Molecular basis of ribosome recognition and mRNA hydrolysis by the E. coli YafQ toxin

Bacterial type II toxin-antitoxin modules are protein–protein complexes whose functions are finely tuned by rapidly changing environmental conditions. E. coli toxin YafQ is suppressed under steady state growth conditions by virtue of its interaction with its cognate antitoxin, DinJ. During stress, DinJ is proteolytically degraded and free YafQ halts translation by degrading ribosome-bound mRNA to slow growth until the stress has passed. Although structures of the ribosome with toxins RelE and YoeB have been solved, it is unclear what residues among ribosome-dependent toxins are essential for mediating both recognition of the ribosome and the mRNA substrate given their low sequence identities. Here we show that YafQ coordinates binding to the 70S ribosome via three surface-exposed patches of basic residues that we propose directly interact with 16S rRNA. We demonstrate that YafQ residues H50, H63, D67 and H87 participate in acid-base catalysis during mRNA hydrolysis and further show that H50 and H63 functionally complement as general bases to initiate the phosphodiester cleavage reaction. Moreover YafQ residue F91 likely plays an important role in mRNA positioning. In summary, our findings demonstrate the plasticity of ribosome-dependent toxin active site residues and further our understanding of which toxin residues are important for function.     

Characterization of Escherichia coli dinJ-yafQ toxin-antitoxin system using insights from mutagenesis data

Escherichia coli dinJ-yafQ operon codes for a functional toxin-antitoxin (TA) system. YafQ toxin is an RNase which, upon overproduction, specifically inhibits the translation process by cleaving cellular mRNA at specific sequences. DinJ is an antitoxin and counteracts YafQ-mediated toxicity by forming a strong protein complex. In the present study we used site-directed mutagenesis of YafQ to determine the amino acids important for its catalytic activity. His50Ala, His63Ala, Asp67Ala, Trp68Ala, Trp68Phe, Arg83Ala, His87Ala, and Phe91Ala substitutions of the predicted active-site residues of YafQ abolished mRNA cleavage in vivo, whereas Asp61Ala and Phe91Tyr mutations inhibited YafQ RNase activity only moderately. We show that YafQ, upon overexpression, cleaved mRNAs preferably 5' to A between the second and third nucleotides in the codon in vivo. YafQ also showed RNase activity against mRNA, tRNA, and 5S rRNA molecules in vitro, albeit with no strong specificity. The endoribonuclease activity of YafQ was inhibited in the complex with DinJ antitoxin in vitro. DinJ-YafQ protein complex and DinJ antitoxin alone selectively bind to one of the two palindromic sequences present in the intergenic region upstream of the dinJ-yafQ operon, suggesting the autoregulation mode of this TA system.     

细菌毒素-抗毒素系统的研究进展

毒素-抗毒素系统(toxin-antitoxin system,TA)由两个共表达的基因组成,其中一个基因编码不稳定的抗毒素蛋白(antitoxin),另一个基因编码稳定的毒素蛋白(toxin).毒素-抗毒素系统最早发现于一些低拷贝的质粒,用来维持低拷贝质粒在菌群中的稳定存在.随后的研究表明,毒素-抗毒素系统广泛存在于细菌,包括一些致病菌的染色体上.在营养缺乏等不良生长条件下,由于基因表达的抑制和蛋白酶的降解作用,不稳定的抗毒素蛋白减少,从而产生游离的毒素蛋白,导致细菌的生长抑制和死亡.毒素-抗毒素系统的生理功能目前还存在争议,有学者认为细茼染色体上的毒素-抗毒素系统可以在不良生长状况下介导细菌的死亡,即细茼程序性细胞死亡(baeterial programmedcell death).但也有证据显示,毒素-抗毒素系统的功能更偏向于应激状态下的生理调节方面,即只起应激状态下的抑菌作用而不是杀菌作用.对细菌生长调控中毒素-抗毒素系统的作用机理进行综述,并探讨毒素-抗毒素系统研究的理论和应用价值.     

Toxin-antitoxin modules as bacterial metabolic stress managers

Bacterial genomes frequently contain operons that encode a toxin and its antidote. These 'toxin-antitoxin (TA) modules' have an important role in bacterial stress physiology and might form the basis of multidrug resistance. The toxins in TA modules act as gyrase poisons or stall the ribosome by mediating the cleavage of mRNA. The antidotes contain an N-terminal DNA-binding region of variable fold and a C-terminal toxin-inhibiting domain. When bound to toxin, the C-terminal domain adopts an extended conformation. In the absence of toxin, by contrast, this domain (and sometimes the whole antidote protein) remains unstructured, allowing its fast degradation by proteolysis. Under silent conditions the antidote inhibits the toxin and the toxin-antidote complex acts as a repressor for the TA operon, whereas under conditions of activation proteolytic degradation of the antidote outpaces its synthesis.     

Escherichia coli dinJ-yafQ genes act as a toxin-antitoxin module

Bacterial toxin-antitoxin (TA) systems are operons that code for a stable toxic protein and a labile antitoxin. TA modules are widespread on the chromosomes of free-living Bacteria and Archaea, where they presumably act as stress response elements. The chromosome of Escherichia coli K-12 encodes four known TA pairs, as well as the dinJ-yafQ operon, which is hypothesized to be a TA module based on operon organization similar to known TA genes. Induction of YafQ inhibited cell growth, but its toxicity was counteracted by coexpression of dinJ cloned on a separate plasmid. YafQ(His)(6) and DinJ proteins coeluted in Ni(2+)-affinity and gel filtration chromatography, implying the formation of a specific and stable YafQ-DinJ protein complex with an estimated molecular mass of c. 37.3 kDa. Induction of YafQ reduced protein synthesis up to 40% as judged by incorporation of [(35)S]-methionine, but did not influence the rates of DNA and RNA synthesis. Structure modelling of E. coli YafQ revealed its structural relationship with bacterial toxins of known structure suggesting that it might act as a sequence-specific mRNA endoribonuclease.     

Bacterial Toxin HigB Associates with Ribosomes and Mediates Translation-dependent mRNA Cleavage at A-rich Sites

Most pathogenic Proteus species are primarily associated with urinary tract infections, especially in persons with indwelling catheters or functional/anatomic abnormalities of the urinary tract. Urinary tract infections caused by Proteus vulgaris typically form biofilms and are resistant to commonly used antibiotics. The Rts1 conjugative plasmid from a clinical isolate of P. vulgaris carries over 300 predicted open reading frames, including antibiotic resistance genes. The maintenance of the Rts1 plasmid is ensured in part by the HigBA toxin-antitoxin system. We determined the precise mechanism of action of the HigB toxin in vivo, which is distinct from other known toxins. We demonstrate that HigB is an endoribonuclease whose enzymatic activity is dependent on association with ribosomes through the 50 S subunit. Using primer extension analysis of several test mRNAs, we showed that HigB cleaved extensively across the entire length of coding regions only at specific recognition sequences. HigB mediated cleavage of 100% of both in-frame and out-of-frame AAA sequences. In addition, HigB cleaved ∼20% of AA sequences in coding regions and occasionally cut single As. Remarkably, the cleavage specificity of HigB coincided with one of the most frequently used codons in the AT-rich Proteus spp., AAA (lysine). Therefore, the HigB-mediated plasmid maintenance system for the Rts1 plasmid highlights the intimate relationship between host cells and extrachromosomal DNA that enables the dynamic acquisition of genes that impart a spectrum of survival advantages, including those encoding multidrug resistance and virulence factors.Toxin-antitoxin (TA)³/addiction/suicide modules typically include an autoregulated operon encoding a labile antitoxin and a more stable toxic protein (1). TA toxins facilitate stress survival (chromosomal) or plasmid maintenance and post-segregational killing (extrachromosomal; reviewed in Refs. 1, 2). Most chromosomal TA toxins inhibit cell growth by reversibly targeting either protein translation or DNA replication; their cognate antitoxins prevent toxin activity during periods of optimal growth but enable finely tuned control of TA module toxicity during relatively short periods of environmental stress. However, prolonged stress leads to a point of no return and cell death (3–5).There are six confirmed chromosomal TA loci in Escherichia coli K12 cells: dinJ-yafQ, relBE, yefM-yoeB, mazEF, chpBI-BK, and hipBA. The toxins MazF and ChpBK are sequence-specific endoribonucleases that cleave free mRNA (6–10). The RelE toxin interacts with the ribosome and induces mRNA cleavage with a preference for the UAG stop codon (11–13). The YafQ toxin is a ribosome-associated endoribonuclease that cleaves in-frame AAA codons that are followed by either an A or G in the subsequent codon (14). The YoeB toxin inhibits translation at the initiation step, apparently by destabilization of the initiation complex (15). HipA toxin is a kinase whose mechanism of action is not known (16, 17).Although the mechanism of action of many E. coli chromosomal and plasmid-derived toxins has been determined, the precise function of the HigB toxin has not been characterized. The higBA TA module is not present in E. coli K12; it resides on the Rts1 plasmid that typically replicates in Proteus spp. and imparts kanamycin resistance as well as temperature-sensitive post-segregational killing at 42 °C (18, 19). Interestingly, one or more chromosomal counterparts of higBA have been reported for several pathogens, including Vibrio cholerae, Streptococcus pneumoniae, E. coli CFT073, and E. coli O157:H7 (20). Some characterization of the two V. cholerae HigBA modules has been performed. First, one of the two higBA modules was shown to possess the general characteristics of TA systems by demonstration of toxin-antitoxin interaction, module organization/regulation, HigB toxicity, and rescue of toxicity with the cognate HigA antitoxin (21). Overexpression of HigB derived from two individual higBA modules encoded in V. cholerae or from Rts1 leads to inhibition of protein synthesis through translation-dependent mRNA cleavage in a manner similar to, but distinct from, RelE (22).HigB is a member of the RelE family of toxins, including RelE, YafQ, and YoeB (20). In this study, we have identified the precise mode of action of HigB from Rts1. HigB associated with the 50 S ribosomal subunit, and this HigB-ribosome complex cleaved within mRNA coding regions at all AAA triplet sequences, both in-frame and out-of-frame. HigB appeared to be responsible for the mRNA cleavage activity of the HigB-ribosome complex because a HigB H92Q mutant lacked mRNA cleavage activity but remained associated with the ribosome. Finally, the cleavage specificity of HigB on plasmid Rts1 coincided with the sequence (AAA, lysine) of either the most abundant or the second most abundant codon in its Proteus host.     

毒素-抗毒素系统介导滞留菌形成机制

滞留菌是一类处于低代谢休眠状态的抗生素耐受细菌亚群，能够在致死性压力应激后存活下来，是抗生素治疗失败和复发性感染的主要原因之一。毒素-抗毒素系统(toxin-antitoxin system, TA)作为压力应激模块普遍存在于各种细菌中，由稳定的毒素和不稳定但可以中和毒素的同源抗毒素组成。压力情况下，第二信使(p)ppGpp激活Lon,随后大多数II型TA系统被激活，诱导滞留菌形成。同样在(p)ppGpp存在的情况下，Obg刺激hokB转录，使毒素积累，抑制细菌DNA复制、转录、翻译等重要的生理过程，驱动细菌形成滞留菌。SOS反应是激活TA系统的另一个主要途径，解除了对tisB转录的抑制，使其在细胞内积累并插入细胞膜，破坏质子动力势，降低胞内ATP水平，诱使休眠和滞留菌形成。讨论TA系统介导滞留菌形成的机制有助于提出新型抗菌策略。     

tRNA is a new target for cleavage by a MazF toxin

Toxin-antitoxin (TA) systems play key roles in bacterial persistence, biofilm formation and stress responses. The MazF toxin from the Escherichia coli mazEF TA system is a sequence- and single-strand-specific endoribonuclease, and many studies have led to the proposal that MazF family members exclusively target mRNA. However, recent data indicate some MazF toxins can cleave specific sites within rRNA in concert with mRNA. In this report, we identified the repertoire of RNAs cleaved by Mycobacterium tuberculosis toxin MazF-mt9 using an RNA-seq-based approach. This analysis revealed that two tRNAs were the principal targets of MazF-mt9, and each was cleaved at a single site in either the tRNA^Pro14 D-loop or within the tRNA^Lys43 anticodon. This highly selective target discrimination occurs through recognition of not only sequence but also structural determinants. Thus, MazF-mt9 represents the only MazF family member known to target tRNA and to require RNA structure for recognition and cleavage. Interestingly, the tRNase activity of MazF-mt9 mirrors basic features of eukaryotic tRNases that also generate stable tRNA-derived fragments that can inhibit translation in response to stress. Our data also suggest a role for tRNA distinct from its canonical adapter function in translation, as cleavage of tRNAs by MazF-mt9 downregulates bacterial growth.     

Production and partial purification of a peptide antibiotic from Staphylococcus epidermidis

When grown on solid or in liquid Brain Heart Infusion at 37°C, Staphylococcus epidermidis NCIB 11536 produced antibiotic activity against a wide range of Gram positive bacteria. Production was influenced by aeration, pH, glucose concentration and specific growth rate. Inhibitory activity could be concentrated by ammonium sulphate precipitation (30–55% saturation). On Sephadex G50 using 0.05 mol/1 sodium phosphate buffer, pH 6.0, two peaks of antibiotic activity were detected. The first peak eluted with the void volume (K_d= 0) and the second peak was retained by the gel (K_d= 0.73–0.77). These two substances did not represent the monomeric and polymeric forms of a staphylococcal bacteriocin. The low mol. wt inhibitor, which was responsible for over 95% of the recovered activity on Sephadex G50, could be partially purified by a combination of gel filtration on Biogel P2 and ion-exchange chromatography on Sephadex C-25. Yields were increased by combining these two steps into a single procedure (duocolumn). The semi-purified inhibitor was desalted using Sep-pak C₁₈ cartridges. Biological activity was resistant to enzymic denaturation except by high concentrations of trypsin (50 units/μg, 3 h, 25°C). This peptide antibiotic is different from any previously described staphylococcal inhibitors.     

Amino acid starvation and colicin D treatment induce A-site mRNA cleavage in Escherichia coli

Escherichia coli possesses a unique RNase activity that cleaves stop codons in the ribosomal aminoacyl-tRNA binding site (A-site) during inefficient translation termination. This A-site mRNA cleavage allows recycling of arrested ribosomes by facilitating recruitment of the tmRNA•SmpB ribosome rescue system. To test whether A-site nuclease activity also cleaves sense codons, we induced ribosome pausing at each of the six arginine codons using three strategies; rare codon usage, arginine starvation, and inactivation of arginine tRNAs with colicin D. In each instance, ribosome pausing induced mRNA cleavage within the target arginine codons, and resulted in tmRNA-mediated SsrA-peptide tagging of the nascent polypeptide. A-site mRNA cleavage did not require the stringent factor ppGpp, or bacterial toxins such as RelE, which mediates a similar nuclease activity. However, the efficiency of A-site cleavage was modulated by the identity of the two codons immediately upstream (5′ side) of the A-site codon. Starvation for histidine and tryptophan also induced A-site cleavage at histidine and tryptophan codons, respectively. Thus, A-site mRNA cleavage is a general response to ribosome pausing, capable of cleaving a variety of sense and stop codons. The induction of A-site cleavage during amino acid starvation suggests this nuclease activity may help to regulate protein synthesis during nutritional stress.     

Growth and translation inhibition through sequence-specific RNA binding by Mycobacterium tuberculosis VapC toxin

The Mycobacterium tuberculosis genome harbors an unusually large number of toxin-antitoxin (TA) modules. Curiously, over half of these are VapBC (virulence-associated protein) family members. Nonetheless, the cellular target, precise mode of action, and physiological role of the VapC toxins in this important pathogen remain unclear. To better understand the function of this toxin family, we studied the features and biochemical properties of a prototype M. tuberculosis VapBC TA system, vapBC-mt4 (Rv0596c-Rv0595c). VapC-mt4 expression resulted in growth arrest, a hallmark of all TA toxins, in Escherichia coli, Mycobacterium smegmatis, and M. tuberculosis. Its expression led to translation inhibition accompanied by a gradual decrease in the steady-state levels of several mRNAs. VapC-mt4 exhibited sequence-specific endoribonuclease activity on mRNA templates at ACGC and AC(A/U)GC sequences. However, the cleavage activity of VapC-mt4 was comparatively weak relative to the TA toxin MazF-mt1 (Rv2801c). Unlike other TA toxins, translation inhibition and growth arrest preceded mRNA cleavage, suggesting that the RNA binding property of VapC-mt4, not RNA cleavage, initiates toxicity. In support of this hypothesis, expression of VapC-mt4 led to an increase in the recovery of total RNA with time in contrast to TA toxins that inhibit translation via direct mRNA cleavage. Additionally, VapC-mt4 exhibited stable, sequence-specific RNA binding in an electrophoretic mobility shift assay. Finally, VapC-mt4 inhibited protein synthesis in a cell-free system without cleaving the corresponding mRNA. Therefore, the activity of VapC-mt4 is mechanistically distinct from other TA toxins because it appears to primarily inhibit translation through selective, stable binding to RNA.     

Shutdown decay of mRNA

Although plasmid-borne and chromosomal toxin-antitoxin (TA) operons have been known for some time, the recent identification of mRNA as the target of at least two different classes of toxins has led to a dramatic renewal of interest in these systems as mediators of stress responses. Members of the MazF/PemK family, the so-called mRNA interferases, are ribonucleases that inhibit translation by destroying cellular mRNAs under stress conditions, while the founder member of the RelE family promotes cleavage of mRNAs through the ribosome. Detailed structures of these enzymes, often in complex with their inhibitors, have provided vital clues to their mechanisms of action. The primary role and regulation of these systems has been the subject of some controversy. One model suggests they play a beneficial role by wiping the slate clean and preventing wasteful energy consumption by the translational apparatus during adaptation to stress conditions, while another favours the idea that their main function is programmed cell death. The two models might not be mutually exclusive if a side-effect of prolonged exposure to toxic RNase activity without de novo synthesis of the inhibitor were a state of dormancy for which we do not yet understand the key to recovery. In this review, I discuss the recent developments in the rapidly expanding field of what I refer to as bacterial shutdown decay.     

Ribosome rescue by tmRNA requires truncated mRNAs

tmRNA targets ribosomes, stalled either on truncated mRNAs or on mRNAs with slowly read sense or stop codons, tags the newly synthesized peptide chains for degradation and allows for their release by a class-1 release factor. We have studied in vitro how the rate of trans-transfer of a peptide from the P-site tRNA to tmRNA and the efficiency by which tmRNA competes with peptide release factors depend on the length of the mRNA downstream from the P-site. We show that the rate and efficiency of tmRNA action decrease rapidly with increasing down stream length and approach zero when it exceeds 15 bases. We demonstrate that tmRNA action is strongly stimulated by RelE cleavage of mRNA in the A site. We conclude that tmRNA action in vivo must always be preceded by mRNA truncation, and suggest that cleavage of ribosome bound mRNAs is a common element in different bacterial stress responses.