Natural Transformation of an Engineered Helicobacter pylori Strain Deficient in Type II Restriction Endonucleases
Natural Transformation of an Engineered Helicobacter pylori Strain Deficient in Type II Restriction Endonucleases
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DOI：
10.1128/jb.00113-12
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发表时间：
2012-07-01
影响因子：
3.2
通讯作者：
Blaser, Martin J.
中科院分区：
文献类型：
--
作者：
Zhang, Xue-Song;Blaser, Martin J.
限制-修饰(RM)系统对于细菌限制外来DNA入侵具有重要意义。自然活性细菌幽门螺杆菌具有高度多样化的菌株特异性II型系统。为了评价菌株特异性限制性内切酶在幽门螺杆菌自然转化中的作用，构建了一个无标记的II型限制性内切酶缺陷(RED)突变体。我们利用SACB介导的反选择方法删除了幽门螺杆菌26695株中编码所有四种活性II型限制性内切酶的基因。供体DNA与大肠杆菌甲基化的外源盒的转化显著增加(CAT和APHA的转化率分别为1.7和2.0log(10))。来自其他幽门螺杆菌菌株的供体DNA对红色菌株的转化也显著增加，达到了与它们共享的II型R-M系统菌株特异性26695的程度。对红色和野生型菌株的比较表明，限制对自然转化过程中DNA片段整合的长度没有影响。在细胞生长或对DNA损伤的敏感性方面也没有差异。总之，这些数据表明，与野生型相比，II型红色突变体在不损失生长或修复能力的情况下增强了竞争能力，促进了幽门螺杆菌突变体的构建和其他基因工程。
Restriction-modification (RM) systems are important for bacteria to limit foreign DNA invasion. The naturally competent bacterium Helicobacter pylori has highly diverse strain-specific type II systems. To evaluate the roles of strain-specific restriction in H. pylori natural transformation, a markerless type II restriction endonuclease-deficient (REd) mutant was constructed. We deleted the genes encoding all four active type II restriction endonucleases in H. pylori strain 26695 using sacB-mediated counter-selection. Transformation by donor DNA with exogenous cassettes methylated by Escherichia coli was substantially (1.7 and 2.0 log(10) for cat and aphA, respectively) increased in the REd strain. There also was significantly increased transformation of the REd strain by donor DNA from other H. pylori strains, to an extent corresponding to their shared type II R-M system strain specificity with 26695. Comparison of the REd and wild-type strains indicates that restriction did not affect the length of DNA fragment integration during natural transformation. There also were no differentials in cell growth or susceptibility to DNA damage. In total, the data indicate that the type II REd mutant has enhanced competence with no loss of growth or repair facility compared to the wild type, facilitating H. pylori mutant construction and other genetic engineering.