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.

中文翻译：

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组蛋白样核糖结构蛋白（H-NS）旁系蛋白StpA激活IE CRISPR-Cas型系统以抵抗大肠杆菌中的自然转化。

CRISPR-Cas系统的工作机制已被深入研究。但是，人们对其监管方式知之甚少。组蛋白样核苷结构蛋白H-NS结合cas基因的启动子（P _cas）并抑制大肠杆菌中的IE CRISPR-Cas系统。尽管H-NS旁系同源蛋白StpA也与P _cas结合，但其在调控CRISPR-Cas系统中的作用尚不清楚。我们以前的工作证明，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IE CRISPR-Cas系统抵抗大肠杆菌自然转化中的功能。。我们首先证明，尽管由于hns缺失，激活的IE型CRISPR-Cas系统干扰了CRISPR-Cas靶向的质粒转移，但stpA失活恢复了自然转化的水平。第二，我们表明灭活stpA降低了P _cas的转录活性。第三，通过比较完整的P _cas和hns和hns stpA null缺失突变体中具有破坏的H-NS结合位点的P _cas的转录活性，我们证明StpA通过与H相同的位点结合来激活cas基因的转录。-P中的_NS。第四，通过用阿拉伯糖诱导型启动子表达StpA，我们证实了StpA的低表达刺激了P _cas的活性。最后，通过定量成熟的CRISPR RNA（crRNA）的水平，我们证明了StpA能够促进crRNA的数量。两者合计，我们的工作建立了StpA作为调节IE CRISPR-Cas系统抵抗大肠杆菌自然转化的转录激活因子。