细菌原型适应性免疫系统,CRISPR-Cas,被认为在研究最好的细菌中被抑制,大肠杆菌K-12。我们在这里表明,大肠杆菌CRISPR-Cas系统是活跃的,并用于抑制其9种缺陷(即,神秘)预言。具体来说,与野生型菌株相比,减少特定干扰RNA(crRNA)的数量会使生长减少40%,增加细胞死亡700%,并阻止持久细胞复苏。通过删除整个13个间隔区(CRISPR阵列)使CRISPR-Cas失活,获得了类似的结果;因此,CRISPR-Cas用于抑制这些隐秘的预言的剩余有害作用,最有可能的是通过CRISPR阵列衍生的crRNA结合到隐秘的progagemRNA,而不是通过隐秘的progageDNA的切割,即,自我定位。始终如一,13个大肠杆菌间隔区中的4个含有与7个隐匿性原噬菌体的mRNA序列互补的区域,CRISPR-Cas的失活增加了隐匿性噬菌体Qin的裂解蛋白YdfD和隐匿性噬菌体DLP-12的裂解蛋白RzoD的mRNA水平。此外,当整个CRISPR-Cas阵列被删除时,通过透射电子显微镜可以清楚地看到裂解,并消除间隔区#12,其编码与DLP-12(包括rzoD)互补区的crRNA,Rac,秦(包括ydfD),和CP4-57神秘的预言,也导致生长抑制和细胞裂解。因此,我们报告了新的结果,即(i)CRISPR-Cas在大肠杆菌中具有活性,(ii)CRISPR-Cas用于驯服隐秘的预言,可能是通过RNAi,即,与活性溶原不同,活跃的CRISPR-Cas和隐秘的预言可能稳定共存。
The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be repressed in the best-studied bacterium, Escherichia coli K-12. We show here that the E. coli CRISPR-Cas system is active and serves to inhibit its nine defective (i.e., cryptic) prophages. Specifically, compared to the wild-type strain, reducing the amounts of specific interfering RNAs (crRNA) decreases growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation. Similar results were obtained by inactivating CRISPR-Cas by deleting the entire 13 spacer region (CRISPR array); hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages, most likely through CRISPR array-derived crRNA binding to cryptic prophage mRNA rather than through cleavage of cryptic prophage DNA, i.e., self-targeting. Consistently, four of the 13 E. coli spacers contain complementary regions to the mRNA sequences of seven cryptic prophages, and inactivation of CRISPR-Cas increases the level of mRNA for lysis protein YdfD of cryptic prophage Qin and lysis protein RzoD of cryptic prophage DLP-12. In addition, lysis is clearly seen via transmission electron microscopy when the whole CRISPR-Cas array is deleted, and eliminating spacer #12, which encodes crRNA with complementary regions for DLP-12 (including rzoD), Rac, Qin (including ydfD), and CP4-57 cryptic prophages, also results in growth inhibition and cell lysis. Therefore, we report the novel results that (i) CRISPR-Cas is active in E. coli and (ii) CRISPR-Cas is used to tame cryptic prophages, likely through RNAi, i.e., unlike with active lysogens, active CRISPR-Cas and cryptic prophages may stably co-exist.