Bindal Gargi, Krishnamurthi Revathy, Seshasayee Aswin Sai Narain, Rath Devashish
Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai, India.
Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai, India.
mSphere. 2017 Nov 22;2(6). doi: 10.1128/mSphere.00483-17. eCollection 2017 Nov-Dec.
Bacterial genomes are rich in horizontally acquired prophages. is an essential gene located in the prophage that is resident in many genomes. Employing a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-based gene silencing approach, we show that RacR is a negative regulator of the divergently transcribed and adjacent operon in K-12. Overexpression of YdaS and YdaT due to RacR depletion leads to cell division defects and decrease in survival. We further show that both YdaS and YdaT can act independently as toxins and that RacR serves to counteract the toxicity by tightly downregulating the expression of these toxins. is an essential gene and one of the many poorly studied genes found on the prophage element that is present in many genomes. Employing a CRISPR-based approach, we have silenced expression to various levels and elucidated its physiological consequences. We show that the downregulation of leads to upregulation of the adjacent operon. Both YdaS and YdaT act as toxins by perturbing the cell division resulting in enhanced cell killing. This work establishes a physiological role for RacR, which is to keep the toxic effects of YdaS and YdaT in check and promote cell survival. We, thus, provide a rationale for the essentiality of in K-12 strains.
细菌基因组富含通过水平转移获得的原噬菌体。RacR是位于原噬菌体中的一个必需基因，该原噬菌体存在于许多细菌基因组中。我们采用基于成簇规律间隔短回文重复序列（CRISPR）-Cas的基因沉默方法，证明RacR是大肠杆菌K-12中 divergent转录且相邻的yda操纵子的负调控因子。由于RacR缺失导致的YdaS和YdaT过表达会导致细胞分裂缺陷和存活率降低。我们进一步证明，YdaS和YdaT都可独立作为毒素发挥作用，而RacR通过严格下调这些毒素的表达来抵消其毒性。RacR是一个必需基因，是许多存在于许多细菌基因组中的原噬菌体元件上发现的研究较少的基因之一。我们采用基于CRISPR的方法，将RacR的表达沉默至不同水平，并阐明了其生理后果。我们发现RacR的下调会导致相邻yda操纵子的上调。YdaS和YdaT都通过干扰细胞分裂作为毒素发挥作用，从而增强细胞杀伤作用。这项工作确定了RacR的生理作用，即控制YdaS和YdaT的毒性作用并促进细胞存活。因此，我们为大肠杆菌K-12菌株中RacR的必需性提供了理论依据。
