CHARACTERIZATION OF THE VIBRIO-CHOLERAE OUTER-MEMBRANE HEME TRANSPORTPROTEIN HUTA - SEQUENCE OF THE GENE, REGULATION OF EXPRESSION, AND HOMOLOGY TO THE FAMILY OF TONB-DEPENDENT PROTEINS

The regulation of hutA, the Vibrio cholerae gene encoding a 77-kDa iro n-regulated outer membrane protein required for heme iron utilization, was characterized, and the DNA sequence of the gene was determined. A hutA::Tn5 lac fusion generated previously (D. P. Henderson and S. M. Payne, Mel. Microbiol. 7:461-469, 1993) was transformed into Fur(-) an d Fur(+) strains of Escherichia coli and V. cholerae. The results of b eta-galactosidase assays on the transformed strains demonstrated that transcription of hutA is regulated by the Fur repressor protein in E. coil and at least partially regulated by Fur in V. cholerae. Analysis of the DNA sequence of hutA indicated that a sequence homologous to th e E. coli consensus Fur box was present in the promoter region of hutA . The amino acid sequence of HutA is homologous to those of several To nB-dependent outer member proteins. However, when the V. cholerae heme utilization system, which requires one or more genes encoded by the r ecombinant plasmid pHUT10 in addition to hutA Carried on a second vect or, was transferred to a wild-type strain and an isogenic tonB mutant of E. coli, the tonB mutant could utilize heme iron as efficiently as the wild-type strain. These data indicate that the V. cholerae heme ut ilization system reconstituted in E. coli does not require a functiona l TonB protein. The tonB mutant transformed with the heme utilization plasmids could not utilize the siderophore ferrichrome as an iron sour ce, indicating that none of the genes encoded on the heme utilization plasmids complements the tonB defect in E. coli. It is possible that a gene(s) encoded by the recombinant heme utilization plasmids encodes a protein serving a TonB-like function in V. cholerae. A region in the carboxy terminus of HutA is homologous to the horse hemoglobin zeta c hain, and the amino acids involved in forming the heme pocket in the z eta chain are conserved in HutA. These data suggest that this region o f HutA is involved in heme binding.