MOD-1 Facility, Virulence Mechanisms Branch, (HFS-025), Division of Virulence Assessment, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD 20708, USA.
Can J Microbiol. 2011 Sep;57(9):714-21. doi: 10.1139/w11-058. Epub 2011 Aug 19.
Vibrio tubiashii expresses virulence factors, such as a vulnificolysin-like hemolysin or cytolysin and a zinc metalloprotease, similar to those of other pathogenic vibrios. In this study, we report the cloning of a novel hemolysin gene of V. tubiashii in Escherichia coli . A V. tubiashii gene library was screened for hemolytic activity on sheep blood agar. Three hemolytic clones pGem:hly1, pGem:hly2, and pGem:hly3 were sequenced, and the sequences showed a strong homology to the ribA gene coding for guanosine triphosphate cyclohydrolase II (GCH II), required for riboflavin biosynthesis and reported to be responsible for hemolytic activity in Helicobacter pylori . The plasmids pGem:hly1 and pGem:hly3 when introduced into E. coli BSV18 (ribA18::Tn5) were able to restore growth of strain BSV18 in a medium without riboflavin and also produced hemolytic activity on blood agar. PCR primers based on the cloned hly-ribA sequence were tested using 23 different Vibrio strains representing 10 different species. Amplification of ribA gene locus only occurred with V. tubiashii strains. In summary, our results indicate that we have cloned a ribA homolog of V. tubiashii that imparts hemolytic activity to E. coli clones, and primers based on this gene locus might be useful as a species-specific identification tool for V. tubiashii.
创伤弧菌表达的毒力因子，如类副溶血性弧菌溶血素或细胞毒素和锌金属蛋白酶，与其他致病性弧菌相似。在本研究中，我们报告了创伤弧菌在大肠杆菌中一种新型溶血素基因的克隆。创伤弧菌基因文库在绵羊血琼脂上筛选溶血活性。三个溶血克隆 pGem:hly1、pGem:hly2 和 pGem:hly3 被测序，其序列与编码鸟苷三磷酸环化水解酶 II（GCH II）的 ribA 基因具有很强的同源性，GCH II 是核黄素生物合成所必需的，并且据报道在幽门螺杆菌中负责溶血活性。当将质粒 pGem:hly1 和 pGem:hly3 导入大肠杆菌 BSV18（ribA18::Tn5）时，能够恢复缺乏核黄素的培养基中 BSV18 的生长，并且在血琼脂上也产生溶血活性。基于克隆的 hly-ribA 序列的 PCR 引物使用代表 10 个不同种的 23 种不同的弧菌菌株进行了测试。只有创伤弧菌菌株扩增了 ribA 基因座。总之，我们的结果表明，我们已经克隆了创伤弧菌的 ribA 同源物，它赋予大肠杆菌克隆溶血活性，并且基于该基因座的引物可能是创伤弧菌的一种有用的种特异性鉴定工具。
