Trieu-Cuot P, Carlier C, Poyart-Salmeron C, Courvalin P
Unité des Agents Antibactériens, CNRS UA 271, Institut Pasteur, France.
Gene. 1991 Jun 15;102(1):99-104. doi: 10.1016/0378-1119(91)90546-n.
The mobilizable shuttle cloning vectors, pAT18 and pAT19, are composed of: (i) the replication origins of pUC and of the broad-host-range enterococcal plasmid pAM beta 1; (ii) an erythromycin-resistance-encoding gene expressed in Gram- and Gram+ bacteria; (iii) the transfer origin of the IncP plasmid RK2; and (iv) the multiple cloning site and the lacZ alpha reporter gene of pUC18 (pAT18) and pUC19 (pAT19). These 6.6-kb plasmids contain ten unique cloning sites that allow screening of derivatives containing DNA inserts by alpha-complementation in Escherichia coli carrying the lacZ delta M15 deletion, and can be efficiently mobilized by self-transferable IncP plasmids co-resident in the E. coli donors. Plasmids pAT18, pAT19 and recombinant derivatives have been successfully transferred by conjugation from E. coli to Bacillus subtilis, Bacillus thuringiensis, Listeria monocytogenes, Enterococcus faecalis, Lactococcus lactis, and Staphylococcus aureus at frequencies ranging from 10(-6) to 10(-9). The presence of a restriction system in the recipient dramatically affects (by three orders of magnitude) the efficiency of conjugal transfer of these vectors from E. coli to Gram+ bacteria.
可移动穿梭克隆载体pAT18和pAT19由以下部分组成：(i)pUC和广宿主范围的肠球菌质粒pAMβ1的复制起点；(ii)在革兰氏阴性菌和革兰氏阳性菌中表达的红霉素抗性编码基因；(iii)IncP质粒RK2的转移起点；以及(iv)pUC18（pAT18）和pUC19（pAT19）的多克隆位点和lacZα报告基因。这些6.6 kb的质粒含有10个独特的克隆位点，可通过α互补在携带lacZΔM15缺失的大肠杆菌中筛选含有DNA插入片段的衍生物，并且可以被共存在大肠杆菌供体中的自我转移IncP质粒有效转移。质粒pAT18、pAT19及其重组衍生物已通过接合从大肠杆菌成功转移至枯草芽孢杆菌、苏云金芽孢杆菌、单核细胞增生李斯特菌、粪肠球菌、乳酸乳球菌和金黄色葡萄球菌，转移频率范围为10^(-6)至10^(-9)。受体中限制系统的存在（相差三个数量级）极大地影响了这些载体从大肠杆菌向革兰氏阳性菌的接合转移效率。
