Vectors for lactobacilli and other Gram-positive bacteria based on the minimal replicon of pRV500 from Lactobacillus sakei
Vectors for lactobacilli and other Gram-positive bacteria based on the minimal replicon of pRV500 from Lactobacillus sakei
复制标题
DOI：
10.1016/j.plasmid.2008.08.002
复制
发表时间：
2008-11-01
期刊：
影响因子：
2.6
通讯作者：
Zagorec, Monique
中科院分区：
文献类型：
--
作者：
Coq, Anne-Marie Crutz-Le;Zagorec, Monique
低拷贝数质粒pRV 500，属于pUC 1287组的θ型质粒，先前从清酒乳杆菌中分离并表征。我们在这里表明，该质粒的复制子也可以在粪肠球菌和枯草芽孢杆菌中复制，但不能在乳酸乳球菌中复制。一个1.25 kb的区域，包括iterons和repA基因是足够的复制，拷贝数控制和相对稳定的维护在L清酒。宿主或质粒携带的因素在维护pUCL 287型质粒的功能的影响进行了讨论。将pRV 500的最小复制子与pBluescript融合构建穿梭E.大肠杆菌/乳酸杆菌克隆载体pRV 610。pRV 610使大肠杆菌中的白色/蓝色lacZ α互补成为可能。杆菌用于选择（红霉素抗性）和复制（iteron和repA基因）的盒各自由独特的限制性位点界定，以便在需要时容易替换。构建了氯霉素或四环素抗性取代红霉素抗性的衍生物。为了允许诱导型基因表达，将铜诱导型启动子置于pRV 613衍生物上。下游报告基因IacZ的表达被证明是由30 PM CuSO 4诱导的。(c)2008年爱思唯尔公司All rights reserved.
The low-copy-number plasmid pRV500, belonging to the pUC1287 group of theta-type plasmids, was previously isolated from Lactobacillus sakei and characterized. We show here that the replicon of this plasmid enables replication also in Enterococcus faecalis and Bacillus subtilis but not in Lactococcus lactis. A 1.25 kb region encompassing the iterons and the repA gene was sufficient for replication, copy-number control and relative stable maintenance in L sakei. Functional implications of host or plasmid-borne factors in the maintenance of pUCL287-type plasmids are discussed. The minimal replicon from pRV500 was fused to pBluescript for constructing the shuttle E. coli/lactobacilli cloning vector pRV610. pRV610 enables the white/blue lacZ alpha-complementation in E. coli. The cassettes for selection (erythromycin resistance) and replication (iterons and repA gene) are each bordered by unique restriction sites for easy replacement if needed. Derivatives in which chloramphenicol or tetracycline resistance replaced erythromycin resistance were constlucted. In order to allow inducible gene expression, a copper-inducible promoter was placed on the pRV613 derivative. Expression of the downstream reporter gene IacZ was shown to be induced by 30 PM CuSO4. (c) 2008 Elsevier Inc. All rights reserved.