Département de Biochimie, de Microbiologie, et de Bio-informatique, Faculté des Sciences et de Génie, Université Laval, Québec City, Québec, Canada.
Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, Québec City, Québec, Canada.
mSphere. 2021 May 19;6(3):e00185-21. doi: 10.1128/mSphere.00185-21.
strain P42S possesses a type II-A CRISPR-Cas system that protects against phage infection and plasmid transformation. The analysis of 293 bacteriophage-insensitive mutants (BIMs) obtained upon exposure to the virulent phage M102AD revealed the acquisition of 399 unique spacers, including several ectopic spacer acquisitions and a few cases of native spacer deletions. The acquisition of multiple spacers was also observed and appears to be mostly due to priming, which has been rarely reported for type II-A systems. Analyses of the acquired spacers indicated that 88% of them are identical to a region of the phage M102AD genome. The remaining 12% of spacers had mismatches with the phage genome, primarily at the 5' end of the spacer, leaving the seed sequence at the 3' end largely intact. When a high multiplicity of infection (MOI) was used in the phage challenge assays, we also observed the emergence of CRISPR BIMs that, in addition to the acquisition of new spacers, displayed a reduced phage adsorption phenotype. While CRISPR-Cas and adsorption resistance work in tandem to protect P42S against phage M102AD, nonidentified antiviral mechanisms are also likely at play in this strain. Bacteria are under the constant threat of viral predation and have therefore developed several defense mechanisms, including CRISPR-Cas systems. While studies on the mode of action of CRISPR-Cas systems have already provided great insights into phage-bacterium interactions, still more information is needed on the biology of these systems. The additional characterization of the type II-A CRISPR-Cas system of P42S in this study provides novel information on the spacer acquisition step, especially regarding protospacer-adjacent motif (PAM) recognition, multiple-spacer acquisition, and priming.
菌株 P42S 拥有 II-A 型 CRISPR-Cas 系统，可抵御噬菌体感染和质粒转化。对暴露于毒性噬菌体 M102AD 后获得的 293 个噬菌体不敏感突变体 (BIM) 的分析揭示了 399 个独特间隔序列的获得，包括几个异位间隔序列的获得和少数情况下的天然间隔序列缺失。还观察到了多个间隔序列的获得，这似乎主要是由于引发作用，而这种作用在 II-A 系统中很少报道。对获得的间隔序列的分析表明，它们中有 88%与噬菌体 M102AD 基因组的一个区域相同。其余 12%的间隔序列与噬菌体基因组存在错配，主要在间隔序列的 5'端，使得 3'端的种子序列基本完整。当在噬菌体挑战试验中使用高感染复数 (MOI) 时，我们还观察到 CRISPR BIM 的出现，除了获得新的间隔序列外，这些 BIM 还表现出噬菌体吸附表型降低。虽然 CRISPR-Cas 和吸附抗性协同作用以保护 P42S 免受噬菌体 M102AD 的侵害，但该菌株中可能还存在未识别的抗病毒机制。细菌不断受到病毒捕食的威胁，因此已经开发出几种防御机制，包括 CRISPR-Cas 系统。虽然关于 CRISPR-Cas 系统作用模式的研究已经为噬菌体-细菌相互作用提供了重要的见解，但仍需要更多关于这些系统生物学的信息。本研究中对 P42S 的 II-A 型 CRISPR-Cas 系统的进一步表征提供了关于间隔序列获得步骤的新信息，特别是关于原间隔序列相邻基序 (PAM) 识别、多个间隔序列获得和引发作用。
