Henderson D P, Payne S M
Department of Microbiology, University of Texas at Austin 78712.
J Bacteriol. 1994 Jun;176(11):3269-77. doi: 10.1128/jb.176.11.3269-3277.1994.
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.
对霍乱弧菌中编码一种77 kDa铁调节外膜蛋白（血红素铁利用所需）的hutA基因的调控进行了表征，并确定了该基因的DNA序列。先前构建的hutA::Tn5 lac融合体（D. P. 亨德森和S. M. 佩恩，《分子微生物学》7:461 - 469，1993）被转化到大肠杆菌和霍乱弧菌的Fur - 和Fur + 菌株中。对转化菌株进行的β - 半乳糖苷酶分析结果表明，hutA的转录在大肠杆菌中受Fur阻遏蛋白调控，在霍乱弧菌中至少部分受Fur调控。对hutA的DNA序列分析表明，在hutA的启动子区域存在一个与大肠杆菌共有Fur框同源的序列。HutA的氨基酸序列与几种依赖TonB的外膜蛋白的序列同源。然而，当除了在第二个载体上携带的hutA外还需要重组质粒pHUT10编码的一个或多个基因的霍乱弧菌血红素利用系统被转移到大肠杆菌的野生型菌株和同基因tonB突变体中时，tonB突变体利用血红素铁的效率与野生型菌株一样高。这些数据表明，在大肠杆菌中重建的霍乱弧菌血红素利用系统不需要功能性的TonB蛋白。用血红素利用质粒转化的tonB突变体不能利用铁载体高铁色素作为铁源，这表明血红素利用质粒上编码的基因都不能弥补大肠杆菌中的tonB缺陷。有可能重组血红素利用质粒编码的一个基因或多个基因在霍乱弧菌中编码一种具有类似TonB功能的蛋白。HutA羧基末端的一个区域与马血红蛋白γ链同源，并且在γ链中形成血红素口袋所涉及的氨基酸在HutA中是保守的。这些数据表明HutA的这个区域参与血红素结合。
