Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Biol Pharm Bull. 2010;33(2):198-203. doi: 10.1248/bpb.33.198.
Using the par to rep region of the 24653 bp plasmid pN315, which is present in Staphylococcus aureus strain N315, we constructed three vectors that can be shuttled between Escherichia coli and S. aureus and maintained stably in S. aureus. Due to plasmid incompatibility, the resident plasmid in S. aureus cells can be replaced via transformation with an entering plasmid, which carries a different drug resistance gene. To evaluate the applicability of this plasmid-based approach for identifying genes essential for S. aureus cell growth, the chromosomal mraY gene, which is involved in peptidoglycan biosynthesis, was deleted in cells harboring a resident plasmid with an intact mraY gene. The resultant disruptant was then transformed with an empty vector. Cells with a chromosomal mraY deletion but lacking the plasmid supplying mraY could not be recovered, suggesting that mraY is indispensable for staphylococcal cell growth or viability. In contrast, other two genes were shown to be dispensable by this system. Thus, the pN315-based plasmids appear to be useful for studying genes essential for S. aureus cell growth.
我们利用存在于金黄色葡萄球菌 N315 菌株中的 24653bp 质粒 pN315 的 par 到 rep 区，构建了三个可在大肠杆菌和金黄色葡萄球菌之间穿梭并在金黄色葡萄球菌中稳定维持的载体。由于质粒不相容性，可通过转化携带不同耐药基因的进入质粒来替换金黄色葡萄球菌细胞中的常驻质粒。为了评估基于质粒的方法在鉴定金黄色葡萄球菌细胞生长所必需基因中的适用性，我们在含有完整 mraY 基因的常驻质粒的细胞中缺失了参与肽聚糖生物合成的染色体 mraY 基因。然后，用空载体转化该缺失突变体。缺乏提供 mraY 的质粒但缺失染色体 mraY 的细胞不能被回收，表明 mraY 对葡萄球菌细胞生长或活力是必需的。相比之下，该系统表明其他两个基因是可有可无的。因此，基于 pN315 的质粒似乎可用于研究金黄色葡萄球菌细胞生长所必需的基因。
