Departamento de Biología Molecular, Universidad de Cantabria and Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), CSIC-Universidad de Cantabria, Santander, Cantabria, Spain.
PLoS Genet. 2020 Apr 29;16(4):e1008750. doi: 10.1371/journal.pgen.1008750. eCollection 2020 Apr.
Plasmids, when transferred by conjugation in natural environments, must overpass restriction-modification systems of the recipient cell. We demonstrate that protein ArdC, encoded by broad host range plasmid R388, was required for conjugation from Escherichia coli to Pseudomonas putida. Expression of ardC was required in the recipient cells, but not in the donor cells. Besides, ardC was not required for conjugation if the hsdRMS system was deleted in P. putida recipient cells. ardC was also required if the hsdRMS system was present in E. coli recipient cells. Thus, ArdC has antirestriction activity against the HsdRMS system and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and presence of Mn2+. ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity was not needed for the antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggered an SOS response in the P. putida recipient cells. Our findings give new insights, and open new questions, into the antirestriction strategies developed by plasmids to counteract bacterial restriction strategies and settle into new hosts.
质粒在自然环境中通过接合转移时，必须越过受体细胞的限制修饰系统。我们证明，由广谱宿主范围质粒 R388 编码的蛋白 ArdC 是大肠杆菌到假单胞菌属接合所必需的。ArdC 在受体细胞中表达是必需的，但在供体细胞中则不是必需的。此外，如果在假单胞菌属受体细胞中缺失 hsdRMS 系统，则不需要 ardC 进行接合。如果 hsdRMS 系统存在于大肠杆菌受体细胞中，也需要 ardC。因此，ArdC 对 HsdRMS 系统具有抗限制活性，从而扩大了 R388 质粒的宿主范围。在没有和存在 Mn2+的情况下，解析了 ArdC 的晶体结构。ArdC 由一个非特异性的 ssDNA 结合 N 端结构域和一个 C 端金属蛋白酶结构域组成，尽管金属蛋白酶活性对于抗限制功能不是必需的。我们还通过 RNA-seq 观察到，ArdC 依赖性接合在假单胞菌属受体细胞中触发了 SOS 反应。我们的发现为质粒对抗细菌限制策略并定居到新宿主中所开发的抗限制策略提供了新的见解，并提出了新的问题。
