Cooney J, Mulvey M, Arbuthnott J P, Foster T J
Microbiology Department, Moyne Institute, Trinity College, Dublin, Ireland.
J Gen Microbiol. 1988 Aug;134(8):2179-88. doi: 10.1099/00221287-134-8-2179.
The gamma-lysin determinant of Staphylococcus aureus strain Smith 5R has been cloned in phage lambda and plasmid vectors in Escherichia coli. Genetic evidence is presented which demonstrates that gamma-lysin requires the co-operative action of two polypeptides expressed by the closely linked hlgA and hlgB genes. Recombinants expressed haemolytic activity in agarose medium but not in agar, a known property of gamma-lysin. Haemolysis was inhibited by antiserum raised against the 32 kDa component of gamma-lysin, but not by anti-alpha-, anti-beta- or anti-delta-lysin serum. Subcloning and transposon Tn5 mutagenesis identified a 3.5 kb region which was necessary for gamma-lysin expression in E. coli. Two genes (hlgA and hlgB) were mapped and their polypeptide products identified. Non-haemolytic Tn5 mutants fell into two groups based upon complementation tests done between extracts of mutants in vitro and also between extracts of mutants and components of gamma-lysin purified from S. aureus culture supernates. Immunoblotting showed that some mutants in group A (defective in expression of hlgA) did not express a 32 kDa polypeptide which was synthesized by the parental haemolytic recombinant and by mutants in group B. Minicell analysis suggested that the products of the hlgB gene were proteins of 38 kDa and 36 kDa. The smaller molecule co-migrates with a protein in a fraction of the S. aureus culture supernate containing component B of gamma-lysin. The 38 kDa polypeptide is probably an unprocessed precursor. Southern hybridization demonstrated that the hlgA and hlgB genes are closely linked in the chromosome of several strains of S. aureus.
金黄色葡萄球菌史密斯5R菌株的γ-溶血素决定簇已在大肠杆菌中克隆到噬菌体λ和质粒载体中。本文提供的遗传学证据表明，γ-溶血素需要紧密连锁的hlgA和hlgB基因表达的两种多肽协同作用。重组体在琼脂糖培养基中表现出溶血活性，但在琼脂中则无此活性，这是γ-溶血素的已知特性。γ-溶血素的32 kDa组分抗血清可抑制溶血，但α-、β-或δ-溶血素抗血清则无此作用。亚克隆和转座子Tn5诱变鉴定出一个3.5 kb区域，该区域是γ-溶血素在大肠杆菌中表达所必需的。定位了两个基因（hlgA和hlgB）并鉴定了它们 的多肽产物。根据体外突变体提取物之间以及突变体提取物与从金黄色葡萄球菌培养上清液中纯化的γ-溶血素组分之间的互补试验，非溶血Tn5突变体分为两组。免疫印迹显示，A组中的一些突变体（hlgA表达缺陷）不表达32 kDa多肽，而亲本溶血重组体和B组中的突变体可合成该多肽。微小细胞分析表明，hlgB基因的产物是38 kDa和36 kDa的蛋白质。较小的分子与含有γ-溶血素组分B的金黄色葡萄球菌培养上清液中的一种蛋白质共迁移。38 kDa多肽可能是未加工的前体。Southern杂交表明，hlgA和hlgB基因在几种金黄色葡萄球菌菌株的染色体中紧密连锁。
