Coordinate regulation of expression of SdsR toxin and its downstream pphA gene by RyeA antitoxin expression in Escherichia coli

RNA-seq analysis of cells with an ryeA or sdsR promoter mutation In Escherichia coli, SdsR and RyeA, a unique pair of mutually cis-encoded sRNAs, act as toxin and antitoxin, respectively. They are located in the same intergenic region, but transcribed in bidirectional way. Expression of SdsR is reciprocally related to that of RyeA; however, it remains unclear how their syntheses are regulated by each other. Here, using sdsR and ryeA promoter mutant strains, we characterized biosynthesis of the two sRNAs during growth and their coordinate regulation. RyeA transcription occurs even when cells enter the stationary phase, but its apparent expression is restricted to exponentially growing cells because of its degradation by SdsR. The appearance of SdsR was similarly delayed due to its RyeA-mediated degradation. We also found that the sdsR promoter was mostly responsible for transcription of the downstream pphA gene encoding a phosphatase and that pphA mRNA was synthesized through transcriptional read-through over the sdsR terminator. Transcription from the sigma70-dependent ryeA promoter inhibited transcription from the ?S-dependent sdsR promoter through transcriptional interference. This transcription inhibition also downregulated pphA expression, but RyeA itself did not downregulate pphA expression. Our results show that expression of the toxin SdsR and its downstream pphA gene are regulated by expression of the antitoxin RyeA, but differently. Overall design: E. coli cells (wild type, ryeA promoter mutant, and sdsR promoter mutant) were grown at 37? for 3 h and 8 h post-inoculation, 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.