avium chromosom e.