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首页> 外文期刊>Applied Microbiology >Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli
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Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

机译：组蛋白样核结构蛋白（H-NS）普拉拉莫治疗STPA激活I-E型CRISPR-CAS系统，免受大肠杆菌的自然转化

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Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (P _(cas) ) and suppresses the type I-E CRISPR-Cas system in Escherichia coli . Although the H-NS paralogue StpA also binds P _(cas) , its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli . We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of P _(cas) . Third, by comparing transcriptional activities of the intact P _(cas) and the P _(cas) with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in P _(cas) . Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of P _(cas) . Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli .IMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli . However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli . In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli . Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.
机译：CRISPR-CAS系统的工作机制已经集中研究。但是，关于它们的监管方式众所周知。组蛋白样核结构蛋白H-NS结合CAS基因的启动子（P _（CAS））并抑制大肠杆菌中的I-E CRISPR-CAS系统。虽然H-NS Paralogue STPA还结合P _（CAS），但其在调节CRISPR-CAS系统方面的作用仍未认定。我们以前的工作确立了大肠杆菌能够在天然转化期间占用双链DNA。在这里，我们调查了STPA在调节I-E CRISPR-CAS系统免受大肠杆菌的自然变换的作用。我们首先记录了虽然由于HNS缺失，虽然活化型I-E CRISPR-CAS系统，但干扰了CRISPR-CAS靶向质粒转移，STPA失活恢复了天然转化水平。其次，我们表明失活的STPA降低了P _（CAS）的转录活性。第三，通过将完整的P _（CAS）和P _（CAS）的转录活性进行比较HNS和HNS STPA缺失突变体中断的H-NS结合位点，我们证明STPA通过结合激活CAS基因的转录在P _（CAS）中的H-NS相同的网站。第四，通过用阿拉伯糖诱导的启动子表达STPA，我们证实在低水平下表达的STPA刺激了P _（CAS）的活性。最后，通过量化成熟克隆RNA（CRRNA）的水平，我们证明了STPA能够促进CRRNA的量。我们的工作建立了STPA作为调节类型的转录激活剂，即抗大肠杆菌的自然转化的CRISPR-CAS系统.Importance STPA通常被认为是核结构蛋白H-NS的分子备份，即报告称IE在大肠杆菌中类型的转录压缩机。然而，STPA在调节I-E CRISPR-CAS系统中的作用仍然难以捉摸。我们以前的作品发现了在大肠杆菌的天然转化期间的双链DNA（DSDNA）入口的新途径。在这项研究中，我们表明，STPA与其普拉拉威治疗I-E CRISPR-CAS系统对抗大肠杆菌的自然转化相反的作用。我们的工作不仅扩大了我们对细胞外核酸的Crispr-CAS介导的适应性免疫的了解，而且还对了解CRISPR-CAS系统的复杂调节机制，还揭示了新的光。此外，Paralogues STPA和H-NS共享DNA结合位点但在转录调节中发挥相反的作用表明组蛋白样蛋白的细菌染色质的高阶压实可以切换原核转录模式。

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