Plasmid transfer and genetic recombination by protoplast fusion in staphylococci
Plasmid transfer and genetic recombination by protoplast fusion in staphylococci
复制标题
葡萄球菌中原生质体融合的质粒转移和基因重组
作者：
F. Götz;S. Ahrné;M. Lindberg
本文介绍了利用原生质体融合技术在金黄色葡萄球菌和凝固酶阴性葡萄球菌中进行质粒转移和基因重组的实验条件。通过在含有0.7至0.8M蔗糖的缓冲培养基中用溶葡萄球菌酶和溶菌酶处理来制备原生质体。在含有琥珀酸盐和牛血清白蛋白的高渗琼脂培养基上完成细胞壁的再生。用聚乙二醇（分子量6，000）处理原生质体混合物后，质粒的转移不仅发生在同一物种的菌株之间，而且发生在不同物种的亲本之间，尽管在后一种情况下频率低约100倍。融合原生质体中染色体基因的纯化需要同时用聚乙二醇和氯化钙处理混合原生质体。建立了一种分离重组子的方法。携带非选择性标记的黑曲霉。将抗生素抗性质粒导入亲本菌株中，并用作检测原生质体融合的初级标记。染色体重组体中发现的克隆与双亲质粒在高频率。该方法似乎有简单的应用程序中构建的菌株具有多个突变性状。
The experimental conditions for plasmid transfer and genetic recombination in Staphylococcus aureus and some coagulase-negative staphylococci by protoplast fusion are described. Protoplasts were prepared by treatment with lysostaphin and lysozyme in a buffered medium with 0.7 to 0.8 M sucrose. Regeneration of cell walls was accomplished on a hypertonic agar medium containing succinate and bovine serum albumin. Transfer of plasmids occurred after treatment of the protoplast mixtures with polyethylene glycol (molecular weight, 6,000) not only between strains of the same species but also between parents of different species, although at approximately 100 times lower frequency in the latter case. Recombination of the chromosomal genes in fused protoplasts required simultaneous treatment of the mixed protoplasts with polyethylene glycol and CaCl2. A method was developed for isolation of recombinants after fusion between mutants of S. areus carrying unselectable markers. Antibiotic resistance plasmids were introduced into the parental strains and used as primary markers to detect protoplast fusion. Chromosomal recombinants were found among the clones with both parental plasmids at a high frequency. The method appears to have simple applications in the construction of strains with multiple mutant characters.