Analysis of regulatory genes for production of hemolysin of Vibrio cholerae.

1.我们将hlyU的内部片段克隆到自杀载体质粒中，该片段被报道为hlyA的正调节因子。将重组质粒导入霍乱弧菌N86中，通过单交换法将hlyU基因破坏。在hlyU缺失的突变体中观察到溶血活性几乎没有降低。我们在几个溶血性霍乱弧菌非O1菌株中使用自杀质粒构建了hlyU缺失突变体。在少数突变株中溶血活性降低，但在大多数菌株中没有观察到破坏hlyU的效果。这表明hlyU并不总是所有菌株中必不可少的正调控因子，在霍乱弧菌中可能存在hlyU以外的未知调控系统。为筛选溶血素产生的调控基因，将霍乱弧菌0.6kb的DNA片段转化到溶血性N86菌株中。获得两株溶血活性降低的菌株（IM 1和IM 2）。利用TIGR基因组数据库分析质粒插入位点的DNA序列。插入位点分别位于IM2的emrD和IM1的ORF内。IM 2中hlyA的mRNA完全减少，而IM 1中则观察到hlyA的mRNA。这些结果表明，emrD周围的区域，这是同源的大肠杆菌的多药耐药基因，与溶血素的生产调节。

1. We cloned the inner fragment of hlyU which is reported as positive regulator for hlyA to a suicide vector plasmid. The recombinant plasmid was introduced to Vibrio cholerae N86 and hlyU was destroyed by single crossover by the plasmid. Little decrease of the hemolytic activity was observed in the hlyU-deleted mutant. We constructed the hlyU-deleted mutant using the suicide plasmid in several hemolytic V.cholerae non-O1 strains. Hemolytic activities in a few mutants was reduced, but, no effect was observed in most of the strains by destroying hlyU.This suggest that hlyU is not always essential positive regulator in all strains and that an unknown regulatory system other than hlyU may exit in V.cholerae.2. To screen unknown gene for regulation of the hemolysin production, suicide vector containing 0.6-kb DNA fragment of whole DNA of V.cholerae was introduced to hemolytic N86 strain. Two strains (IM1 and IM2) which reduced in hemolytic activities were obtained. The DNA sequences of the insertion sites of plasmids were analyzed using TIGR genome data base. The insertion sites were inside of emrD in IM2 and a ORF in IM1, respectively. mRNAs of hlyA in IM2 was completely diminished and that of IM1 was observed. These results suggest that the region around emrD, which is homologous to the multi-drug resistant gene of Escherichia coli, is related to regulation of hemolysin production.