Escherichia coli rnlA and rnlB Compose a Novel Toxin–Antitoxin System

Abstract

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 ...

The ClpXP Protease Is Responsible for the Degradation of the Epsilon Antidote to the Zeta Toxin of the Streptococcal pSM19035 Plasmid

“…There are also known TA systems that use the two-component protease ClpP, which cooperates with the ATPase-active chaperones ClpA or ClpX (11,12). Moreover, degradation of the antidote can be carried out by more than one protease (12)(13)(14)(15). In contrast to the E. coli antitoxins, knowledge of the proteolysis of antitoxins from other bacterial species is scarce.…”

AbpA and AbpB provide anti-phage activity in <i>Escherichia coli</i>

“…(MH1 araD + ) were used as wild types (Koga et al, 2011). TY0832 (MH1 ΔabpA::kan) and TY0833 (MH1 ΔabpB::kan) were constructed by GT7-mediated transduction of a kanamycin-resistance marker from strains JW2609 (BW25113 ΔyfjL::kan) and JW2608 (BW25113 ΔyfjK::kan), which were provided by the National BioResource Project (NIG, Japan).…”

“…TA systems are broadly conserved in plasmids and bacterial chromosomes (Gerdes et al, 2005), and are implicated in many physiological roles, such as plasmid maintenance (Ogura and Hiraga, 1983;Yarmolinsky, 1995), stress response (Gerdes et al, 2005;Wang et al, 2011), bacterial persistence against antibiotics (Maisonneuve et al, 2011(Maisonneuve et al, , 2013Amato et al, 2013;Germain et al, 2013) and biofilm formation Wang et al, 2011). We have demonstrated that the two toxins RnlA and LsoA in TA systems are activated after T4 phage infection and result in shutting off gene expression by rapidly degrading mRNAs to block T4 propagation (Kai et al, 1996;Yonesaki, 2005, 2012;Koga et al, 2011).…”

Rapid Degradation of Host mRNAs by Stimulation of RNase E Activity by Srd of Bacteriophage T4

“…After boiling the samples, bound proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [10% gel for full-length RNase E-FLAG, 12% gel for RNase E(1-598)-FLAG, or 15% gel for Srd-His and His-IscR] and electroblotted onto Immuno-Blot PVDF membrane (Bio-Rad). Western blot analysis was performed as previously described (Koga et al 2011). Pull-down and immunoprecipitation analyses were performed at least three times, and a representative result is shown in Figure 5.…”

“…coli K-12 strains MH1 (sup 0 araD139 hsdR DlacX74 rpsL), TY0807 (MH1 araD + ) (Koga et al 2011), and YT10 (MH1 zce726::Tn10) were used as wild types. YT10 or YT20 (YT10 ams1) was constructed by T4 GT7 phage transduction (Wilson et al 1979) of the tetracycline-resistance marker from GW10 (Wachi et al 1997) or by GT7 phage transduction of the tetracycline-resistance marker together with a temperature-sensitive mutation of ams1 from GW20 (Wachi et al 1997).…”