Characterization of the Vibrio cholerae outer membrane heme transport protein 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 iron-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, Mol. Microbiol. 7:461-469, 1993) was transformed into Fur- and Fur+ strains of Escherichia coli and V. cholerae. The results of beta-galactosidase assays on the transformed strains demonstrated that transcription of hutA is regulated by the Fur repressor protein in E. coli and at least partially regulated by Fur in V. cholerae. Analysis of the DNA sequence of hutA indicated that a sequence homologous to the 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 TonB-dependent outer member proteins. However, when the V. cholerae heme utilization system, which requires one or more genes encoded by the recombinant plasmid pHUT10 in addition to hutA carried on a second vector, 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 utilization system reconstituted in E. coli does not require a functional TonB protein. The tonB mutant transformed with the heme utilization plasmids could not utilize the siderophore ferrichrome as an iron source, 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 gamma chain, and the amino acids involved in forming the heme pocket in the gamma chain are conserved in HutA. These data suggest that this region of HutA is involved in heme binding.
Henderson
D P
DP
Department of Microbiology, University of Texas at Austin 78712.
Payne
S M
SM
D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D006418:Heme / Q000378:metabolism*; D006454:Hemoglobins / Q000235:genetics; D006736:Horses / Q000235:genetics; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D012097:Repressor Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014734:Vibrio cholerae / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D006454:Hemoglobins; C087477:HutA protein, Vibrio cholerae; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; D006418:Heme