Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli
Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli
复制标题
类组蛋白类核结构蛋白 (H-NS) 旁系同源物 StpA 激活 I-E 型 CRISPR-Cas 系统以对抗大肠杆菌中的自然转化
DOI：
10.1128/aem.00731-20
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发表时间：
2020-07-01
影响因子：
4.4
通讯作者：
Qiu, Juanping
中科院分区：
文献类型：
--
作者：
Sun, Dongchang;Mao, Xudan;Qiu, Juanping
StpA通常被认为是核苷酸结构蛋白H-NS的分子备份，H-NS被报道为大肠杆菌中I-E型CRISPR-Cas系统的转录阻遏物。然而，StpA在调节I-E型CRISPR-Cas系统中的作用仍然难以捉摸。我们的前期工作揭示了双链DNA（dsDNA）在大肠杆菌自然转化过程中进入的新途径。杆菌在这项研究中，我们发现StpA在调节I-E型CRISPR-Cas系统对抗E.杆菌我们的工作不仅扩展了我们对CRISPR-Cas介导的针对细胞外核酸的适应性免疫的知识，而且还为理解CRISPR-Cas系统的复杂调控机制提供了新的思路。此外，发现旁系同源物StpA和H-NS共享一个DNA结合位点，但在转录调控中发挥相反的作用，表明组蛋白样蛋白对细菌染色质的高阶压缩可以切换原核转录模式。CRISPR-Cas系统的工作机制已被深入研究。然而，人们对它们是如何被监管的知之甚少。组蛋白样类核结构蛋白H-NS结合cas基因的启动子（Pcas）并抑制大肠杆菌中的I-E型CRISPR-Cas系统。虽然H-NS parasitic StpA也结合Pcas，但其在调节CRISPR-Cas系统中的作用仍未确定。我们以前的工作建立了E.大肠杆菌能够在自然转化过程中吸收双链DNA。在这里，我们研究了StpA在调节I-E型CRISPR-Cas系统抵抗E.杆菌我们首先记录了，尽管由于hns缺失，激活的I-E型CRISPR-Cas系统干扰了CRISPR-Cas靶向质粒转移，但stpA失活恢复了天然转化的水平。其次，我们发现stpA失活降低了Pcas的转录活性。第三，通过比较完整的Pcas和Pcas与H-NS结合位点被破坏的hns和hns stpA空缺失突变体的转录活性，我们证明了StpA通过结合到Pcas中与H-NS相同的位点来激活cas基因的转录。第四，通过用阿拉伯糖诱导型启动子表达StpA，我们证实了低水平表达的StpA刺激Pcas的活性。最后，通过定量成熟CRISPR RNA（crRNA）的水平，我们证明了StpA能够促进crRNA的量。综上所述，我们的工作确定了StpA在调节I-E型CRISPR-Cas系统对抗大肠杆菌的自然转化中充当转录激活剂。杆菌重要性StpA通常被认为是类核结构蛋白H-NS的分子备份，H-NS被报道为大肠杆菌中I-E型CRISPR-Cas系统的转录阻遏物。然而，StpA在调节I-E型CRISPR-Cas系统中的作用仍然难以捉摸。我们的前期工作揭示了双链DNA（dsDNA）在大肠杆菌自然转化过程中进入的新途径。杆菌在这项研究中，我们发现StpA在调节I-E型CRISPR-Cas系统对抗E.杆菌我们的工作不仅扩展了我们对CRISPR-Cas介导的针对细胞外核酸的适应性免疫的知识，而且还为理解CRISPR-Cas系统的复杂调控机制提供了新的思路。此外，发现旁系同源物StpA和H-NS共享一个DNA结合位点，但在转录调控中发挥相反的作用，表明组蛋白样蛋白对细菌染色质的高阶压缩可以切换原核转录模式。
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. ABSTRACT 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 (Pcas) and suppresses the type I-E CRISPR-Cas system in Escherichia coli. Although the H-NS paralogue StpA also binds Pcas, 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 Pcas. Third, by comparing transcriptional activities of the intact Pcas and the Pcas 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 Pcas. Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of Pcas. 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.