Functional identification of the fatty acid reductase components encoded in the luminescence operon of Vibrio fischeri
Functional identification of the fatty acid reductase components encoded in the luminescence operon of Vibrio fischeri
复制标题
费氏弧菌发光操纵子编码的脂肪酸还原酶成分的功能鉴定
作者：
M. Boylan;A. Graham;E. Meighen
从发光菌ATCC7744中获得了一个DNA克隆，将其插入到pBR322中，在大肠杆菌中表达发光。通过对重组质粒在大肠杆菌中合成的蛋白质进行脂肪酸酰化，鉴定了参与光反应脂肪醛底物生物合成的多肽。除了酶切位点上的一些差异和对光的表达需要较低的温度外，该克隆的区域与已报道的费氏弧菌MJ1发光系统(Engebrecht等人，Cell 32：773-781)相似。在含有重组质粒的大肠杆菌提取物中可以检测到脂肪酸还原酶活性，而在亲本菌株的提取物中检测不到脂肪酸还原酶活性。用[~3H]十四酸体内标记，我们发现克隆系统合成的酰化多肽与标记的多肽(34、42和54千道尔顿)相对应，并且只有在诱导发光后才能被检测到。这些结果直接证明了编码脂肪酸还原酶多肽的基因是费氏弧菌发光系统中发光操纵子的组成部分。
A clone of DNA, obtained from the luminescent bacterium Vibrio fischeri ATCC 7744 and inserted into pBR322, was found to express luminescence in Escherichia coli. Polypeptides involved in biosynthesis of the fatty aldehyde substrate for the light reaction were identified by fatty acid acylation of proteins synthesized in E. coli from the recombinant plasmid. The cloned region was similar to that reported for the V. fischeri MJ1 luminescence system (Engebrecht et al., Cell 32:773-781), except for some differences in endonuclease restriction sites and the requirement of a lower temperature for the expression of light in our cloned system. Fatty acid reductase activity could be detected in extracts of E. coli harboring the recombinant plasmid but not in extracts of the parental V. fischeri strain. Using in vivo labeling with [3H]tetradecanoic acid, we showed that the acylated polypeptides synthesized in the cloned system corresponded to the labeled polypeptides in V. fischeri (34, 42, and 54 kilodaltons) and that they could only be detected after induction of luminescence. These results provide direct evidence that the genes coding for the fatty acid reductase polypeptides are an integral part of the luminescence operon in the V. fischeri luminescence system.