Structure–function studies of Escherichia coli RnlA reveal a novel toxin structure involved in bacteriophage resistance

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 Å. RnlA is composed of three independent domains: NTD (N ‐t erminal d omain), NRD (N r epeated d omain) and DBD (D md‐b inding d omain), 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.     

A promiscuous antitoxin of bacteriophage T4 ensures successful viral replication

Bacteria are constantly threatened by predation from bacteriophage parasites and, in response, have evolved an array of resistance mechanisms. These resistance mechanisms then place greater selection pressure on the infecting bacteriophages, which develop counter-strategies in a perpetual 'arms race' between virus and host. Toxin-antitoxin (TA) loci are widespread in bacteria and can confer multiple benefits, including resistance to bacteriophages. The study by Otsuka and Yonesaki, published in this issue of Molecular Microbiology, describes a new plasmid-encoded TA system, lsoAB, which confers resistance to a dmd(-) mutant of bacteriophage T4 through the activity of the LsoA toxin. Infections with wild-type T4, however, are unaffected as the Dmd protein acts as an alternative antitoxin to LsoA, thus preventing its anti-bacteriophage activity. Dmd has also been shown to negate the activity of a related toxin, RnlA. This is a striking result indicating that Dmd can act as a promiscuous antitoxin, binding and inhibiting multiple toxin partners, when antitoxin activity is generally considered to be limited to a single cognate toxin. This study is an exciting addition to both the bacteriophage resistance and TA fields, and suggests a greater role for TA system-based resistance and counter-resistance in the world's oldest predator-prey relationship.     

Dmd of bacteriophage T4 functions as an antitoxin against Escherichia coli LsoA and RnlA toxins

Enterohaemorrhagic 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.     

细菌毒素-抗毒素系统在噬菌体流产感染中的作用北大核心CSCD

细菌常受到数量众多的噬菌体感染,宿主细菌在和噬菌体竞赛中进化出多样化的分子策略,流产感染(abortive infection,Abi)是其中之一。毒素-抗毒素系统(toxin-antitoxin system,TA)会在细菌受到压力胁迫时表达并介导细菌的低代谢甚至休眠,还能直接减少子代噬菌体形成。此外,部分毒素序列和结构与Cas蛋白高度同源,噬菌体甚至会编码抗毒素类似物来阻遏对应毒素的活性。这表明流产感染中细菌死亡过程导致的噬菌体感染失败与TA功能高度重合,TA可能是噬菌体侵染宿主的主要阻力和防御力量之一。文中基于TA系统的分类和功能,对参与噬菌体流产感染的TA系统进行了综述,并预测具有流产功能的TA系统和其在抗生素开发和疾病治疗中的应用前景。这有助于认识细菌-噬菌体相互作用,并指导噬菌体治疗和合成生物学。     

Mycobacteriophage Fruitloop gp52 inactivates Wag31 (DivIVA) to prevent heterotypic superinfection

Bacteriophages engage in complex dynamic interactions with their bacterial hosts and with each other. Bacteria have numerous mechanisms to resist phage infection, and phages must co‐evolve by overcoming bacterial resistance or by choosing an alternative host. Phages also compete with each other, both during lysogeny by prophage‐mediated defense against viral attack and by superinfection exclusion during lytic replication. Phages are enormously diverse genetically and are replete with small genes of unknown function, many of which are not required for lytic growth, but which may modulate these bacteria–phage and phage–phage dynamics. Using cellular toxicity of phage gene overexpression as an assay, we identified the 93‐residue protein gp52 encoded by Cluster F mycobacteriophage Fruitloop. The toxicity of Fruitloop gp52 overexpression results from interaction with and inactivation of Wag31 (DivIVA), an essential Mycobacterium smegmatis protein organizing cell wall biosynthesis at the growing cellular poles. Fruitloop gene 52 is expressed early in lytic growth and is not required for normal Fruitloop lytic replication but interferes with Subcluster B2 phages such as Hedgerow and Rosebush. We conclude that Hedgerow and Rosebush are Wag31‐dependent phages and that Fruitloop gp52 confers heterotypic superinfection exclusion by inactivating Wag31.     

A Moderate Toxin,GraT, Modulates Growth Rate and Stress Tolerance of Pseudomonas putida

Chromosomal toxin-antitoxin (TA) systems are widespread among free-living bacteria and are supposedly involved in stress tolerance. Here, we report the first TA system identified in the soil bacterium Pseudomonas putida. The system, encoded by the loci PP1586-PP1585, is conserved in pseudomonads and belongs to the HigBA family. The new TA pair was named GraTA for the growth rate-affecting ability of GraT and the antidote activity of GraA. The GraTA system shares many features common to previously described type II TA systems. The overexpression of GraT is toxic to the antitoxin deletion mutants, since the toxin''s neutralization is achieved by binding of the antitoxin. Also, the graTA operon structure and autoregulation by antitoxin resemble those of other TA loci. However, we were able to delete the antitoxin gene from the chromosome, which shows the unusually mild toxicity of innate GraT compared to previously described toxins. Furthermore, GraT is a temperature-dependent toxin, as its growth-regulating effect becomes more evident at lower temperatures. Besides affecting the growth rate, GraT also increases membrane permeability, resulting in higher sensitivity to some chemicals, e.g., NaCl and paraquat. Nevertheless, the active toxin helps the bacteria survive under different stressful conditions and increases their tolerance to several antibiotics, including streptomycin, kanamycin, and ciprofloxacin. Therefore, our data suggest that GraT may represent a new class of mild chromosomal regulatory toxins that have evolved to be less harmful to their host bacterium. Their moderate toxicity might allow finer growth and metabolism regulation than is possible with strong growth-arresting or bactericidal toxins.     

Against the mainstream: the membrane‐associated type I toxin BsrG from Bacillus subtilis interferes with cell envelope biosynthesis without increasing membrane permeability

Toxin‐antitoxin loci, which encode a toxic protein alongside with either RNA or a protein able to counteract the toxicity, are widespread among archaea and bacteria. These loci are implicated in persistence, and as addiction modules to ensure stable inheritance of plasmids and phages. In type I toxin‐antitoxin systems, a small RNA acts as an antitoxin, which prevents the synthesis of the toxin. Most type I toxins are small hydrophobic membrane proteins generally assumed to induce pores, or otherwise permeabilise the cytoplasmic membrane and, as a result, induce cell death by energy starvation. Here we show that this mode of action is not a conserved property of type I toxins. The analysis of the cellular toxicity caused by Bacillus subtilis prophage SPβ‐encoded toxin BsrG revealed that, surprisingly, it neither dissipates membrane potential nor affects cellular ATP‐levels. In contrast, BsrG strongly interferes with the cell envelope biosynthesis, causes membrane invaginations together with delocalisation of the cell wall synthesis machinery and triggers autolysis. Furthermore, efficient inhibition of protein biosynthesis is observed. These findings question the simplistic assumption that small membrane targeting toxins generally act by permeabilising the membrane.     

A cis‐acting antitoxin domain within the chromosomal toxin–antitoxin module EzeT of Escherichia coli quenches toxin activity

Toxin–antitoxin (TA) systems are widespread genetic modules in the genomes of bacteria and archaea emerging as key players that modulate bacterial physiology. They consist of two parts, a toxic component that blocks an essential cellular process and an antitoxin that inhibits this toxic activity during normal growth. According to the nature of the antitoxin and the mode of inhibition, TA systems are subdivided into different types. Here, we describe the characterization of a type II‐like TA system in Escherichia coli called EzeT. While in conventional type II systems the antitoxin is expressed in trans to form an inactive protein–protein complex, EzeT consists of two domains combining toxin and cis‐acting antitoxin functionalities in a single polypeptide chain. We show that the C‐terminal domain of EzeT is homologous to zeta toxins and is toxic in vivo. The lytic phenotype could be attributed to UDP‐N‐acetylglucosamine phosphorylation, so far only described for type II epsilon/zeta systems from Gram‐positive streptococci. Presence of the N‐terminal domain inhibits toxicity in vivo and strongly attenuates kinase activity. Autoinhibition by a cis‐acting antitoxin as described here for EzeT‐type TA systems can explain the occurrence of single or unusually large toxins, further expanding our understanding of the TA system network.     

Ribonucleases in bacterial toxin–antitoxin systems

Toxin–antitoxin (TA) systems are widespread in bacteria and archaea and play important roles in a diverse range of cellular activities. TA systems have been broadly classified into 5 types and the targets of the toxins are diverse, but the most frequently used cellular target is mRNA. Toxins that target mRNA to inhibit translation can be classified as ribosome-dependent or ribosome-independent RNA interferases. These RNA interferases are sequence-specific endoribonucleases that cleave RNA at specific sequences. Despite limited sequence similarity, ribosome-independent RNA interferases belong to a limited number of structural classes. The MazF structural family includes MazF, Kid, ParE and CcdB toxins. MazF members cleave mRNA at 3-, 5- or 7-base recognition sequences in different bacteria and have been implicated in controlling cell death (programmed) and cell growth, and cellular responses to nutrient starvation, antibiotics, heat and oxidative stress. VapC endoribonucleases belong to the PIN-domain family and inhibit translation by either cleaving tRNA^fMet in the anticodon stem loop, cleaving mRNA at -AUA(U/A)-hairpin-G- sequences or by sequence-specific RNA binding. VapC has been implicated in controlling bacterial growth in the intracellular environment and in microbial adaptation to nutrient limitation (nitrogen, carbon) and heat shock. ToxN shows structural homology to MazF and is also a sequence-specific endoribonuclease. ToxN confers phage resistance by causing cell death upon phage infection by cleaving cellular and phage RNAs, thereby interfering with bacterial and phage growth. Notwithstanding our recent progress in understanding ribonuclease action and function in TA systems, the environmental triggers that cause release of the toxin from its cognate antitoxin and the precise cellular function of these systems in many bacteria remain to be discovered. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis

Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369–375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.     

细菌毒素-抗毒素系统的功能

毒素-抗毒素(Toxin-Antitoxin,TA)系统广泛存在于原核生物和古细菌的染色体和质粒中。此系统由2个共表达的基因组成,分别编码稳定的毒素蛋白和易降解的抗毒素,毒素通常发挥毒性作用抑制细菌生长,而抗毒素则可中和毒性,二者相互作用对细菌生长状态起精密调节作用。根据TA的组成和抗毒素的性质,目前已经发现有6型TA,这些TA系统在细菌中发挥的作用一直是近年来学者们研究的热点,文中对细菌TA的功能研究进展进行了综述。     

BREX is a novel phage resistance system widespread in microbial genomes

The perpetual arms race between bacteria and phage has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems, which include the CRISPR‐Cas and restriction‐modification systems, have proven to be invaluable in the biotechnology and dairy industries. Here, we report on a six‐gene cassette in Bacillus cereus which, when integrated into the Bacillus subtilis genome, confers resistance to a broad range of phages, including both virulent and temperate ones. This cassette includes a putative Lon‐like protease, an alkaline phosphatase domain protein, a putative RNA‐binding protein, a DNA methylase, an ATPase‐domain protein, and a protein of unknown function. We denote this novel defense system BREX (Bacteriophage Exclusion) and show that it allows phage adsorption but blocks phage DNA replication. Furthermore, our results suggest that methylation on non‐palindromic TAGGAG motifs in the bacterial genome guides self/non‐self discrimination and is essential for the defensive function of the BREX system. However, unlike restriction‐modification systems, phage DNA does not appear to be cleaved or degraded by BREX, suggesting a novel mechanism of defense. Pan genomic analysis revealed that BREX and BREX‐like systems, including the distantly related Pgl system described in Streptomyces coelicolor, are widely distributed in ~10% of all sequenced microbial genomes and can be divided into six coherent subtypes in which the gene composition and order is conserved. Finally, we detected a phage family that evades the BREX defense, implying that anti‐BREX mechanisms may have evolved in some phages as part of their arms race with bacteria.     

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.     

Analysis of Non-Typeable Haemophilous influenzae VapC1 Mutations Reveals Structural Features Required for Toxicity and Flexibility in the Active Site

Bacteria have evolved mechanisms that allow them to survive in the face of a variety of stresses including nutrient deprivation, antibiotic challenge and engulfment by predator cells. A switch to dormancy represents one strategy that reduces energy utilization and can render cells resistant to compounds that kill growing bacteria. These persister cells pose a problem during treatment of infections with antibiotics, and dormancy mechanisms may contribute to latent infections. Many bacteria encode toxin-antitoxin (TA) gene pairs that play an important role in dormancy and the formation of persisters. VapBC gene pairs comprise the largest of the Type II TA systems in bacteria and they produce a VapC ribonuclease toxin whose activity is inhibited by the VapB antitoxin. Despite the importance of VapBC TA pairs in dormancy and persister formation, little information exists on the structural features of VapC proteins required for their toxic function in vivo. Studies reported here identified 17 single mutations that disrupt the function of VapC1 from non-typeable H. influenzae in vivo. 3-D modeling suggests that side chains affected by many of these mutations sit near the active site of the toxin protein. Phylogenetic comparisons and secondary mutagenesis indicate that VapC1 toxicity requires an alternative active site motif found in many proteobacteria. Expression of the antitoxin VapB1 counteracts the activity of VapC1 mutants partially defective for toxicity, indicating that the antitoxin binds these mutant proteins in vivo. These findings identify critical chemical features required for the biological function of VapC toxins and PIN-domain proteins.     

The RES domain toxins of RES‐Xre toxin‐antitoxin modules induce cell stasis by degrading NAD+

Type II toxin‐antitoxin (TA) modules, which are important cellular regulators in prokaryotes, usually encode two proteins, a toxin that inhibits cell growth and a nontoxic and labile inhibitor (antitoxin) that binds to and neutralizes the toxin. Here, we demonstrate that the res‐xre locus from Photorhabdus luminescens and other bacterial species function as bona fide TA modules in Escherichia coli. The 2.2 Å crystal structure of the intact Pseudomonas putida RES‐Xre TA complex reveals an unusual 2:4 stoichiometry in which a central RES toxin dimer binds two Xre antitoxin dimers. The antitoxin dimers each expose two helix‐turn‐helix DNA‐binding domains of the Cro repressor type, suggesting the TA complex is capable of binding the upstream promoter sequence on DNA. The toxin core domain shows structural similarity to ADP‐ribosylating enzymes such as diphtheria toxin but has an atypical NAD⁺‐binding pocket suggesting an alternative function. We show that activation of the toxin in vivo causes a depletion of intracellular NAD⁺ levels eventually leading to inhibition of cell growth in E. coli and inhibition of global macromolecular biosynthesis. Both structure and activity are unprecedented among bacterial TA systems, suggesting the functional scope of bacterial TA toxins is much wider than previously appreciated.     

Modelling the stability of Stx lysogens

Shiga-toxin-converting bacteriophages (Stx phages) are temperate phages of Escherichia coli, and can cause severe human disease. The spread of shiga toxins by Stx phages is directly linked to lysogen stability because toxins are only synthesized and released once the lytic cycle is initiated. Lysogens of Stx phages are known to be less stable than those of the related lambda phage; this is often described in terms of a 'hair-trigger' molecular switch from lysogeny to lysis. We have developed a mathematical model to examine whether known differences in operator regions and binding affinities between Stx phages and lambda phage can account for the lower stability of Stx lysogens. The Stx phage 933W has only two binding sites in its left operator region (compared to three in phage lambda), but this has a minimal effect on 933W lysogen stability. However, the relatively weak binding affinity between repressor molecules and the second binding site in the right operator is found to significantly reduce the stability of its lysogens, and may account for the hair-trigger nature of the switch. Reduced lysogen stability can lead to increased frequency of genetic recombination in bacterial genomes. The development of the mathematical model has considerable utility in understanding the behaviour and evolution of the molecular switch, with implications for phage-related diseases.     

Cry11Aa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loop alpha-8 of domain II

Bacillus thuringiensis subs israelensis produces Cry toxins active against mosquitoes. Receptor binding is a key determinant for specificity of Cry toxins composed of three domains. We found that exposed loop alpha-8 of Cry11Aa toxin, located in domain II, is an important epitope involved in receptor interaction. Synthetic peptides corresponding to exposed regions in domain II (loop alpha-8, beta-4 and loop 3) competed binding of Cry11Aa to membrane vesicles from Aedes aegypti midgut microvilli. The role of loop alpha-8 of Cry11A in receptor interaction was demonstrated by phage display and site-directed mutagenesis. We isolated a peptide-displaying phage (P5.tox), that recognizes loop alpha-8 in Cry11Aa, interferes interaction with the midgut receptor and attenuates toxicity in bioassay. Loop alpha-8 mutants affected in toxicity and receptor binding were characterized.