RNase LS was originally identified as a potential antagonist of bacteriophage T4 infection. When T4 dmd is defective, RNase LS activity rapidly increases after T4 infection and cleaves T4 mRNAs to antagonize T4 reproduction. Here we show that rnlA, a structural gene of RNase LS, encodes a novel toxin, and that rnlB (formally yfjO), located immediately downstream of rnlA, encodes an antitoxin against RnlA. Ectopic expression of RnlA caused inhibition of cell growth and rapid degradation of mRNAs in DrnlAB cells. On the other hand, RnlB neutralized these RnlA effects. Furthermore, overexpression of RnlB in wild-type cells could completely suppress the growth defect of a T4 dmd mutant, that is, excess RnlB inhibited RNase LS activity. Pull-down analysis showed a specific interaction between RnlA and RnlB. Compared to RnlA, RnlB was extremely unstable, being degraded by ClpXP and Lon proteases, and this instability may increase RNase LS activity after T4 infection. All of these results suggested that rnlA-rnlB define a new toxin-antitoxin (TA) system. B ACTERIAL toxin-antitoxin (TA) systems are composed of a stable toxin and an unstable antitoxin (reviewed in Engelberg-Kulka and Glaser 1999). There are two different types of TA systems depending on the nature of antitoxin. In the type I systems, antitoxin is a small regulatory RNA that blocks the translation of toxin (Gerdes and Wagner 2007). In the type II systems, both toxin and antitoxin are proteins and antitoxin neutralizes toxin by direct interaction (Zhang et al. 2003a). When expression from type II TA loci is impaired by various kinds of stresses, such as amino acid starvation or translational inhibition by antibiotics (Christensen et al. 2001;Sat et al. 2001), antitoxin is rapidly decreased and consequently the level of toxin unbound (UB) with antitoxin is increased, leading to the activation of toxin (reviewed in Gerdes et al. 2005).RNase LS contributes to mRNA turnover in Escherichia coli, although its effect seems modest in comparison to that of a major RNase, RNase E (Otsuka and Yonesaki 2005). Recently we found one important role for this RNase in the physiology of E. coli cells: it targets cyaA mRNA (encoding adenylate cyclase) to reduce its expression (Iwamoto et al. 2008). Interestingly, the activity of RNase LS becomes much stronger after T4 infection ( We surveyed the E. coli DNA sequence in the vicinity of rnlA and found a promoter-like sequence, the open reading frame (ORF) of rnlA, the ORF of the downstream gene rnlB (formerly yfjO), and a terminator-like sequence consistently aligned in this order, suggesting that rnlA and rnlB form an operon. In addition, the terminal region in the rnlA ORF and the start region of the rnlB ORF overlap by 7 bp, implying an intimate coupling in their expression. These features prompted us to inquire whether rnlB is involved in RNase LS activity. In this study, we demonstrate that RnlB suppresses RNase LS activity. We also demonstrated that expression of RnlA in the absence of RnlB degrades E. coli bulk ...

SummaryEnterohaemorrhagic Escherichia coli O157:H7 harbours a cryptic plasmid, pOSAK1, that carries only three ORFs: mobA (involved in plasmid mobilization), ORF1 and ORF2. Predicted proteins encoded by these two ORFs were found to share a weak homology with RnlA and RnlB, respectively, a toxin-antitoxin system encoded on the E. coli K-12 chromosome. Here, we report that lsoA (ORF1) encodes a toxin and lsoB (ORF2) an antitoxin. In spite of the homologies, RnlB and LsoB functioned as antitoxins against only their cognate toxins and not interchangeably with each other. Interestingly, T4 phage Dmd suppressed the toxicities of both RnlA and LsoA by direct interaction, the first example of a phage with an antitoxin against multiple toxins.

Bacteriophages have strict host specificity and the step of adsorption is one of key factors for determining host specificity. Here, we systematically examined the interaction between the Escherichia coli receptors lipopolysaccharide (LPS) and outer membrane protein C (OmpC), and the long tail fibers of bacteriophage T4. Using a variety of LPS mutants, we demonstrated that T4 has no specificity for the sugar sequence of the outer core (one of three LPS regions) in the presence of OmpC but, in the absence of OmpC, can adsorb to a specific LPS which has only one or two glucose residues without a branch. These results strengthen the idea that T4 adsorbs to E. coli via two distinct modes, OmpC‐dependent and OmpC‐independent, suggested by previous reports (Prehm et al. 1976; Yu and Mizushima 1982). Isolation and characterization of the T4 mutants Nik (No infection to K‐12 strain), Nib (No infection to B strain), and Arl (altered recognition of LPS) identified amino acids of the long tail fiber that play important roles in the interaction with OmpC or LPS, suggesting that the top surface of the distal tip head domain of T4 long tail fibers interacts with LPS and its lateral surface interacts with OmpC.

The dmd gene of bacteriophage T4 is required for the stability of late-gene mRNAs. When this gene is mutated, late genes are globally silenced because of rapid degradation of their mRNAs. Our previous work suggested that a novel Escherichia coli endonuclease, RNase LS, is responsible for the rapid degradation of mRNAs. In this study, we demonstrated that rnlA (formerly yfjN) is essential for RNase LS activity both in vivo and in vitro. In addition, we investigated a role of RNase LS in the RNA metabolism of E. coli cells under vegetative growth conditions. A mutation in rnlA reduced the decay rate of many E. coli mRNAs, although there are differences in the mutational effects on the stabilization of different mRNAs. In addition, we found that a 307-nucleotide fragment with an internal sequence of 23S rRNA accumulated to a high level in rnlA mutant cells. These results strongly suggest that RNase LS plays a role in the RNA metabolism of E. coli as well as phage T4.

Gene uvsX of phage T4 controls genetic recombination and the repair of DNA damage. We have recently purified the gene product, and here describe its properties. The protein has a single‐stranded DNA‐dependent ATPase activity. It binds efficiently to single‐ and double‐stranded DNAs at 0 degrees C in a cooperative manner. At 30 degree C the double‐stranded DNA‐protein complex was stable, but the single‐stranded DNA‐protein complex dissociated rapidly. The instability of the latter complex was reduced by ATP. The protein renatured heat‐denatured double‐stranded DNA, and assimilated linear single‐stranded DNA into homologous superhelical duplexes to produce D‐loops. The reaction is stimulated by gene 32 protein when the uvsX protein is limiting. With linear double‐stranded DNA and homologous, circular single‐stranded DNA, the protein catalyzed single‐strand displacement in the 5′ to 3′ direction with the cooperation of gene 32 protein. All reactions required Mg2+, and all except DNA binding required ATP. We conclude that the uvsX protein is directly involved in strand exchange and is analogous to the recA protein of Escherichia coli. The differences between the uvsX protein and the recA protein, and the role of gene 32 protein in single‐strand assimilation and single‐strand displacement are briefly discussed.