Deregulation of growth-phase-dependent expression of SdsR causes cell death in Escherichia coli

RNA-seq analysis of cells with pulsed overexpression of SdsR Most small noncoding RNAs (sRNAs) are known to base pair with target mRNAs and regulate their stability or translation with the help of Hfq to trigger various changes in cell metabolism. SdsR (also known as RyeB), which is a member of the RpoS regulon, was reported as an abundant sRNA that represses tolC and mutS in E. coli. It is known to be specific to the stationary phase and is not expressed during the exponential phase, but no previous study had examined the importance of this growth phase-dependent regulation for cell growth and survival. In this study, we examined how forced expression of SdsR during the exponential phase alters cell growth and survival. We found that ectopic expression of SdsR during the exponential phase triggered a significant and Hfq-dependent cell death. This SdsR-driven cell death was alleviated by overexpression of RyeA, an sRNA transcribed on the opposite DNA strand, suggesting that SdsR/RyeA may represent a novel type of toxin/antitoxin (T/A) system in which both the toxin and the antitoxin are sRNAs. To identify target genes involved in the observed SdsR-driven cell death, we first performed RNA-seq analysis and used the RNA-seq data to predict which SdsR-targeted mRNAs could contribute to SdsR-driven cell death. We then examined whether repression of translation of each predicted target mRNA could cause the observed SdsR-driven growth defects, and found that the SdsR-driven cell death seen in our system was mainly caused by the SdsR-mediated repression of yhcB, which encodes an inner membrane protein. E. coli cells containing pHM4T, pSdsR, or pSdsR(45-103) were grown at 37℃ to an OD600 of 1.5~1.7. A final volume of 1 mM IPTG was added to the cell culture, followed by another 10 min and total RNAs were extracted using an RNeasy mini kit (Qiagen). For DNase treatment of 10 μg of total RNA, a Turbo DNA free kit (Ambion) was used according to the manufacturer’s guidelines. All RNA sequencing and alignment procedures were conducted by ChunLab. The Ribo-Zero rRNA removal kit (Epicentre) was used for ribosomal RNA depletion according to manufacturer instructions. Libraries for Illumina sequencing were generated using a TruSeq Stranded mRNA sample prep kit (Illumina) according to the manufacturer’s protocol. RNA sequencing was performed on the Illumina HiSeq 2500 platform using single-end 50 bp sequencing. The sequence data for the reference genome were retrieved form the NCBI database. Quality-filtered reads were aligned to the reference genome sequence using Bowtie2. The relative transcript abundance was measured in Relative Log Expression (RLE). To screen for mRNAs whose levels differed by more than two-fold versus that wild-type cells, we filtered for mRNAs with EdgeR p values < 0.05.