CRISPR-Cas-Mediated Gene Silencing Reveals RacR To Be a Negative Regulator of YdaS and YdaT Toxins in Escherichia coli K-12

Abstract

Bacterial genomes are rich in horizontally acquired prophages. <i>racR</i> is an essential gene located in the <i>rac</i> prophage that is resident in many <i>Escherichia coli</i> 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 <i>ydaS-ydaT</i> operon in <i>Escherichia coli</i> 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. <b>IMPORTANCE</b><i>racR</i> is an essential gene and one of the many poorly studied genes found on the <i>rac</i> prophage element that is present in many <i>Escherichia coli</i> genomes. Employing a CRISPR-based approach, we have silenced <i>racR</i> expression to various levels and elucidated its physiological consequences. We show that the downregulation of <i>racR</i> leads to upregulation of the adjacent <i>ydaS-ydaT</i> 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 <i>racR</i> in <i>Escherichia coli</i> K-12 strains.