East A K, Dyke K G
Department of Biochemistry, University of Oxford, UK.
J Gen Microbiol. 1989 Apr;135(4):1001-15. doi: 10.1099/00221287-135-4-1001.
The plasmid-encoded beta-lactamase genes of six strains of Staphylococcus aureus were cloned and shown to be expressed in Escherichia coli. The cloned genes were re-introduced into S. aureus via a shuttle vector, and expressed beta-lactamase. However, clones containing only the small amount of DNA found necessary for expression of ampicillin resistance in E. coli did not express beta-lactamase in S. aureus. Much larger pieces of DNA from the original plasmid were necessary to obtain expression in S. aureus. Some of the six strains of S. aureus synthesized beta-lactamase constitutively and some released only a small proportion of the enzyme into the medium. Both these characteristics were maintained in the clones so it is concluded that they are features either of the gene itself or of the surrounding DNA. The cloned genes were sequenced and the putative amino acid sequences of the beta-lactamases were compared. There are several differences between the sequences and in particular one change in the N-terminal region, at a position believed to be especially important for export of proteins from the cell, is thought to have a key effect on whether or not the enzyme is found in the medium.
对六株金黄色葡萄球菌的质粒编码β-内酰胺酶基因进行了克隆，并证明其在大肠杆菌中表达。通过穿梭载体将克隆的基因重新导入金黄色葡萄球菌中，并表达β-内酰胺酶。然而，仅含有在大肠杆菌中表达氨苄青霉素抗性所需少量DNA的克隆在金黄色葡萄球菌中不表达β-内酰胺酶。需要来自原始质粒的大得多的DNA片段才能在金黄色葡萄球菌中获得表达。六株金黄色葡萄球菌中的一些菌株组成性地合成β-内酰胺酶，而一些菌株仅将一小部分酶释放到培养基中。这两个特征在克隆中都得以保留，因此得出结论，它们是基因本身或周围DNA的特征。对克隆的基因进行了测序，并比较了β-内酰胺酶的推定氨基酸序列。序列之间存在几个差异，特别是在N端区域有一个变化，据信该位置对于蛋白质从细胞中输出特别重要，被认为对酶是否存在于培养基中有关键影响。
