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 ΔrnlAB 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.
BACTERIAL 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 (Ueno and Yonesaki 2001; Otsuka and Yonesaki 2005); it rapidly degrades T4 late mRNAs to prevent their expression, and consequently blocks the propagation of T4 phage when an RNase LS inhibitor encoded by T4 dmd gene is defective. Therefore, RNase LS plays an important role as a potential antagonist of T4 infection. rnlA is the structural gene for RNase LS and purified His-tagged RnlA has an endoribonucleolytic activity that Dmd can inhibit (Otsuka and Yonesaki 2005; Otsuka et al. 2007).
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 mRNA almost indiscriminately. Our results indicate that the rnlAB operon is a new type II TA system, in which RnlA is the toxin and RnlB the antitoxin against RnlA.
MATERIALS AND METHODS
Phages and bacterial strains:
Wild-type bacteriophage T4 is T4D. The amSF16 mutant contains an amber mutation in the dmd gene (Kai et al. 1996; Ueno and Yonesaki 2001). E. coli K-12 strain MH1 (sup0 araD139 hsdR ΔlacX74 rpsL) was described previously (Otsuka and Yonesaki 2005). TY0807 (MH1 araD+) was constructed by T4 GT7 phage transduction (Wilson et al. 1979) of araD+ from W3110 into MH1 and was screened for the ability to grow on M9 minimal medium containing arabinose as a sole carbon source. TY0802 (MH1 ΔrnlAB) and TY0809 (TY0807 ΔrnlAB) were constructed as described (Datsenko and Wanner 2000). Briefly, a fragment containing a chloramphenicol-resistance cassette flanked with the sequences upstream of rnlA and downstream of rnlB was amplified by PCR with pKD3 as a template and the primers 5′-attgtagagtttccccatatgtttctatgggatccaggaacatatgaatatcctccttag and 5′-gttaatatcatgccaaaagggcgaattctatactggttcgtgtgtaggctggagctgctt. The fragment was introduced into W3110 harboring pKD46, which encodes λ-phage Red. Chloramphenicol-resistant colonies were screened by PCR with primer 1 (5′-atgtttctatgggatccagg) and primer 2 (5′-gctatttgatcatattggac) to select ΔrnlAB∷cat cells. After ΔrnlAB∷cat was transferred into MH1 or TY0807 by T4 GT7 phage transduction (Wilson et al. 1979), the chloramphenicol-resistance cassette was removed by yeast Flp recombinase expressed from pCP20 (Cherepanov and Wackernagel 1995) to construct TY0802 and TY0809. BW25113, BW25113 ΔclpA∷kan, BW25113 ΔclpX∷kan, and BW25113 Δlon∷kan were kindly provided by the National BioResource Project (National Institute of Genetics, Kyoto, Japan).
Plasmids constructed in this work are listed in Table 1. To clone the rnlAB ORFs, a DNA fragment was amplified by PCR with W3110 DNA as a template using primers 1 and 2 and ligated into the EcoRV site in pBluescript II SK+ to construct pMK02, in which rnlAB was oriented in the direction opposite of the lac promoter. The rnlA promoter region was amplified by PCR with W3110 DNA as a template using the primers 5′-cgatcgatgttgctgcttgg and 5′-cgaagcccagcccttgaccc. The resulting DNA fragment was digested with ClaI and BamHI and ligated into pBR322. The EcoRI site of this plasmid was disrupted by EcoRI digestion, blunted with T4 DNA polymerase (Nippon Gene), and self-ligated. The BamHI–SalI fragment in the constructed plasmid was replaced with the BamHI–SalI fragment from pMK02 to construct a plasmid containing the rnlAB ORFs with an rnlA promoter region to yield pMK05.
To remove rnlA, pMK05 was digested with BamHI and SphI, blunted with T4 DNA polymerase, and self-ligated to construct pMK08. In addition, a DNA fragment containing the rnlB ORF alone was amplified by PCR with pMK05 as a template using the primers 5′-cgggatccaaggacttatatattg and primer 2, digested with BamHI and EcoRI, and substituted for the rnlAB region in pMK05 to construct pMK09. A DNA fragment containing rnlB was excised with BamHI and HindIII from pMK09 and subcloned into pJK289 (Kato and Ikeda 1996) to construct pMK33.
A nonsense codon was introduced into rnlB in pMK05 by PCR using the primer 5′-ctgtcgttatgtgaaccggcttcgag (mutagenic sequence underlined) and the rnlA region in the resulting plasmid was removed in the same manner as pMK08 to construct pMK14.
To construct pMK19, a DNA fragment was amplified by PCR with pMK05 as a template using primers 3 (5′-cgggatccatgtttgaaatcaccgg) and 4 (5′-gctgcaggttcgtttagaaag), digested with BamHI and PstI, and ligated into pQE-80L (Qiagen). pMK20 was constructed in the following way: A duplex formed between 5′-aattaaagaggagaaag and 5′-gatcctttctcctcttt was ligated into pQE-80L previously digested with EcoRI and BamHI to remove the 6×His-tag region. The resulting plasmid was digested with BamHI and PstI and ligated with an rnlB-containing fragment obtained by PCR using pMK05 as a template with primers 3 and 4.
To construct pBAD24–rnlA, a DNA fragment was amplified by PCR with pMK05 as a template using the primers 5′-ggaggaattcaccatgacaatcaggagttacaaaaac and 5′-agtgctgcagtcataatttcaacctagctacgc, digested with EcoRI and PstI, and ligated into pBAD24 (Guzman et al. 1995). A DNA fragment containing N-terminal Flag-tag (8 amino acids: DYKDDDDK) fused to RnlA was amplified by PCR with pMK05 as a template using the primers 5′-ccatggtaccagattacaaggatgacgacgataagacaatcaggagttacaaaaac (Flag peptide sequence underlined) and 5′-aactgcagaactcaaacaatatataag, digested with KpnI and PstI, and ligated into pBAD24. The fragment containing flag–rnlA was excised from the resulting plasmid with BamHI and PstI and ligated into pBAD33 (Guzman et al. 1995) to construct pBAD33F–rnlA.
To construct pHU102, a DNA fragment was amplified by PCR with W3110 genomic DNA as a template using the primers 5′-gcgagctcatgacggctgaattg and 5′-ggggtaccttacatcattacgac, digested with SacI and KpnI, and ligated into pQE-80L.
mRNA functional decay rate:
Cells were grown at 30° in M9-glycerol medium, supplemented with 0.3% casamino acids, 1 μg/ml thiamine and 20 μg/ml tryptophan, and rifampicin was added to 500 μg/ml. Time 0 was set 2 min after the addition of rifampicin. [35S]methionine/cysteine (American Radiolabeled Chemicals, St. Louis; >37 TBq/mmol) was added to a 100-μl culture to 3.7 MBq/ml at various times for 3 min. Labeled proteins from equal portions of cell culture were analyzed by electrophoresis through a 12.5% polyacrylamide gel containing 0.1% SDS and the half-life of bulk mRNA was calculated by plotting against time the sum of intensities for all bands present between the top and bottom of each lane. An autograph was taken with a Bio-Image analyzer (Fuji BAS-1800). The band intensities were quantified using the ImageJ program.
Northern blot analysis:
Cells were grown at 30° in LB medium. A 1.5-ml aliquot of cell culture was quickly chilled on ice and cells were harvested by centrifugation. Total RNA extraction and northern blotting were carried out according to Kai et al. (1996). Radioactive probes for ompA and lpp mRNAs were prepared according to Ueno and Yonesaki (2004). An autograph was taken with a Bio-Image analyzer (Fuji BAS-1800). The band intensities were quantified using the ImageJ program.
Pull-down of Flag–RnlA and His–RnlB:
S30 fractions of cell extract were prepared as described (Nirenberg 1963; Stanley and Wahba 1967) and an aliquot containing 20 mg protein was mixed with 20 μl of Ni-NTA agarose beads (Qiagen) or anti-Flag M2 agarose beads (Sigma-Aldrich) by end-over-end rotation for 8 hr at 4°. The agarose beads were sedimented at 15,000 rpm for 1 min at 4° and washed five times with 1 ml of TMCK-T buffer [10 mm Tris-HCl (pH 7.5), 10 mm magnesium acetate, 30 mm KCl, 0.02% Tween 20] containing 40 mm imidazole for Ni-NTA agarose or TMCK-T buffer containing 0.1 m NaCl for anti-Flag M2 agarose. Bound proteins were eluted (EL) from Ni-NTA agarose with 50 μl of TMCK-T buffer containing 400 mm imidazole or from anti-Flag M2 agarose with 50 μl of TMCK-T buffer containing 0.1 m NaCl and 0.2 mg/ml Flag peptide (Sigma-Aldrich).
Western blot analysis:
Proteins were separated by electrophoresis through a 15% polyacrylamide gel containing 0.1% SDS and transferred onto a PVDF membrane. Membranes were blocked for 60 min at room temperature with 5% skim milk in TBS-Tween [20 mm Tris-HCl (pH 8.0), 150 mm NaCl, 0.05% Tween-20] and incubated overnight at 4° with a mouse anti-His antibody (GE Healthcare, 1:3000) or mouse anti-Flag antibody (Sigma, 1:5000). Next, membranes were incubated for 60 min at room temperature with HRP-coupled sheep anti-mouse antibody (GE Healthcare, 1:10,000). Protein bands were detected with Immobilon Western Chemiluminescent Substrate (Millipore) and an LAS image analyzer (Fujifilm).
Mutations in rnlB:
To construct an rnlB mutant, we attempted to replace the chromosomal rnlB with rnlB∷kan in a plasmid with a temperature-sensitive DNA replication system (Link et al. 1997). Although this technique was easy and effective for disrupting chromosomal rnlA (Otsuka and Yonesaki 2005), it was difficult to obtain an rnlB∷kan mutant with this technique and the only candidate was found to have a frame-shifting deletion at nucleotide position 267 in the rnlA ORF in addition to rnlB∷kan. This strain completely lacked RNase LS activity, like rnlA∷kan cells. In addition, we found that JW5418 (ΔrnlB∷kan) from the National BioResource Project also lacked RNase LS activity because it had a deletion of five nucleotides at the C-terminal end of the rnlA ORF. In contrast, when cells were previously transformed with pMK33 carrying rnlB, the chromosomal rnlB could be efficiently replaced with rnlB∷kan. These observations suggested that rnlA in the absence of rnlB was deleterious to cells.
Effects of rnlA expression on the growth of ΔrnlAB cells:
The above result suggested that RnlA is toxic to cells and RnlB neutralizes the toxicity of RnlA. To investigate the toxicity of RnlA, TY0807 (wild type) and TY0809 (ΔrnlAB) cells were transformed with pBAD24–rnlA and transformants were cultivated on LB agar plates containing various concentrations of l-arabinose (Figure 1A). TY0809 cells grew as normally as TY0807 cells did in the absence of l-arabinose. However, the colony size of TY0809 was much smaller than that of TY0807 in the presence of 0.01% l-arabinose and the growth of TY0809 was almost completely inhibited in the presence of 0.1% l-arabinose. On the other hand, the induction of RnlA hardly affected the growth of TY0807, suggesting that endogenous RnlB counteracted the RnlA toxicity. Similarly, when TY0809 cells were cotransformed with pBAD24–rnlA and pMK33, the latter of which expressed RnlB constitutively, impairment of cell growth by pBAD24–rnlA in the presence of l-arabinose was not observed (Figure 1B).
Effects of rnlA on mRNA stability in the absence of rnlB:
Because RnlA is an essential component of RNase LS and has an endoribonuclease activity in vitro (Otsuka et al. 2007), RnlA expression was anticipated to stimulate the degradation of E. coli mRNAs. To investigate this possibility, we measured functional mRNA half-lives with or without induction of RnlA (Figure 2A) by estimating the ability of mRNA to direct protein synthesis after transcription has been blocked. TY0809 cells harboring pBAD24–rnlA were grown to midlog phase and supplemented or not with 0.2% l-arabinose. After incubation for 4 min, rifampicin, a transcriptional inhibitor, was added to measure the functional half-lives of mRNAs. The half-life in the absence of l-arabinose was 8.1 min and was reduced to 3.8 min in the presence of l-arabinose. To examine the decay of individual transcripts, we measured the stability of lpp and ompA transcripts. These two transcripts are known to be stable species (Ueno and Yonesaki 2004) and were therefore chosen to facilitate measurement of mRNA decay accelerated by RnlA. After induction of rnlA, ompA mRNA was significantly reduced at 8 min and was almost undetectable at 32 min. Reduction of lpp mRNA was also significant after 16 min (Figure 2B). To confirm that mRNA degradation activity was increased after induction of rnlA, we examined the decay rate of these mRNAs. At 4 min after induction of rnlA, rifampicin was added to cell cultures and total RNAs were extracted at various times for Northern blotting. The half-lives were 28.4 min for lpp mRNA and 20.1 min for ompA mRNA, respectively, without induction of rnlA. The half-lives became shorter after induction of rnlA, 8.5 min for lpp mRNA and 3.7 min for ompA mRNA, respectively (Figure 2C).
Effect of rnlB on RNase LS activity:
The above results implied that RnlB suppresses RnlA or RNase LS activity. To clarify this possibility, we adopted a T4 dmd mutant to monitor the RNase LS activity. dmd encodes an inhibitor of RNase LS (Otsuka et al. 2007) and, when dmd is defective, RNase LS rapidly degrades T4 mRNA at late stages of infection, terminating T4 development (Kai et al. 1996). Cells were spread on an agar plate, a T4 suspension was spotted on the cells and T4 growth was visualized after overnight incubation (Figure 3). TY0802 (ΔrnlAB) cells showed plaques or a zone of clearing when either wild-type T4 or a dmd mutant was spotted, indicating vigorous growth of T4. MH1 (wild type) cells harboring no plasmid or a vector alone allowed vigorous growth of wild-type T4 but restricted the growth of T4 dmd; the efficiency of plating was reduced to 10−2 and the plaque size was sharply reduced, indicating that T4 growth was inhibited by RNase LS (Kai et al. 1996). MH1 cells with pMK08 carrying rnlB, like TY0802 cells, supported the growth of a dmd mutant as well as of wild-type T4, suggesting that RNase LS activity was impaired by RnlB probably overexpressed from the plasmid. Consistently, this effect of pMK08 on the growth of T4 dmd was completely eliminated by introducing a stop codon into the rnlB ORF (pMK14).
We further confirmed the suppressive role of RnlB for RNase LS activity using pMK20 carrying lacI and rnlB; the former was constitutively expressed and the latter was placed downstream of the lac promoter. MH1 harboring pMK20 was grown to midlog phase and rnlB was induced for 30 min with various concentrations of IPTG. The cells were infected with T4 phage and the number of progeny phage per infected cell (burst size) was measured after incubating for an additional 70 min (Table 2). The burst size of wild-type T4 was 100–110 in the range of IPTG concentrations adopted in this experiment. On the other hand, a dmd mutant could not propagate in the absence of IPTG, the burst size being <1. When IPTG was added to 5–50 μm, the burst size increased as IPTG concentration increased; at 50 μm, the burst size reached ∼100, comparable to the burst size of wild-type phage, suggesting that suppression of RNase LS activity was nearly complete. To assess the inhibitory effect of RnlB on RNase LS activity in term of mRNA cleavage, total RNA was extracted from MH1 cells harboring pMK20 at 20 min after infection by a dmd mutant and analyzed for cleavage in soc mRNA by primer extension analysis (Figure 4). In agreement with previous experiments (Kai and Yonesaki 2002; Otsuka and Yonesaki 2005), we identified RNase LS-specific cleavages in soc mRNA at nucleotide positions 135, 144, 145, 153, 172, 185, and 207 from the 5′ terminus. These RNase LS-specific cleavages decreased with increasing IPTG concentration and the amount of full-length soc mRNA increased in parallel. On the other hand, the cleavage at nucleotide position 59 generated by RNase E (Otsuka et al. 2003) was not affected by induction of RnlB. These results suggested that overexpression of RnlB specifically represses RNase LS activity.
Association of RnlA and RnlB:
To investigate an interaction between RnlA and RnlB, we performed a pull-down assay. For this purpose, we constructed pBAD33F–rnlA encoding Flag-tagged RnlA and pMK19 encoding His-tagged RnlB to facilitate their detection. Flag–RnlA showed toxicity similar to RnlA in TY0809 and His–RnlB could neutralize the toxicity, indicating that both tagged proteins are functional (Figure 5A). S30 extract was prepared from TY0809 cells harboring pBAD33F–rnlA and pMK19 or pHU102 encoding His–Rng as a control and mixed with Ni-NTA beads or anti-Flag M2 beads. After washing with buffer, bound proteins were eluted with imidazole or Flag peptide. In a pull-down experiment with Ni-NTA beads, Flag–RnlA was efficiently recovered in the bound protein fraction (EL) together with His–RnlB, but not with His–Rng (Figure 5B). In a reciprocal experiment with anti-Flag M2 beads, His–RnlB, but not His–Rng, could be recovered in the bound protein fraction together with Flag–RnlA (Figure 5C). These results suggested that RnlA and RnlB were specifically associated with each other.
Stabilities of RnlA and RnlB:
The relation between RnlA and RnlB reminded us of a type II TA system. Because type II TA systems are composed of a stable toxin and an unstable antitoxin, we investigated stabilities of RnlA and RnlB. TY0809 harboring pBAD33F–rnlA and pMK19 were grown to midlog phase and the two proteins were co-induced with l-arabinose and IPTG. After tetracycline was added to stop further translation, the amounts of RnlA and RnlB were monitored by Western blotting and their half-lives were measured. The half-lives were 27.6 min for Flag–RnlA and 2.1 min for His–RnlB, respectively (Figure 6A, left half). T4 infection rapidly shuts off host gene expression (Svenson and Karlstrom 1976; Kashlev et al. 1993; Ueno and Yonesaki 2004) and we accordingly anticipated that rapid degradation of RnlB also occurred upon T4 infection. In fact, His–RnlB was degraded rapidly after infection of a T4 dmd phage, while Flag–RnlA remained stable (Figure 6A, right half). To identify protease(s) responsible for rapid degradation of RnlB, we examined the effects of Lon and Clp, which are major proteases in E. coli (Figure 6B). Because the decay of RnlB was not significant in the first 5 min with the BW25113 strain adopted for this experiment, the half-life of RnlB was calculated on the basis of its amounts after 5 min and was 6.7 min in wild type and 4.5 min in ΔclpA. On the other hand, it was prolonged to 14 min in ΔclpX and 23 min in Δlon.
Several lines of evidence clearly demonstrate that rnlAB is a new type II TA system in E. coli. First, RnlA was severely toxic in ΔrnlAB cells, which could be suppressed by RnlB (Figure 1). Second, RnlA in the absence of RnlB accelerated mRNA decay (Figure 2). Third, RnlA and RnlB formed a complex (Figure 5). Finally, RnlB was unstable while RnlA was stable (Figure 6).
Eight type II TA loci in E. coli (relBE, dinJ yafQ, yefM yoeB, prlF yhaV, mazEF, chpBS, hicAB, and mqsR ygiT) encode mRNA interferases whose ectopic induction inhibits translation by mRNA cleavage. On the basis of their mechanism of action, the mRNA interferases can be divided into two groups: RelE, YafQ, and YoeB are associated with the ribosome and cleave mRNA at the ribosomal A site, while MazF, ChpB, HicA, YhaV, and MqsR cleave mRNA independently of the ribosome (Pedersen et al. 2003; Zhang et al. 2003b, 2005b; Schmidt et al. 2007; Christensen-Dalsgaard and Gerdes 2008; Jørgensen et al. 2009; Prysak et al. 2009; Yamaguchi et al. 2009; Zhang and Inouye 2009). RelE has been the most extensively characterized ribosome-dependent mRNA interferase. Although RelE cannot cleave naked RNA in vitro, RelE associated with the ribosomes cleave mRNA positioned at the A site between the second and third nucleotides of the codon, both in vivo and in vitro (Galvani et al. 2001; Christensen and Gerdes 2003; Pedersen et al. 2003). MazF has been the most characterized ribosome-independent mRNA interferase (Zhang et al. 2003b, 2005a; Muñoz-Gómez et al. 2004). However, ribosomal translocation can occasionally stimulate cleavage by MazF when a target site is shielded by secondary structure and the ribosomes disrupt the structure during translation (Christensen-Dalsgaard and Gerdes 2008). RNase LS cleaves RNA both independently of and dependently on translation. In addition, recent reports suggest that some translation-independent cleavages are stimulated by translation (Kai and Yonesaki 2002; Otsuka and Yonesaki 2005), suggesting the similarity of RNase LS action with that of MazF.
In most TA systems, an antitoxin gene is located upstream of its cognate toxin gene. However, the opposite order of antitoxin and toxin genes, as with rnlA–rnlB, is not exceptional and is found in hicAB (Makarova et al. 2006; Jørgensen et al. 2009) and mqsR ygiT (Yamaguchi et al. 2009). In the case of rnlAB, a promoter within the rnlA ORF was identified as an rnlB promoter in addition to an rnlA promoter upstream of rnlA. The β-galactosidase reporter assays demonstrated that the rnlB promoter is approximately eightfold stronger than the rnlA promoter. In addition, transcription from the rnlA promoter is negatively regulated by IscR (Otsuka et al. 2010). Therefore, the level of RnlB may exceed that of RnlA.
The half-life of RnlB was 2 min, much shorter than the 27 min of RnlA (Figure 6A). Because a mutation in clpX or lon, but not in clpA, significantly stabilized RnlB (Figure 6B), ClpXP and Lon, but not ClpAP, may be involved in the rapid degradation of RnlB. T4 infection results in remarkable increase of RNase LS activity (Otsuka and Yonesaki 2005). T4 has mechanisms to terminate host gene expression at the level of translation as well as transcription immediately after infection (Svenson and Karlstrom 1976; Kashlev et al. 1993; Ueno and Yonesaki 2004). Therefore, it is reasonable to observe that unstable RnlB disappears rapidly after T4 infection, resulting in increased RNase LS activity. In the absence of Dmd, T4 cannot propagate because of rapid degradation of late mRNAs by RNase LS (Kai et al. 1996) or RNase LS antagonizes T4 growth. Similarly, other type II TA systems can be assumed to be antiphage systems because termination of host gene expression by a phage such as T4 results in disappearance of antitoxin and activation of toxin, namely mRNA interferase that can block phage gene expression. In this connection, it is worth noting that phage P1 propagates more efficiently in the absence of mazF than in its presence (Hazan and Engelberg-Kulka 2004).
We cordially thank John W. Drake at the National Institute of Environmental Health Sciences for invaluable help with the manuscript. We thank the staff of the Radioisotope Research Center at Toyonaka, Osaka University, for the facilitation of our research, because all of our experiments using radioisotopes were carried out at the center. We thank the National BioResource Project (National Institute of Genetics, Kyoto, Japan): E. coli, for distributing E. coli gene disruptants. This work was supported in part by a grant from the Yakult Bio-Science Foundation and by a grant from the program Grants-in-Aid for Scientific Research (category C) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
↵1 Present address: Department of Systems Microbiology, Center for Systems Biology, Denmark Technical University, DK-2800 Lyngby, Denmark.
Communicating editor: S. Gottesman
- Received August 4, 2010.
- Accepted October 20, 2010.
- Copyright © 2011 by the Genetics Society of America