USE OF A NOVEL CASSETTE TO LABEL PHENOTYPICALLY A CRYPTIC PLASMID OF BACILLUS-SUBTILIS AND MAP LOCI INVOLVED IN ITS STABLE MAINTENANCE
USE OF A NOVEL CASSETTE TO LABEL PHENOTYPICALLY A CRYPTIC PLASMID OF BACILLUS-SUBTILIS AND MAP LOCI INVOLVED IN ITS STABLE MAINTENANCE
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DOI：
10.1099/00221287-136-5-905
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发表时间：
1990-05-01
期刊：
影响因子：
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通讯作者：
THOMAS, CM
中科院分区：
文献类型：
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作者：
GLEAVE, AP;MOUNTAIN, A;THOMAS, CM
为了便于研究革兰氏阳性菌的隐质粒的维持，我们构建了一个新的卡带cAPG1000 (5.0 kb)，它携带一个选择性标记（来自金黄色葡萄球菌质粒pC194的氯霉素耐药性）和一个可筛选标记（来自芽孢杆菌噬菌体SPO2克隆启动子表达的脓杆菌假单胞菌TOL质粒的xtlE基因），其两侧有终止子以防止卡带激活或转录与插入位点相邻的抑制位点。为了证明该磁带的有效性，我们从cAPG1000插入和插入/删除衍生物的特性中绘制了一个8.6 kb的枯草芽孢杆菌内生冷冻质粒（pod2000）稳定维持所需的位点。我们已经确定了复制区域以及分离和结构稳定性所需的单独区域。分离机制是非常有效的，因为它允许没有可检测的损失，尽管事实上，携带质粒的细菌有一个大大增加的平均世代时间。
In order to facilitate studies on the maintenance of cryptic plasmids from Gram-positive bacteria we have constructed a novel cassette cAPG1000 (5.0 kb) which carries both a selectable marker (chloramphenicol resistance from Staphylococcus aureus plasmid pC194) and a screenable marker (the xtlE gene from the TOL plasmid of Pseudomonas putida expressed from a cloned promoter of Bacillus phage SPO2) and which is flanked by terminators to prevent transcription from the cassette activating or inhibiting loci adjacent to the site of insertion. To demonstrate the usefulness of this cassette we have mapped loci required for stable maintenance of an 8.6 kb crytic plasmid endogenous to Bacillus subtilis (pPOD2000) from the properties of cAPG1000 insertion and insertion/deletion derivatives. We have identified the replication region as well as separate regions required for segregational and structural stability. The segregational mechanism is very efficient since it allows no detectable loss despite the fact that bacteria carrying the plasmid have a greatly increased mean generation time.