Specialized transduction:: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M-bovis BCG and M-smegmatis
Specialized transduction:: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M-bovis BCG and M-smegmatis
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DOI：
10.1099/00221287-148-10-3007
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发表时间：
2002-10-01
期刊：
影响因子：
2.8
通讯作者：
Jacobs, WR
中科院分区：
文献类型：
--
作者：
Bardarov, S;Bardarov, S;Jacobs, WR
作者开发了一种简单而高效的系统，用于在快速和缓慢生长的分枝杆菌中产生等位基因交换。在该方法中，将被选择标记破坏的感兴趣的基因克隆到条件复制（温度敏感）穿梭质粒中以产生专门的转导分枝杆菌噬菌体。分枝杆菌噬菌体基因组中的温度敏感性突变允许在30 ℃的允许温度下复制，但阻止在37 ℃的非允许温度下复制。在非允许温度下的转导导致重组底物高效递送至受体群体中的几乎所有细胞。靶基因中的缺失突变用侧翼为γ δ-res（解离酶识别靶）位点的抗肿瘤抗性基因标记。已经经历同源重组事件的转导子可以方便地在含有抗生素的培养基上选择。为了证明该遗传系统的效用，在牛分枝杆菌BCG、结核分枝杆菌三株和耻垢分枝杆菌的三个亚株中产生了七种不同的靶向基因破坏。分离lysA、nadBC、panC、panCD、leuCD、Rv 3291 c和Rv 0867 c基因或操纵子中的突变体作为抗寄生虫（并且在某些情况下营养缺陷型）转导子。使用编码γ δ-解离酶（tnpR）的质粒，可以去除抗性基因，产生未标记的缺失突变。从本研究中观察到的高频率的等位基因交换事件可以得出结论，特化转导是一种非常有效的分枝杆菌遗传操作技术，是构建分枝杆菌等基因菌株的首选方法。结核、BCG或M.它们的区别在于确定的突变。
The authors have developed a simple and highly efficient system for generating allellic exchanges in both fast- and slow-growing mycobacteria. In this procedure a gene of interest, disrupted by a selectable marker, is cloned into a conditionally replicating (temperature-sensitive) shuttle phasmid to generate a specialized transducing mycobacteriophage. The temperature-sensitive mutations in the mycobacteriophage genome permit replication at the permissive temperature of 30 degreesC but prevent replication at the nonpermissive temperature of 37 degreesC. Transduction at a non-permissive temperature results in highly efficient delivery of the recombination substrate to virtually all cells in the recipient population. The deletion mutations in the targeted genes are marked with antibiotic-resistance genes that are flanked by gammadelta-res (resolvase recognition target) sites. The transductants which have undergone a homologous recombination event can be conveniently selected on antibiotic-containing media. To demonstrate the utility of this genetic system seven different targeted gene disruptions were generated in three substrains of Mycobacterium bovis BCG, three strains of Mycobacterium tuberculosis, and Mycobacterium smegmatis. Mutants in the lysA, nadBC, panC, panCD, leuCD, Rv3291c and Rv0867c genes or operons were isolated as antibiotic-resistant (and in some cases auxotrophic) transductants. Using a plasmid encoding the gammadelta-resolvase (tnpR), the resistance genes could be removed, generating unmarked deletion mutations. It is concluded from the high frequency of alliellic exchange events observed in this study that specialized transduction is a very efficient technique for genetic manipulation of mycobacteria and is a method of choice for constructing isogenic strains of M. tuberculosis, BCG or M. smegmatis which differ by defined mutations.