Poyart C, Trieu-Cuot P
Laboratoire de Microbiologie, Unité INSERM 411, Faculté de Médecine Necker-Enfants Malades, Paris, France.
FEMS Microbiol Lett. 1997 Nov 15;156(2):193-8. doi: 10.1111/j.1574-6968.1997.tb12726.x.
A low-copy-number vector designated pTCV-lac has been constructed to provide a convenient system to analyze regulatory elements in Gram-positive bacteria. The main components of this vector are: (i) the origins of replication of pACYC184 and of the broad-host-range enterococcal plasmid pAM beta 1, (ii) erythromycin- and kanamycin-resistance-encoding genes for selection in Gram-negative and Gram-positive bacteria, (iii) the transfer origin of the IncP plasmid RK2, and (iv) a promoterless beta-galactosidase-encoding lacZ gene with a Gram-positive ribosome binding site. This 12 kb plasmid is present in Gram-positive hosts in three to five copies per chromosome equivalent and contains three unique cloning sites (EcoRI, SmaI, BamHI) for cloning of DNA inserts upstream of the lacZ gene. Plasmid pTCV-lac and derivatives carrying different promoter fragments have been transferred by conjugation from an Escherichia coli IncP mobilizing donor strain to Bacillus subtilis, Listeria monocytogenes, Enterococcus faecalis, and Streptococcus agalactiae. These plasmids were structurally stable in these hosts and the corresponding promoter activities, quantitated by the determination of the beta-galactosidase specific activities, were found to cover at least a 100-fold range in beta-galactosidase values. These results indicate that pTCV-lac should be useful for analysis of gene regulation in a wide range of Gram-positive bacteria.
已构建了一种低拷贝数载体，命名为pTCV-lac，用于提供一个便捷系统来分析革兰氏阳性菌中的调控元件。该载体的主要组成部分包括：（i）pACYC184的复制起点和广宿主范围的肠球菌质粒pAMβ1的复制起点；（ii）用于在革兰氏阴性菌和革兰氏阳性菌中进行筛选的红霉素和卡那霉素抗性编码基因；（iii）IncP质粒RK2的转移起点；（iv）一个无启动子的β-半乳糖苷酶编码lacZ基因，带有革兰氏阳性核糖体结合位点。这个12 kb的质粒在革兰氏阳性宿主中以每染色体当量三到五个拷贝的形式存在，并包含三个独特的克隆位点（EcoRI、SmaI、BamHI），用于在lacZ基因上游克隆DNA插入片段。携带不同启动子片段的质粒pTCV-lac及其衍生物已通过接合从大肠杆菌IncP可动员供体菌株转移至枯草芽孢杆菌、单核细胞增生李斯特菌、粪肠球菌和无乳链球菌。这些质粒在这些宿主中结构稳定，通过测定β-半乳糖苷酶的比活性来定量的相应启动子活性，其β-半乳糖苷酶值的范围至少覆盖100倍。这些结果表明，pTCV-lac对于分析多种革兰氏阳性菌中的基因调控应该是有用的。
