Abstract
The expression of iron-regulated systems in gram-negative bacteria is generally controlled by the Fur protein, which represses the transcription of iron-regulated promoters by using Fe
2+
as a cofactor. Mutational analysis of the
Campylobacter jejuni fur
gene was carried out by generation of a set of mutant copies of
fur
which had a kanamycin or chloramphenicol resistance gene introduced into the regions encoding the N and C termini of the Fur protein. The mutated genes were recombined into the
C. jejuni
NCTC 11168 chromosome, and putative mutants were confirmed by Southern hybridization.
C. jejuni
mutants were obtained only when the resistance genes were transcribed in the same orientation as the
fur
gene. The
C. jejuni fur
mutant grew slower than the parental strain. Comparison of protein profiles of fractionated
C. jejuni
cells grown in low- or high-iron medium indicated derepressed expression of three iron-regulated outer membrane proteins with molecular masses of 70, 75, and 80 kDa. Characterization by N-terminal amino acid sequencing showed the 75-kDa protein to be identical to CfrA, a
Campylobacter coli
siderophore receptor homologue, whereas the 70-kDa protein was identified as a new siderophore receptor homologue. Periplasmic fractions contained four derepressed proteins with molecular masses of 19, 29, 32, and 36 kDa. The 19-kDa protein has been previously identified, but its function is unknown. The cytoplasmic fraction contained two iron-repressed and two iron-induced proteins with molecular masses of 26, 55, 31, and 40 kDa, respectively. The two iron-repressed proteins have been previously identified as the oxidative stress defense proteins catalase (KatA) and alkyl hydroperoxide reductase (AhpC). AhpC and KatA were still iron regulated in the
fur
mutant, suggesting the presence of Fur-independent iron regulation. Further analysis of the
C. jejuni
iron and Fur regulons by using two-dimensional gel electrophoresis demonstrated the total number of iron- and Fur-regulated proteins to be lower than for other bacterial pathogens.