Heme utilization in Vibrio cholerae and analysis of domains involved in the specificity of TonB for TonB-dependent receptors

Vibrio cholerae has multiple iron transport systems, one of which involves heme uptake through the outer membrane receptor HutA. This study demonstrates that V. cholerae encodes two additional TonB-dependent heme receptors, HutR and HasR. HutR has significant homology to HutA and to other bacterial outer membrane heme receptors, and the role of HutR in hemin utilization and its localization in the outer membrane were confirmed. The hutR gene was co-transcribed with the upstream gene ptrB, and expression from the ptrB promoter was negatively regulated by iron. HasR is most similar to the hemophore-utilizing heme receptors from Pseudomonas aeruginosa and Serratia marcescens. A mutant defective in all three heme receptors was unable to utilize hemin as an iron source. HutA and HutR functioned with either V. cholerae TonB1 or TonB2. In contrast, hemin uptake through HasR was TonB2- dependent. Efficient utilization of hemoglobin as an iron source required HutA and TonB1. The triple heme receptor mutant exhibited no defect in its ability to compete with its Vib- parental strain in an infant mouse model of infection, indicating that additional iron sources are present in vivo. V. cholerae utilized heme derived from marine invertebrate hemoglobins, suggesting that heme may be available to V. cholerae growing in the marine environment. Although E. coli TonB and V. cholerae TonB1 exhibit different specificities for outer membrane receptors, these TonB proteins are similar enough that functional chimeras can be created between them. The activities of the chimeric TonB proteins demonstrated that the C-terminal one-third of TonB constitutes a functional domain responsible for receptor specificity. A Pro238Thr substitution in V. cholerae TonB1 resulted in the ability of TonB1 to recognize a wider range of receptors, indicating that very C-terminal end of TonB1 determines receptor specificity. Domain-switching experiments between E. coli ChuA and V. cholerae HutA showed that the TonB box heptapeptide at the Nterminus of these receptors does not contain specificity determinants. Instead, specificity was controlled by the residue immediately preceding the TonB box. Taken together, these data suggest that functional interactions take place between the C-terminus of TonB and the very N-terminal domain of TonB-dependent receptors.