Chen Q, Actis L A, Tolmasky M E, Crosa J H
Department of Molecular Microbiology and Immunology, School of Medicine, Oregon Health Sciences University, Portland 97201-3098.
J Bacteriol. 1994 Jul;176(14):4226-34. doi: 10.1128/jb.176.14.4226-4234.1994.
We have isolated a recombinant clone harboring the chromosomal aroC gene, encoding chorismate synthase, from Vibrio anguillarum 775 by complementation of the Escherichia coli aroC mutant AB2849 which was transfected with a cosmid gene bank of the plasmidless V. anguillarum H775-3. The nucleotide sequence was determined, and an open reading frame that corresponds to a protein of 372 amino acids was found. The calculated mass of 40,417 Da was correlated with the size of the V. anguillarum aroC product detected in vitro. The homology of the V. anguillarum aroC gene to the aroC genes of E. coli and Salmonella typhi is 68% at the nucleotide level and 78% at the protein level. The expression of the aroC transcript is not regulated by iron, as determined by Northern (RNA) blot hybridization analysis. After insertion of an antibiotic resistance gene cassette within the cloned aroC gene, an aroC mutant of V. anguillarum was generated by allelic exchange. This mutant is deficient in the production of 2,3-dihydroxybenzoic acid (2,3-DHBA). Our bioassay and complementation experiments with this mutant demonstrate that the chromosome-mediated 2,3-DHBA is a precursor of the pJM1 plasmid-mediated siderophore anguibactin.
我们通过用无质粒的鳗弧菌H775的黏粒基因文库转染大肠杆菌aroC突变体AB2849进行互补，从鳗弧菌775中分离出一个携带编码分支酸合酶的染色体aroC基因的重组克隆。测定了核苷酸序列，发现一个对应于372个氨基酸的蛋白质的开放阅读框。计算出的40417道尔顿的质量与体外检测到的鳗弧菌aroC产物的大小相关。鳗弧菌aroC基因与大肠杆菌和伤寒沙门氏菌的aroC基因在核苷酸水平上的同源性为68%，在蛋白质水平上为78%。通过Northern（RNA）印迹杂交分析确定，aroC转录本的表达不受铁的调节。在克隆的aroC基因内插入抗生素抗性基因盒后，通过等位基因交换产生了鳗弧菌的aroC突变体。该突变体在2,3-二羟基苯甲酸（2,3-DHBA）的产生上存在缺陷。我们用该突变体进行的生物测定和互补实验表明，染色体介导的2,3-DHBA是pJM1质粒介导的铁载体anguibactin的前体。
