Ando T, Xu Q, Torres M, Kusugami K, Israel D A, Blaser M J
Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine and VA Medical Center, Nashville, TN, USA.
Mol Microbiol. 2000 Sep;37(5):1052-65. doi: 10.1046/j.1365-2958.2000.02049.x.
Helicobacter pylori cells are naturally competent for the uptake of both plasmid and chromosomal DNA. However, we demonstrate that there are strong barriers to transformation of H. pylori strains by plasmids derived from unrelated strains. We sought to determine the molecular mechanisms underlying these barriers. Transformation efficiency was assessed using pHP1, an Escherichia coli-H. pylori shuttle vector conferring kanamycin resistance. Transformation of 33 H. pylori strains was attempted with pHP1 purified from either E. coli or H. pylori, and was successfully introduced into only 11 strains. Digestion of H. pylori chromosomes with different restriction endonucleases (REs) showed that DNA methylation patterns vary substantially among strains. The strain most easily transformed, JP26, was found to have extremely low endogenous RE activity and to lack a restriction-modification (R-M) system, homologous to MboI, which is highly conserved among H. pylori strains. When we introduced this system to JP26, pHP1 from MboI.M+ JP26, but not from wild-type JP26, transformed MboI R-M+ JP26 and heterologous MboI R-M+ wild-type H. pylori strains. Parallel studies with pHP1 from dam+ and dam- E. coli strains confirmed these findings. These data indicate that the endogenous REs of H. pylori strains represent a critical barrier to interstrain plasmid transfer among H. pylori.
幽门螺杆菌细胞天然具有摄取质粒DNA和染色体DNA的能力。然而，我们证明，来自不相关菌株的质粒对幽门螺杆菌菌株的转化存在强大障碍。我们试图确定这些障碍背后的分子机制。使用pHP1评估转化效率，pHP1是一种赋予卡那霉素抗性的大肠杆菌-幽门螺杆菌穿梭载体。用从大肠杆菌或幽门螺杆菌中纯化的pHP1尝试转化33株幽门螺杆菌菌株，仅11株成功导入。用不同的限制性内切酶（REs）消化幽门螺杆菌染色体表明，菌株间的DNA甲基化模式差异很大。最容易转化的菌株JP26，其内源RE活性极低，并且缺乏与MboI同源的限制修饰（R-M）系统，该系统在幽门螺杆菌菌株中高度保守。当我们将该系统引入JP26时，来自MboI.M+ JP26的pHP1能够转化MboI R-M+ JP26和异源MboI R-M+野生型幽门螺杆菌菌株，而来自野生型JP26的pHP1则不能。用来自dam+和dam-大肠杆菌菌株的pHP1进行的平行研究证实了这些发现。这些数据表明，幽门螺杆菌菌株的内源REs是幽门螺杆菌菌株间质粒转移的关键障碍。
