Structure and activity of AbiQ,a lactococcal endoribonuclease belonging to the type III toxin–antitoxin system

AbiQ is a phage resistance mechanism found on a native plasmid of Lactococcus lactis that abort virulent phage infections. In this study, we experimentally demonstrate that AbiQ belongs to the recently described type III toxin–antitoxin systems. When overexpressed, the AbiQ protein (ABIQ) is toxic and causes bacterial death in a bacteriostatic manner. Northern and Western blot experiments revealed that the abiQ gene is transcribed and translated constitutively, and its expression is not activated by a phage product. ABIQ is an endoribonuclease that specifically cleaves its cognate antitoxin RNA molecule in vivo. The crystal structure of ABIQ was solved and site‐directed mutagenesis identified key amino acids for its anti‐phage and/or its RNase function. The AbiQ system is the first lactococcal abortive infection system characterized to date at a structural level.     

AbiQ, an Abortive Infection Mechanism from Lactococcus lactis

Lactococcus lactis W-37 is highly resistant to phage infection. The cryptic plasmids from this strain were coelectroporated, along with the shuttle vector pSA3, into the plasmid-free host L. lactis LM0230. In addition to pSA3, erythromycin- and phage-resistant isolates carried pSRQ900, an 11-kb plasmid from L. lactis W-37. This plasmid made the host bacteria highly resistant (efficiency of plaquing <10⁻⁸) to c2- and 936-like phages. pSRQ900 did not confer any resistance to phages of the P335 species. Adsorption, cell survival, and endonucleolytic activity assays showed that pSRQ900 encodes an abortive infection mechanism. The phage resistance mechanism is limited to a 2.2-kb EcoRV/BclI fragment. Sequence analysis of this fragment revealed a complete open reading frame (abiQ), which encodes a putative protein of 183 amino acids. A frameshift mutation within abiQ completely abolished the resistant phenotype. The predicted peptide has a high content of positively charged residues (pI = 10.5) and is, in all likelihood, a cytosolic protein. AbiQ has no homology to known or deduced proteins in the databases. DNA replication assays showed that phage c21 (c2-like) and phage p2 (936-like) can still replicate in cells harboring AbiQ. However, phage DNA accumulated in its concatenated form in the infected AbiQ⁺ cells, whereas the AbiQ⁻ cells contained processed (mature) phage DNA in addition to the concatenated form. The production of the major capsid protein of phage c21 was not hindered in the cells harboring AbiQ.     

RNase HI stimulates the activity of RnlA toxin in Escherichia coli

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.     

Assembly Dynamics and Stability of the Pneumococcal Epsilon Zeta Antitoxin Toxin (PezAT) System from Streptococcus pneumoniae

The pneumococcal epsilon zeta antitoxin toxin (PezAT) system is a chromosomally encoded, class II toxin antitoxin system from the human pathogen Streptococcus pneumnoniae. Neutralization of the bacteriotoxic protein PezT is carried out by complex formation with its cognate antitoxin PezA. Here we study the stability of the inhibitory complex in vivo and in vitro. We found that toxin release is impeded in Escherichia coli and Bacillus subtilis due to the proteolytic resistance of PezA once bound to PezT. These findings are supported by in vitro experiments demonstrating a strong thermodynamic stabilization of both proteins upon binding. A detailed kinetic analysis of PezAT assembly revealed that these particular features of PezAT are based on a strong, electrostatically guided binding mechanism leading to a stable toxin antitoxin complex with femtomolar affinity. Our data show that PezAT complex formation is distinct to all other conventional toxin antitoxin modules and a controlled mode of toxin release is required for activation.     

A Common Origin for the Bacterial Toxin-Antitoxin Systems parD and ccd,Suggested by Analyses of Toxin/Target and Toxin/Antitoxin Interactions

Bacterial toxin-antitoxin (TA) systems encode two proteins, a potent inhibitor of cell proliferation (toxin) and its specific antidote (antitoxin). Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets.     

Translation affects YoeB and MazF messenger RNA interferase activities by different mechanisms

Prokaryotic toxin–antitoxin loci encode mRNA cleaving enzymes that inhibit translation. Two types are known: those that cleave mRNA codons at the ribosomal A site and those that cleave any RNA site specifically. RelE of Escherichia coli cleaves mRNA at the ribosomal A site in vivo and in vitro but does not cleave pure RNA in vitro. RelE exhibits an incomplete RNase fold that may explain why RelE requires its substrate mRNA to presented by the ribosome. In contrast, RelE homologue YoeB has a complete RNase fold and cleaves RNA independently of ribosomes in vitro. Here, we show that YoeB cleavage of mRNA is strictly dependent on translation of the mRNA in vivo. Non-translated model mRNAs were not cleaved whereas the corresponding wild-type mRNAs were cleaved efficiently. Model mRNAs carrying frameshift mutations exhibited a YoeB-mediated cleavage pattern consistent with the reading frameshift thus giving strong evidence that YoeB cleavage specificity was determined by the translational reading frame. In contrast, site-specific mRNA cleavage by MazF occurred independently of translation. In one case, translation seriously influenced MazF cleavage efficiency, thus solving a previous apparent paradox. We propose that translation enhances MazF-mediated cleavage of mRNA by destabilization of the mRNA secondary structure.     

The Clostridium difficile cell wall protein CwpV confers phase‐variable phage resistance

Bacteriophages are present in virtually all ecosystems, and bacteria have developed multiple antiphage strategies to counter their attacks. Clostridium difficile is an important pathogen causing severe intestinal infections in humans and animals. Here we show that the conserved cell‐surface protein CwpV provides antiphage protection in C. difficile. This protein, for which the expression is phase‐variable, is classified into five types, each differing in their repeat‐containing C‐terminal domain. When expressed constitutively from a plasmid or the chromosome of locked ‘ON’ cells of C. difficile R20291, CwpV conferred antiphage protection. Differences in the level of phage protection were observed depending on the phage morphological group, siphophages being the most sensitive with efficiency of plaquing (EOP) values of < 5 × 10^?7 for phages ?CD38‐2, ?CD111 and ?CD146. Protection against the myophages ?MMP01 and ?CD52 was weaker, with EOP values between 9.0 × 10^?3 and 1.1 × 10^?1. The C‐terminal domain of CwpV carries the antiphage activity and its deletion, or part of it, significantly reduced the antiphage protection. CwpV does not affect phage adsorption, but phage DNA replication is prevented, suggesting a mechanism reminiscent of superinfection exclusion systems normally encoded on prophages. CwpV thus represents a novel ubiquitous host‐encoded and phase‐variable antiphage system in C. difficile.     

Crystal structure of apo and copper bound HP0894 toxin from Helicobacter pylori 26695 and insight into mRNase activity

The toxin–antitoxin (TA) systems widely spread among bacteria and archaea are important for antibiotic resistance and microorganism virulence. The bacterial kingdom uses TA systems to adjust the global level of gene expression and translation through RNA degradation. In Helicobacter pylori, only two TA systems are known thus far. Our previous studies showed that HP0894–HP0895 acts as a TA system and that HP0894 exhibits intrinsic RNase activity. However, the precise molecular basis for interaction with substrate or antitoxin and the mechanism of mRNA cleavage remain unclear. Therefore, in an attempt to shed some light on the mechanism behind the TA system of HP0894–HP0895, here we present the crystal structures of apo- and metal-bound H. pylori 0894 at 1.28 Å and 1.89 Å, respectively. Through the combined approach of structural analysis and structural homology search, the amino acids involved in mRNase active site were monitored and the reorientations of different residues were discussed in detail. In the mRNase active site of HP0894 toxin, His84 acts as a catalytic residue and reorients itself to exhibit this type of activity, acting as a general acid in an acid–base catalysis reaction, while His47 and His60 stabilize the transition state. Lys52, Glu58, Asp64 and Arg80 have phosphate binding and specific sequence recognition. Glu58 also acts as a general base, and substrate reorientation is caused by Phe88. Based on experimental findings, a model for antitoxin binding could be suggested.     

Viral Evasion of a Bacterial Suicide System by RNA–Based Molecular Mimicry Enables Infectious Altruism

Abortive infection, during which an infected bacterial cell commits altruistic suicide to destroy the replicating bacteriophage and protect the clonal population, can be mediated by toxin-antitoxin systems such as the Type III protein–RNA toxin-antitoxin system, ToxIN. A flagellum-dependent bacteriophage of the Myoviridae, ΦTE, evolved rare mutants that “escaped” ToxIN-mediated abortive infection within Pectobacterium atrosepticum. Wild-type ΦTE encoded a short sequence similar to the repetitive nucleotide sequence of the RNA antitoxin, ToxI, from ToxIN. The ΦTE escape mutants had expanded the number of these “pseudo-ToxI” genetic repeats and, in one case, an escape phage had “hijacked” ToxI from the plasmid-borne toxIN locus, through recombination. Expression of the pseudo-ToxI repeats during ΦTE infection allowed the phage to replicate, unaffected by ToxIN, through RNA–based molecular mimicry. This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system. Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.     

An ADP‐ribosyltransferase Alt of bacteriophage T4 negatively regulates the Escherichia coli MazF toxin of a toxin–antitoxin module

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.     

Characterization of the two-component abortive phage infection mechanism AbiT from Lactococcus lactis

During the production of fermented dairy products, virulent bacteriophages infecting Lactococcus lactis can delay or stop the milk acidification process. A solution to this biological problem consists of introducing natural phage barriers into the strains used by the dairy industry. One such hurdle is called abortive infection (Abi) and causes premature cell death with no or little phage progeny. Here, we describe the isolation and characterization of a novel Abi mechanism encoded by plasmid pED1 from L. lactis. The system is composed of two constitutively cotranscribed genes encoding putative proteins of 127 and 213 amino acids, named AbiTi and AbiTii, respectively. Site-directed mutagenesis indicated that a hydrophobic region at the C-terminal extremity of AbiTi is essential to the antiphage phenotype. The AbiT system is effective against phages of the 936 and P335 species (efficiency of plaquing between 10(-5) and 10(-7)) and causes a 20-fold reduction in the efficiency to form centers of infection as well as a 10- to 12-fold reduction in the burst size. Its efficacy could be improved by raising the plasmid copy number, but changing the intrinsic ratio of AbiTi and AbiTii did not greatly affect the antiphage activity. The monitoring of the intracellular phage infection process by DNA replication, gene expression, and electron microscopy as well as the study of phage mutants by genome mapping indicated that AbiT is likely to act at a later stage of the phage lytic cycle.     

Plasmid-Determined Systems for Restriction and Modification Activity and Abortive Infection in Streptococcus cremoris

Streptococcus cremoris strain IL964 possessed a restriction and modification (R/M) activity which resulted in a bacteriophage efficiency of plating of 5 × 10⁻⁶. Phage sensitivity of protoplast-induced plasmid-cured derivatives indicated that two plasmids called pIL103 (5.7 kilobases) and pIL107 (15.2 kilobases) were each coding for one R/M system. Plasmid pIL103-encoded R/M was ascertained by transfer into the plasmid-free, R⁻/M⁻ strain IL1403 of S. lactis, using protoplast cotransformation. This procedure failed for pIL107 because of some degree of incompatibility between pIL107 and the indicator plasmid pHV1301 used in cotransformation experiments. We also observed that plasmid pIL105 (8.7 kilobases) which showed no incidence on phage sensitivity in the parental strain IL964, mediated abortive infection in strain IL1403. In 97% of the infected cells, the phage infection was abortive, while in the remaining 3% phages were produced with a decreased burst size (50 instead of 180).     

Characterization of the Phd repressor-antitoxin boundary

The P1 plasmid addiction operon (a classic toxin-antitoxin system) encodes Phd, an unstable 73-amino-acid repressor-antitoxin protein, and Doc, a stable toxin. It was previously shown by deletion analysis that the N terminus of Phd was required for repressor activity and that the C terminus was required for antitoxin activity. Since only a quarter of the protein or less was required for both activities, it was hypothesized that Phd might have a modular organization. To further test the modular hypothesis, we constructed and characterized a set of 30 point mutations in the third and fourth quarters of Phd. Four mutations (PhdA36H, V37A, I38A, and F44A) had major defects in repressor activity. Five mutations (PhdD53A, D53R, E55A, F56A, and F60A) had major defects in antitoxin activity. As predicted by the modular hypothesis, point mutations affecting each activity belonged to disjoint, rather than overlapping, sets and were separated rather than interspersed within the linear sequence. A final deletion experiment demonstrated that the C-terminal 24 amino acid residues of Phd (preceded by a methionine) retained full antitoxin activity.