High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes
High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes
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DOI：
10.1111/j.1574-6968.1997.tb13882.x
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发表时间：
1997-10-15
影响因子：
2.1
通讯作者：
Smith, CP
中科院分区：
文献类型：
--
作者：
Flett, F;Mersinias, V;Smith, CP
许多链霉菌，包括天蓝色链霉菌 A3(2)，都拥有有效的甲基特异性限制系统，可以为异源 DNA 的引入提供有效的屏障。我们使用两种大肠杆菌供体（标准甲基化熟练菌株 S17-1 和甲基化缺陷供体 ET12567(pUB307)）比较了不同类型质粒向 S. coelicolor 和 S. lividans 66 的属间接合转移效率。我们证明甲基化缺陷供体可以产生比标准供体多> 10(4)倍的天蓝色链球菌外缀合物。对于通过位点特异性重组整合到宿主染色体中的 pSET152 衍生物来说，接合混合物中高达 10% 的链霉菌孢子继承了该质粒。接合程序足够有效，可以获得具有“自杀”递送质粒的接合子，因此提供了在甲基DNA限制性链霉菌和可能的其他细菌中进行基因破坏的简单途径。
Many streptomycetes, including S. coelicolor A3(2), possess a potent methyl-specific restriction system which can present an effective barrier to the introduction of heterologous DNA. We have compared the efficiency of intergeneric conjugal transfer of different types of plasmids to S. coelicolor and S. lividans 66 using two E. coli donors: the standard, methylation proficient strain S17-1, and the methylation deficient donor, ET12567(pUB307). We demonstrate that the methylation deficient donor can yield > 10(4)-fold more S. coelicolor exconjugants than the standard donor. In the case of pSET152 derivatives, which integrate into the host chromosome by site-specific recombination, up to 10% of streptomycete spores in the conjugation mixture inherit the plasmid. The conjugation procedure is efficient enough to obtain exconjugants with 'suicide' delivery plasmids and therefore provides a simple route for conducting gene disruptions in methyl DNA-restricting streptomycetes, and possibly other bacteria.