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The toxin-antitoxin (TA) genetic modules control various bacterial events, such as plasmid maintenance, persister cell formation, and phage defense. They also exist in mobile genetic elements, including prophages, however, their physiological roles remain poorly understood. Here, we demonstrate that hokW-sokW, a putative TA locus encoded in Sakai prophage 5 (Sp5) in enterohemorrhagic Escherichia coli O157: H7 Sakai strain, functions as a type I TA system. Bacterial growth assays showed that the antitoxic activity of sokW RNA against HokW toxin partially requires an endoribonuclease, RNase III, and an RNA chaperone, Hfq. We also demonstrated that hokW-sokW assists Sp5-mediated lysis of E. coli cells when prophage induction is promoted by the DNA-damaging agent mitomycin C (MMC). We found that MMC treatment diminished sokW RNA and increased both the expression level and inner membrane localization of HokW in a RecA-dependent manner. Remarkably, the number of released Sp5 phages decreased by half in the absence of hokW-sokW. These results suggest that hokW-sokW plays a novel role as a TA system that facilitates the release of Sp5 phage progeny through E. coli lysis.

The toxin-antitoxin (TA) genetic modules control various bacterial events, such as plasmid maintenance, persister cell formation, and phage defense. They also exist in mobile genetic elements, including prophages; however, their physiological roles remain poorly understood. Here, we demonstrate that hokW-sokW, a putative TA locus encoded in Sakai prophage 5 (Sp5) in enterohemorrhagic Escherichia coli O157: H7 Sakai strain, functions as a type I TA system. Bacterial growth assays showed that the antitoxic activity of sokW RNA against HokW toxin partially requires an endoribonuclease, RNase III, and an RNA chaperone, Hfq. We also demonstrated that hokW-sokW assists Sp5-mediated lysis of E. coli cells when prophage induction is promoted by the DNA-damaging agent mitomycin C (MMC). We found that MMC treatment diminished sokW RNA and increased both the expression level and inner membrane localization of HokW in a RecA-dependent manner. Remarkably, the number of released Sp5 phages decreased by half in the absence of hokW-sokW. These results suggest that hokW-sokW plays a novel role as a TA system that facilitates the release of Sp5 phage progeny through E. coli lysis.

While several metallic implants with bioactive coatings have been developed thus far for treating bone deformations or deterioration, a multifunctional coating with the desired mechanical and antibacterial properties has not been demonstrated. This study aimed to reveal the effect of the composition of hydroxyapatite (HAp)/gray titania coatings on the mechanical and antibacterial properties for biomedical applications. Suspension plasma spray (SPS) aided successful deposition of HAp/gray titania coatings on the surface of titanium substrates. The microstructure of coatings with different compositions was then characterized using scanning electron microscopy, X-ray diffraction, and Raman spectroscopy to identify the crystal structure. All results consistently demonstrated that SPS could transform Ti2O3 into TiO2 with mixed Magneli phases, such as Ti4O7 and Ti3O5, which could typically demonstrate photocatalytic activity. Hardness, Young's modulus, and interfacial strength of composite coatings commonly increased with an increase in the weight percentage of TiO2. A multi-modal damage assessment combining acoustic emission (AE), infrared ray camera (IR), and digital-image-correlation (DIC) was performed to monitor the damage process of HAp composite coating, which successfully revealed initiations of microcracks and nonlinear deformation at interface until fracture. Antibacterial test performed for examining the cytotoxic effects against E. coli under LED light irradiation conditions revealed that SPS HAp/gray titania coating could significantly enhance the antibacterial properties. Enhanced antibacterial properties can be attributed to an increase in the number of Magneli phases and better bacterial adhesion was attributed to hydrophilic properties conferred by submicron-sized particles. Hence, SPS can help fabricate visible light-responsive antibacterial coating, which can be used for medical devices.

Bacteriophages are the most abundant and diverse biological entities on Earth. Phages exhibit strict host specificity that is largely conferred by adsorption. However, the mechanism underlying this phage host specificity remains poorly understood. In this study, we examined the interaction between outer membrane protein C (OmpC), one of the Escherichia coli receptors, and the long tail fibers of bacteriophage T4. T4 phage uses OmpC of the K-12 strain, but not of the O157 strain, for adsorption, even though OmpCs from the two E. coli strains share 94% homology. We identified amino acids P177 and F182 in loop 4 of the K-12 OmpC as essential for T4 phage adsorption in the copresence of loops 1 and 5. Analyses of phage mutants capable of adsorbing to OmpC mutants demonstrated that amino acids at positions 937 and 942 of the gp37 protein, which is present in the distal tip (DT) region of the T4 long tail fibers, play an important role in adsorption. Furthermore, we created a T4 phage mutant library with artificial modifications in the DT region and isolated and characterized multiple phage mutants capable of adsorbing to OmpC of the O157 strain or lipopolysaccharide of the K-12 strain. These results shed light on the mechanism underlying the phage host specificity mediated by gp37 and OmpC and may be useful in the development of phage therapy via artificial modifications of the DT region of T4 phage. IMPORTANCE Understanding the host specificity of phages will lead to the development of phage therapy. The interaction between outer membrane protein C (OmpC), one of the Escherichia coli receptors, and the gp37 protein present in the distal tip (DT) region of the long tail fibers of T4 bacteriophages largely determines their host specificity. Here, we elucidated the amino acid residues important for the interaction between gp37 and OmpC. This result suggests that the shapes of both proteins at the binding interface play important roles in their interactions, which are likely mediated by multiple residues of both binding partners. Additionally, we successfully isolated multiple phage mutants capable of adsorbing to a variety of E. coli receptors using a mutant T4 phage library with artificial modifications in the DT region, providing a foundation for the alteration of the host specificity.

A competitive bacteria-phage coevolution is often referred to as an “arms race.” On the one hand, bacteria have evolved a variety of defense mechanisms against phages such as inhibition of phage adsorption and cleavage of phage nucleic acids through the restriction-modification system or the CRISPR-Cas system. On the other hand, phages have also devised many means to protect themselves from bacterial defense mechanisms. The abortive infection (Abi) system, one of the bacterial defense mechanisms, inhibits phage propagation by killing phage-infected cells before the phage life cycle is completed. Abi can be achieved through the toxin-antitoxin (TA) system, a widespread genetic module in prokaryotes composed of a toxin and the corresponding antitoxin. In this system, the toxin inhibits an essential cellular process to arrest cell growth, while the antitoxin neutralizes the toxicity of a cognate toxin. MazF and RnlA toxins of the Escherichia coli TA systems and ToxN toxin of the plant pathogen Pectobacterium atrosepticum TA system have been well studied and demonstrated to possess endoribonuclease activities required for Abi. In this chapter, I will discuss the functions, structures, and regulations of their endoribonucleases. I will also discuss how these endoribonucleases induce Abi and how phages counteract Abi.

Antimicrobial peptides are potential molecules for the development of novel antibiotic agents. The ZorO toxin of a type I toxin&ndash, antitoxin system in Escherichia coli O157:H7 is composed of 29 amino acids and its endogenous expression inhibits E. coli growth. However, little is known about its inhibitory mechanism. In this study, we demonstrate that the ZorO localized in the inner membrane affects the plasma membrane integrity and potential when expressed in E. coli cells, which triggers the production of cytotoxic hydroxyl radicals. We further show that five internal amino acids (Ala&ndash, Leu&ndash, Arg&ndash, Leu, ALLRL) of ZorO are necessary for its toxicity. This result prompted us to address the potential of the synthetic ALLRL peptide as an antimicrobial. Exogenously-added ALLRL peptide to Gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis, and a fungus, Candida albicans, trigger cell membrane damage and exhibit growth defect, while having no effect on Gram-negative bacterium, E. coli. The ALLRL peptide retains its activity under the physiological salt concentrations, which is in contrast to natural antimicrobial peptides. Importantly, this peptide has no toxicity against mammalian cells. Taken together, an effective and short peptide, ALLRL, would be an attractive antimicrobial to Gram-positive bacteria and C. albicans.

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.

Bacteria have obtained a variety of resistance mechanisms including toxin-antitoxin (TA) systems against bacteriophages (phages), whereas phages have also evolved to overcome bacterial anti-phage mechanisms. Dmd from T4 phage can suppress the toxicities of homologous toxins LsoA and RnlA from Escherichia coli, representing the first example of a phage antitoxin against multiple bacterial toxins in known TA systems. Here, the crystal structure of LsoA-Dmd complex showed Dmd is inserted into the deep groove between the N-terminal repeated domain (NRD) and the Dmd-binding domain (DBD) of LsoA. The NRD shifts significantly from a 'closed' to an 'open' conformation upon Dmd binding. Site-directed mutagenesis of Dmd revealed the conserved residues (W31 and N40) are necessary for LsoA binding and the toxicity suppression as determined by pull-down and cell toxicity assays. Further mutagenesis identified the conserved Dmd-binding residues (R243, E246 and R305) of LsoA are vital for its toxicity, and suggested Dmd and LsoB may possess different inhibitory mechanisms against LsoA toxicity. Our structure-function studies demonstrate Dmd can recognize LsoA and inhibit its toxicity by occupying the active site possibly via substrate mimicry. These findings have provided unique insights into the defense and counter-defense mechanisms between bacteria and phages in their co-evolution.

Toxin-antitoxin (TA) systems are widely conserved in prokaryotic plasmids and chromosomes and are linked to many roles in cell physiology, including plasmid maintenance, stress response, persistence and protection from phage infection. A TA system is composed of a stable toxin and a labile antitoxin that inhibits a harmful effect of the cognate toxin. When gene expression from the TA loci is repressed under certain conditions such as nutrient starvation, the toxin is freed from the rapidly degrading antitoxin and obstructs an essential cellular process, such as DNA replication, translation and peptidoglycan synthesis, which subsequently causes growth arrest. TA systems are classified into five types according to the nature and the function of antitoxins, and the activity of toxins is tightly regulated in a variety of ways. This short-review highlights several novel regulatory mechanisms for Escherichia coli toxins that we recently discovered.

Summary Prokaryotic toxin–antitoxin (TA) systems are linked to many roles in cell physiology, such as plasmid maintenance, stress response, persistence and protection from phage infection, and the activities of toxins are tightly regulated. Here, we describe a novel regulatory mechanism for a toxin of Escherichia coli TA systems. The MazF toxin of MazE-MazF, which is one of the best characterized type II TA systems, was modified immediately after infection with bacteriophage T4. Mass spectrometry demonstrated that the molecular weight of this modification was 542 Da, corresponding to a mono-ADP-ribosylation. This modification disappeared in cells infected with T4 phage lacking Alt, which is one of three ADP-ribosyltransferases encoded by T4 phage and is injected together with phage DNA upon infection. In vivo and in vitro analyses confirmed that T4 Alt ADP-ribosylated MazF at an arginine residue at position 4. Finally, the ADP-ribosylation of MazF by Alt resulted in the reduction of MazF RNA cleavage activity in vitro, suggesting that it may function to inactivate MazF during T4 infection. This is the first example of the chemical modification of an E. coli toxin in TA systems to regulate activity.

Escherichia coli messenger RNAs (mRNAs) are rapidly degraded immediately after bacteriophage T4 infection, and the host RNase E contributes to this process. Here, we found that a previously uncharacterized factor of T4 phage, Srd (Similarity with rpoD), was involved in T4-induced host mRNA degradation. The rapid decay of ompA and lpp mRNAs was partially alleviated and a decay intermediate of lpp mRNA rapidly accumulated in cells infected with T4 phage lacking srd. Exogenous expression of Srd in uninfected cells significantly accelerated the decay of these mRNAs. In addition, lpp(T) RNA, with a sequence identical to the decay intermediate of lpp mRNA and a triphosphate at 5′-end, was also destabilized by Srd. The destabilization of these RNAs by Srd was not observed in RNase E-defective cells. The initial cleavage of a primary transcript by RNase E can be either direct or dependent on the 5′-end of transcript. In the latter case, host RppH is required to convert the triphosphate at 5′-end to a monophosphate. lpp(T) RNA, but not lpp and ompA mRNAs, required RppH for Srd-stimulated degradation, indicating that Srd stimulates both 5′-end-dependent and -independent cleavage activities of RNase E. Furthermore, pull-down and immunoprecipitation analyses strongly suggested that Srd physically associates with the N-terminal half of RNase E containing the catalytic moiety and the membrane target sequence. Finally, the growth of T4 phage was significantly decreased by the disruption of srd. These results strongly suggest that the stimulation of RNase E activity by T4 Srd is required for efficient phage growth.

The Escherichia coli RnlA-RnlB toxin–antitoxin system is related to the anti-phage mechanism. Under normal growth conditions, an RnlA toxin with endoribonuclease activity is inhibited by binding of its cognate RnlB antitoxin. After bacteriophage T4 infection, RnlA is activated by the disappearance of RnlB, resulting in the rapid degradation of T4 mRNAs and consequently no T4 propagation when T4 dmd encoding a phage antitoxin against RnlA is defective. Intriguingly, E. coli RNase HI, which plays a key role in DNA replication, is required for the activation of RnlA and stimulates the RNA cleavage activity of RnlA. Here, we report an additional role of RNase HI in the regulation of RnlA-RnlB system. Both RNase HI and RnlB are associated with NRD (one of three domains of RnlA). The interaction between RnlB and NRD depends on RNase HI. Exogenous expression of RnlA in wild-type cells has no effect on cell growth because of endogenous RnlB and this inhibition of RnlA toxicity requires RNase HI and NRD. These results suggest that RNase HI recruits RnlB to RnlA through NRD for inhibiting RnlA toxicity and thus plays two contrary roles in the regulation of RnlA-RnlB system.

Bacteria have a variety of resistance mechanisms for surviving bacteriophage infections. Here, we describe a novel anti-phage mechanism in Escherichia coli. Cells harboring a plasmid with the genes abpA and abpB, formerly yfjL and yfjK, blocked the propagation of bacteriophages belonging to three families: T4, T2, T7 and λ phages. Both genes were necessary for the inhibition of phage propagation, and deletion of either chromosomal gene resulted in a 20% increase of progeny compared to wild-type cells. Neither overexpression nor deficiency of AbpA and AbpB had any apparent effect on E. coli growth. We isolated seven suppressor mutants of T4 phage that grew weakly on cells overexpressing AbpA and AbpB, and found that their mutations were all located in gene 41, which encodes a replicative DNA helicase that is essential for DNA replication. Furthermore, we demonstrated that AbpA and AbpB inhibited DNA replication and late gene expression of T4 phage. Similarly, DNA replication of T7 and λ phages was also inhibited by AbpA and AbpB. These results strongly suggest that E. coli AbpA and AbpB target DNA replication of phages to block their propagation.

Escherichia coli RnlA-RnlB is a newly identified toxin-antitoxin (TA) system that plays a role in bacteriophage resistance. RnlA functions as a toxin with mRNA endoribonuclease activity and the cognate antitoxin RnlB inhibits RnlA toxicity in E. coli cells. Interestingly, T4 phage encodes the antitoxin Dmd, which acts against RnlA to promote its own propagation, suggesting that RnlA-Dmd represents a novel TA system. Here, we have determined the crystal structure of RnlA refined to 2.10 angstrom. RnlA is composed of three independent domains: NTD (N-terminal domain), NRD (N repeated domain) and DBD (Dmd-binding domain), which is an organization not previously observed among known toxin structures. Small-angle X-ray scattering (SAXS) analysis revealed that RnlA forms a dimer in solution via interactions between the DBDs from both monomers. The in vitro and in vivo functional studies showed that among the three domains, only the DBD is responsible for recognition and inhibition by Dmd and subcellular location of RnlA. In particular, the helix located at the C-terminus of DBD plays a vital role in binding Dmd. Our comprehensive studies reveal the key region responsible for RnlA toxicity and provide novel insights into its structure-function relationship.

Bacteria have obtained a variety of resistance mechanisms including toxin-antitoxin (TA) systems against bacteriophages (phages), whereas phages have also evolved to overcome bacterial anti-phage mechanisms. Dmd from T4 phage can suppress the toxicities of homologous toxins LsoA and RnlA from Escherichia coli, representing the first example of a phage antitoxin against multiple bacterial toxins in known TA systems. Here, the crystal structure of LsoA-Dmd complex showed Dmd is inserted into the deep groove between the N-terminal repeated domain (NRD) and the Dmd-binding domain (DBD) of LsoA. The NRD shifts significantly from a 'closed' to an 'open' conformation upon Dmd binding. Site-directed mutagenesis of Dmd revealed the conserved residues (W31 and N40) are necessary for LsoA binding and the toxicity suppression as determined by pull-down and cell toxicity assays. Further mutagenesis identified the conserved Dmd-binding residues (R243, E246 and R305) of LsoA are vital for its toxicity, and suggested Dmd and LsoB may possess different inhibitory mechanisms against LsoA toxicity. Our structure-function studies demonstrate Dmd can recognize LsoA and inhibit its toxicity by occupying the active site possibly via substrate mimicry. These findings have provided unique insights into the defense and counter-defense mechanisms between bacteria and phages in their co-evolution.

Prokaryotic toxin-antitoxin (TA) systems are linked to many roles in cell physiology, such as plasmid maintenance, stress response, persistence and protection from phage infection, and the activities of toxins are tightly regulated. Here, we describe a novel regulatory mechanism for a toxin of Escherichia coli TA systems. The MazF toxin of MazE-MazF, which is one of the best characterized type II TA systems, was modified immediately after infection with bacteriophage T4. Mass spectrometry demonstrated that the molecular weight of this modification was 542 Da, corresponding to a mono-ADP-ribosylation. This modification disappeared in cells infected with T4 phage lacking Alt, which is one of three ADP-ribosyltransferases encoded by T4 phage and is injected together with phage DNA upon infection. In vivo and in vitro analyses confirmed that T4 Alt ADP-ribosylated MazF at an arginine residue at position 4. Finally, the ADP-ribosylation of MazF by Alt resulted in the reduction of MazF RNA cleavage activity in vitro, suggesting that it may function to inactivate MazF during T4 infection. This is the first example of the chemical modification of an E. coli toxin in TA systems to regulate activity.

The Escherichia coli endoribonuclease LS was originally identified as a potential antagonist of bacteriophage T4. When the T4 dmd gene is defective, RNase LS cleaves T4 mRNAs and antagonizes T4 reproduction. This RNase also plays an important role in RNA metabolisms in E. coli. rnlA is an essential gene for RNase LS activity, but the transcriptional regulation of this gene remains to be elucidated. An Fe-S cluster protein, IscR, acts as a transcription factor and controls the expression of genes that are necessary for Fe-S cluster biogenesis. Here, we report that overexpression of IscR suppressed RNase LS activity, causing the loss of antagonist activity against phage T4. This suppressive effect did not require the ligation of Fe-S cluster into IscR. β-Galactosidase reporter assays showed that transcription from an rnlA promoter increased in iscR-deleted cells compared to wild-type cells, and gel-mobility shift assays revealed specific binding of IscR to the rnlA promoter region. RT–PCR analysis demonstrated that endogenous rnlA mRNA was reduced by overexpression of IscR and increased by deletion of iscR. From these results, we conclude that IscR negatively regulates transcription of rnlA and represses RNase LS activity.

Bacteria have a variety of resistance mechanisms for surviving bacteriophage infections. Here, we describe a novel anti-phage mechanism in Escherichia coli. Cells harboring a plasmid with the genes abpA and abpB, formerly yfjL and yfjK, blocked the propagation of bacteriophages belonging to three families: T4, T2, T7 and λ phages. Both genes were necessary for the inhibition of phage propagation, and deletion of either chromosomal gene resulted in a 20% increase of progeny compared to wild-type cells. Neither overexpression nor deficiency of AbpA and AbpB had any apparent effect on E. coli growth. We isolated seven suppressor mutants of T4 phage that grew weakly on cells overexpressing AbpA and AbpB, and found that their mutations were all located in gene 41, which encodes a replicative DNA helicase that is essential for DNA replication. Furthermore, we demonstrated that AbpA and AbpB inhibited DNA replication and late gene expression of T4 phage. Similarly, DNA replication of T7 and λ phages was also inhibited by AbpA and AbpB. These results strongly suggest that E. coli AbpA and AbpB target DNA replication of phages to block their propagation.

A type II toxin-antitoxin system in Escherichia coli, rnlA-rnlB, functions as an anti-phage mechanism. RnlA is a toxin with an endoribonuclease activity and the cognate RnlB inhibits RnlA toxicity in E. coli cells. After bacteriophage T4 infection, RnlA is activated by the disappearance of RnlB, resulting in the rapid degradation of T4 mRNAs and consequently no T4 propagation, when T4 dmd is defective: Dmd is an antitoxin against RnlA for promoting own propagation. Previous studies suggested that the activation of RnlA after T4 infection was regulated by multiple components. Here, we provide the evidence that RNase HI is an essential factor for activation of RnlA. The dmd mutant phage could grow on ΔrnhA (encoding RNase HI) cells, in which RnlA-mediated mRNA cleavage activity was defective. RNase HI bound to RnlA in vivo and enhanced the RNA cleavage activity of RnlA in vitro. In addition, ectopic expression of RnlA in ΔrnlAB ΔrnhA cells has less effect on cell toxicity and RnlA-mediated mRNA degradation than in ΔrnlAB cells. This is the first example of a direct factor for activation of a toxin.

Escherichia coli ribonuclease LS is a potential antagonist of bacteriophage T4. When the T4 dmd gene is defective, RNase LS cleaves T4 mRNAs and antagonizes T4 reproduction. Our previous work demonstrated that E. coli rnlA is essential for RNase LS activity. Here we show that His-tagged RnlA cleaves T4 soc RNA at one of the sites also cleaved by RNase LS in a cell extract. The cleavage activities of His-tagged RnlA and the RNase LS activity in a cell extract were inhibited by Dmd encoded by T4 phage. Fractionation of the RNase LS activity in a cell extract showed that it sedimented through a sucrose density gradient as a 1000-kDa complex that included RnlA. Pull-down experiments revealed more than 10 proteins associated with His-tagged RnlA. Among these, triose phosphate isomerase exhibited a remarkable affinity to RnlA. These results suggest that RnlA plays a central role in RNase LS activity and that its activity is regulated by multiple components.

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.

Thedmdmutant of bacteriophage T4 has a defect in growth because of rapid degradation of late-gene mRNAs, presumably caused by mutant-specific cleavages of RNA. Some such cleavages can occur in an allele-specific manner, depending on the translatability of RNA or the presence of a termination codon. Other cleavages are independent of translation. In the present study, by introducing plasmids carrying varioussocalleles, we could detect cleavages ofsocRNA in uninfected cells identical to those found indmdmutant-infected cells. We isolated fiveEscherichia colimutant strains in which thedmdmutant was able to grow. One of these strains completely suppressed thedmdmutant-specific cleavages ofsocRNA. The loci of theE. colimutations and the effects of mutations in known RNase-encoding genes suggested that an RNA cleavage activity causing thedmdmutant-specific mRNA degradation is attributable to a novel RNase. In addition, we present evidence that 5′-truncatedsocRNA, a stable form in T4-infected cells regardless of the presence of admdmutation, is generated by RNase E.

The bacteriophage T4 e gene encodes lysozyme (e-lysozyme), which releases progeny phage after normal infection of Escherichia coli cells. A mutation in the spackle gene suppresses the defect in e-lysozyme (Emrich, 1968). The spackle gene was mapped between genes 41 and 61, but its precise location has not previously been determined. In the current study, we constructed an amber mutant of gene 61.3, amST14, by site-directed mutagenesis. The gene 61.3 mutant shares phenotypes with spackle mutants: The amST14 mutant forms large plaques with sharp edges and exhibits truncated lysis inhibition, and furthermore, the mutation can suppress the defect in e-lysozyme activity. In addition, cloned gene 61.3 can rescue (by homologous recombination) as well as complement the S12 mutation in the spackle gene. These results strongly suggest that gene 61.3 is the spackle gene. Indeed, the S12 mutant has one base deletion of five in a consecutive A tract in the gene 61.3 coding region, substituting an unrelated 6-amino acid sequence for the 9 C-terminal amino acids in the gene 61.3 protein. The gene 61.3 protein is predicted to localize in the periplasmic space after cleavage of a signal sequence.