Publications by Year: 1994

Henderson DP, Payne SM. 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. J Bacteriol. 176 (11) :3269-77.Abstract
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.
Payne SM. Detection, isolation, and characterization of siderophores. Methods Enzymol. 235 :329-44.
Henderson DP, Payne SM. Vibrio cholerae iron transport systems: roles of heme and siderophore iron transport in virulence and identification of a gene associated with multiple iron transport systems. Infect Immun. 62 (11) :5120-5.Abstract
Vibrio cholerae iron transport mutants were tested for their ability to cause disease in an infant mouse model. The mice were challenged with either the wild-type strain, a vibriobactin synthesis mutant, a heme utilization mutant, or double mutants containing both the vibriobactin synthesis defect and the heme utilization defect. When mice were challenged with 10(7) bacteria, the ability of the double mutant to survive in the intestines was greatly reduced and that of the heme utilization mutant was slightly reduced compared with that of the wild type or the vibriobactin synthesis mutant. When the inoculum size was reduced 10-fold, all of the iron transport mutants failed to colonize the intestines and failed to cause diarrhea in the mice, whereas the wild-type strain was not cleared and elicited a diarrheal response. These data indicate that disruption of either the heme utilization or the vibriobactin uptake system reduces the ability of V. cholerae to cause disease. One of the heme utilization mutants, DHH1, was found to be defective also in utilization of vibriobactin and ferrichrome, mimicking the Escherichia coli TonB- phenotype. This mutant was the least virulent of the iron transport mutants tested. Transformation of DHH1 with the recombinant plasmid pHUT4 restored the abilities to use hemin, vibriobactin, and ferrichrome as iron sources, suggesting that pHUT4 encodes a gene(s) involved globally in the iron transport systems. Hybridization of Vibrio DNA with the V. cholerae heme utilization genes demonstrated the presence of DNA homologous to the genes encoding the outer membrane protein HutA and the inner membrane protein HutB in all the V. cholerae strains tested. The probe containing hutA, but not that containing hutB, also hybridized to DNA from Vibrio parahaemolyticus.