Most small noncoding RNAs (sRNAs) are known to base pair with target mRNAs and regulate mRNA stability or translation to trigger various changes in the cell metabolism of Escherichia coli. The SdsR sRNA is expressed specifically during the stationary phase and represses tolC and mutS expression. However, it was not previously known whether the growth-phase-dependent regulation of SdsR is important for cell growth. Here, we ectopically expressed SdsR during the exponential phase and examined cell growth and survival. We found that ectopic expression of SdsR led to a significant and Hfq-dependent cell death with accompanying cell filamentation. This SdsR-driven cell death was alleviated by overexpression of RyeA, an sRNA transcribed on the opposite DNA strand, suggesting that SdsR/RyeA is a novel type of toxin-antitoxin (T/A) system in which both the toxin and the antitoxin are sRNAs. We defined the minimal region required for the SdsR-driven cell death. We also performed RNA-seq analysis and identified 209 genes whose expression levels were altered by more than twofold following pulse expression of ectopic SdsR at exponential phase. Finally, we found that that the observed SdsR-driven cell death was mainly caused by the SdsR-mediated repression of yhcB, which encodes an inner membrane protein.

RpoS is a key regulator of general stress responses in Escherichia coli. Its expression is post-transcriptionally up-regulated by the small RNAs (sRNAs), ArcZ, DsrA and RprA, through sRNA-rpoS mRNA interactions. Although overexpression of the sRNA, CyaR, was reported to down-regulate rpoS expression, how CyaR regulates rpoS has not been determined. Here, we report that CyaR represses rpoS expression by base-pairing with a region next to the ArcZ binding site in the 5ʹ UTR of rpoS mRNA and that CyaR expression itself is down-regulated by ArcZ through sRNA-sRNA interaction. The short form of ArcZ, but not the full-length form, can base-pair with CyaR. This ArcZ-CyaR interaction triggers degradation of CyaR by RNase E, alleviating the CyaR-mediated rpoS repression. These results suggest that ArcZ not only participates in rpoS translation as an activator, but also acts as a regulator of the reciprocally acting CyaR, maximizing its rpoS-activating effect.

In Escherichia coli , SdsR and RyeA, a unique pair of mutually cis -encoded small RNAs (sRNAs), act as toxin and antitoxin, respectively. SdsR and RyeA expression are reciprocally regulated; however, how each regulates the synthesis of the other remains unclear. Here, we characterized the biosynthesis of the two sRNAs during growth and investigated their coordinate regulation using sdsR and ryeA promoter mutant strains. We found that RyeA transcription occurred even upon entry of cells into the stationary phase, but its apparent expression was restricted to exponentially growing cells because of its degradation by SdsR. Likewise, the appearance of SdsR was delayed owing to its RyeA-mediated degradation. We also found that the sdsR promoter was primarily responsible for transcription of the downstream pphA gene encoding a phosphatase and that pphA mRNA was synthesized by transcriptional read-through over the sdsR terminator. Transcription from the σ 70 -dependent ryeA promoter inhibited transcription from the σ S -dependent sdsR promoter through transcriptional interference. This transcriptional inhibition also downregulated pphA expression, but RyeA itself did not downregulate pphA expression.

In Escherichia coli, initiation of chromosomal replication occurs at the replication origin, oriC. The binding of various proteins to the replication origin regulates the initiation of chromosomal DNA synthesis. Previously, Cnu, which was also named YdgT, was identified as an oriC-binding protein. 1 Cnu shares extensive sequence similarity to the nucleoid-associated Hha/YmoA protein family. Cnu and Hha together form a complex with H-NS and the complex binds oriC. 1 Cnu interacts with H-NS and StpA (H-NS paralogue) and either Cnu-StpA or Hha-StpA interaction can protect StpA from Lon-mediated proteolysis. 2 As both the cnu and hha mutation lead to the reduction of origin content, Cnu is supposed to participate in the regulation of the replication initiation. 1 Considering that replication initiation is coupled to growth rate, cnu expression should be regulated in accordance to growth stages. However, it has not known yet how cnu expression is regulated. In an effort to address this question, we examined promoter regions responsible for cnu transcription. For this purpose, we used primer extension analysis to determine 5 0 ends of cnu mRNA. Total RNAs prepared from exponentially growing or stationary-phase E. coli cells were used for primer extension analysis ( Figure 1). We found six major primer extension products whose 5 0 ends correspond to −59/−60, −96, −118/−119, −136, −143, and − 184 relative to the ATG start codon of Cnu. A homology search of the upstream sequences disclosed three promoter regions P1, P2, and P3, whose predicted transcription start sites well match to −59/−60, −136, and − 184, respectively. Since the other primer extension products to positions −96, −118/−119, and − 143 were not at the predicted transcription start sites, they might be produced from degradation products of the upstream transcripts. Therefore, the primer extension analysis suggests that cnu transcription occurs at the three tandem promoters. P3 transcription was prominent in the exponential phase, while P1 was active in the stationary phase. P2 transcription is functional in both the exponential and stationary phases.We then examined further in detail how cnu expression is regulated during growth. Total RNAs were prepared from cell cultures at different growth stages and subjected to primer extension analysis ( Figure 2). P3 was highly active in the mid-exponential phase (5 h growth), but its (a) (b) (c) Figure 1. Determination of 5 0 ends of cnu mRNA. (a) Identification of primer extension products. JM109 cells containing plasmid pHL355-36 were used for the preparation of total cellular RNAs. The 32 P-labeled primer Ext was used for primer extension. The products were analyzed by 8% denaturing polyacrylamide gel electrophoresis. The sequencing ladders (G, A, T, and C) were prepared with primer Ext. The numbers on the left side of the gel indicate the 5 0 end positions. E and S stand for exponentially growing and stationary-phase cells, respectively. (B) Schematic map of the cnu gene and its tandem promoters. (C) Sequences o...