CLONING AND EXPRESSION IN ESCHERICHIA-COLI AND STAPHYLOCOCCUS-AUREUS OF THE BETA-LYSIN DETERMINANT FROM STAPHYLOCOCCUS-AUREUS - EVIDENCE THAT BACTERIOPHAGE CONVERSION OF BETA-LYSIN ACTIVITY IS CAUSED BY INSERTIONAL INACTIVATION OF THE BETA-LYSIN DETERMINANT
CLONING AND EXPRESSION IN ESCHERICHIA-COLI AND STAPHYLOCOCCUS-AUREUS OF THE BETA-LYSIN DETERMINANT FROM STAPHYLOCOCCUS-AUREUS - EVIDENCE THAT BACTERIOPHAGE CONVERSION OF BETA-LYSIN ACTIVITY IS CAUSED BY INSERTIONAL INACTIVATION OF THE BETA-LYSIN DETERMINANT
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DOI：
10.1016/0882-4010(86)90040-9
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发表时间：
1986-12-01
影响因子：
3.8
通讯作者：
BIRKBECK, TH
中科院分区：
文献类型：
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作者：
COLEMAN, DC;ARBUTHNOTT, JP;BIRKBECK, TH
利用噬菌体置换载体lambda.L47.1在大肠杆菌K-12中克隆了金黄色葡萄球菌CN6708的β-裂解素决定簇(Hib)。通过将Hib+重组噬菌体的片段克隆到表达载体pACYC184和pBR322中，并通过构建和分析几个亚克隆、体外缺失和Tn5插入突变，将Hib决定簇定位于1250个碱基对的DNA序列。含有Hib+质粒的大肠杆菌细胞可以很容易地检测到位于细胞质中的β-溶血素和鞘磷脂酶活性。在大肠杆菌小细胞中检测到由Hib决定簇编码的两个相对分子质量分别为38000和33000的多肽，而用特异性抗-β-赖氨酸血清进行免疫印迹实验仅检测到33000个道尔顿蛋白。与克隆的Hib决定簇和转化葡激酶噬菌体vphi.13的DNA的杂交分析表明，金黄色葡萄球菌的噬菌体转化为β-溶酶活性的丧失是由于插入了vphi。13DNA进入或邻近β-溶素决定簇。用穿梭质粒将克隆的Hib决定簇转移到β-溶血素阴性的金黄色葡萄球菌中，其中野生型染色体决定簇通过溶源转换被灭活。在含有克隆决定簇的金黄色葡萄球菌上清液中很容易检测到β-溶酶活性。
The beta-lysin determinant(Hib) from Staphylococcus aureus CN6708 was cloned in Escherichia coli K-12 using the bacteriophage replacement vector .lambda.L47.1. The Hib determinant was localised to a 1250 base pair DNA sequence by cloning fragments from a Hib+ recombinant phage into the plasmid vectors pACYC184 and pBR322 in E. coli K-12, and by the subsequent construction and analysis of several sub-clones, in vitro deletion and Tn5 insertin mutations. E. coli cells harbouring Hib+ plasmids expressed readily detectable levels of .beta.-lysin and sphingomyelinase activity, which were located in the cytoplasm. Two polypeptides of molecular weight 38 000 and 33 000 which were encoded by the Hib determinant were detected in E. coli minicells, but only the 33 000 dalton protein was detected in immunoblotting experiments with specific anti-.beta.-lysin serum. Hybridisation analysis with probes made from the cloned Hib determinant and from DNA of the staphylokinase-converting phage .vphi.13, indicated that bacteriophage conversion of S. aureus to loss of .beta.-lysin activity is due to insertion of .vphi. 13 DNA into or adjacent to the .beta.-lysin determinant. A shuttle plasmid was used to transfer the cloned Hib determinant into a .beta.-lysin negative strain of S. aureus where the wild-type chromosomal determinant was inactivated by lysogenic conversion. Beta-lysin activity was readily detected in supernatants of S. aureus harbouring the cloned determinant.