Department of Medicine, New York University School of Medicine and VA Medical Center, New York, New York, USA.
J Bacteriol. 2012 Jul;194(13):3407-16. doi: 10.1128/JB.00113-12. Epub 2012 Apr 20.
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 counterselection. 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.
限制修饰（RM）系统对于细菌限制外源 DNA 入侵非常重要。自然感受态细菌幽门螺杆菌具有高度多样化的菌株特异性 II 型系统。为了评估菌株特异性限制在幽门螺杆菌自然转化中的作用，构建了一种无标记的 II 型限制内切酶缺陷（REd）突变体。我们使用 sacB 介导的反向选择，在幽门螺杆菌菌株 26695 中缺失编码所有四种活性 II 型限制内切酶的基因。用大肠杆菌甲基化的供体 DNA 转化，REd 菌株的转化效率显著提高（cat 和 aphA 的转化效率分别提高了 1.7 和 2.0 个对数）。来自其他幽门螺杆菌菌株的供体 DNA 对 REd 菌株的转化效率也显著提高，与它们与 26695 共享的 II 型 R-M 系统菌株特异性相对应。REd 菌株和野生型菌株的比较表明，限制并不影响自然转化过程中 DNA 片段整合的长度。细胞生长或对 DNA 损伤的敏感性也没有差异。总的来说，数据表明，与野生型相比，II 型 Red 突变体具有增强的感受态，而不会丧失生长或修复能力，这有利于幽门螺杆菌突变体的构建和其他遗传工程。
