ABSTRACTThe ferric enterobactin (FeEnt) receptor CfrA is present in the majority ofCampylobacter jejuniisolates and is responsible for high-affinity iron acquisition. Our recent work and that of others strongly suggested the existence of another FeEnt uptake system inCampylobacter. Here we have identified and characterized a new FeEnt receptor (designated CfrB) using bothin vitroandin vivosystems. CfrB, a homolog ofC. jejuniNCTC 11168 Cj0444, shares approximately 34% of amino acid identity with CfrA. Alignment of complete CfrB sequences showed that the CfrB is highly conserved inCampylobacter. Immunoblotting analysis using CfrB-specific antiserum demonstrated that CfrB was dramatically induced under iron-restricted conditions and was produced in the majority ofCampylobacter coli(41 out of 45) and in someC. jejuni(8 out of 32) primary strains from various sources and from geographically diverse areas. All of the CfrB-producingC. colistrains also produced CfrA, which was rarely observed in the testedC. jejunistrains. IsogeniccfrB,cfrA, andcfrA cfrBdouble mutants were constructed in 43 diverseCampylobacterstrains. Growth promotion assays using these mutants demonstrated that CfrB has a major role in FeEnt iron acquisition inC. coli. Chicken colonization experiments indicated that inactivation of thecfrBgene alone greatly reduced and even abolishedCampylobactercolonization of the intestines. A growth assay using CfrB-specific antiserum strongly suggested that specific CfrB antibodies could block the function of CfrB and diminish FeEnt-mediated growth promotion under iron-restricted conditions. Together, this work reveals the complexity of FeEnt systems in the two closely relatedCampylobacterspecies and demonstrates the important role of the new FeEnt receptor CfrB inCampylobacteriron acquisition andin vivocolonization.