Yet another way that phage λ manipulates its Escherichia coli host: λrexB is involved in the lysogenic–lytic switch

The life cycle of phage λ has been studied extensively. Of particular interest has been the process leading to the decision of the phage to switch from lysogenic to lytic cycle. The principal participant in this process is the λcI repressor, which is cleaved under conditions of DNA damage. Cleaved λcI no longer acts as a repressor, allowing phage λ to switch from its lysogenic to lytic cycle. The well‐known mechanism responsible for λcI cleavage is the SOS response. We have recently reported that the Escherichia coli toxin‐antitoxin mazEF pathway inhibits the SOS response; in fact, the SOS response is permitted only in E. coli strains deficient in the expression of the mazEF pathway. Moreover, in strains lysogenic for prophage λ, the SOS response is enabled by the presence of λrexB. λRexB had previously been found to inhibit the degradation of the antitoxin MazE, thereby preventing the toxic action of MazF. Thus, phage λ rexB gene not only safeguards the prophage state by preventing death of its E. coli host but is also indirectly involved in the lysogenic–lytic switch.     

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.     

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.     

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.     

Coverage of diarrhoea‐associated Escherichia coli isolates from different origins with two types of phage cocktails

Eighty‐nine T4‐like phages from our phage collection were tested against four collections of childhood diarrhoea‐associated Escherichia coli isolates representing different geographical origins (Mexico versus Bangladesh), serotypes (69 O, 27 H serotypes), pathotypes (ETEC, EPEC, EIEC, EAEC, VTEC, Shigella), epidemiological settings (community and hospitalized diarrhoea) and years of isolation. With a cocktail consisting of 3 to 14 T4‐like phages, we achieved 54% to 69% coverage against predominantly EPEC isolates from Mexico, 30% to 53% against mostly ETEC isolates from a prospective survey in Bangladesh, 24% to 61% against a mixture of pathotypes isolated from hospitalized children in Bangladesh, and 60% coverage against Shigella isolates. In comparison a commercial Russian phage cocktail containing a complex mixture of many different genera of coliphages showed 19%, 33%, 50% and 90% coverage, respectively, against the four above‐mentioned collections. Few O serotype‐specific phages and no broad‐host range phages were detected in our T4‐like phage collection. Interference phenomena between the phage isolates were observed when constituting larger phage cocktails. Since the coverage of a given T4‐like phage cocktail differed with geographical area and epidemiological setting, a phage composition adapted to a local situation is needed for phage therapy approaches against E. coli pathogens.     

Phage T4‐induced DNA breaks activate a tRNA repair‐defying anticodon nuclease

The natural role of the conserved bacterial anticodon nuclease (ACNase) RloC is not known, but traits that set it apart from the homologous phage T4‐excluding ACNase PrrC could provide relevant clues. PrrC is silenced by a genetically linked DNA restriction‐modification (RM) protein and turned on by a phage‐encoded DNA restriction inhibitor. In contrast, RloC is rarely linked to an RM protein, and its ACNase is regulated by an internal switch responsive to double‐stranded DNA breaks. Moreover, PrrC nicks the tRNA substrate, whereas RloC excises the wobble nucleotide. These distinctions suggested that (i) T4 and related phage that degrade their host DNA will activate RloC and (ii) the tRNA species consequently disrupted will not be restored by phage tRNA repair enzymes that counteract PrrC. Consistent with these predictions we show that Acinetobacter baylyi RloC expressed in Escherichia coli is activated by wild‐type phage T4 but not by a mutant impaired in host DNA degradation. Moreover, host and T4 tRNA species disrupted by the activated ACNase were not restored by T4's tRNA repair system. Nonetheless, T4's plating efficiency was inefficiently impaired by AbaRloC, presumably due to a decoy function of the phage encoded tRNA target, the absence of which exacerbated the restriction.     

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.     

ACA‐specific RNA sequence recognition is acquired via the loop 2 region of MazF mRNA interferase

MazF is an mRNA interferase that cleaves mRNAs at a specific RNA sequence. MazF from E. coli (MazF‐ec) cleaves RNA at A and CA. To date, a large number of MazF homologs that cleave RNA at specific three‐ to seven‐base sequences have been identified from bacteria to archaea. MazF‐ec forms a dimer, in which the interface between the two subunits is known to be the RNA substrate‐binding site. Here, we investigated the role of the two loops in MazF‐ec, which are closely associated with the interface of the MazF‐ec dimer. We examined whether exchanging the loop regions of MazF‐ec with those from other MazF homologs, such as MazF from Myxococcus xanthus (MazF‐mx) and MazF from Mycobacterium tuberculosis (MazF‐mt3), affects RNA cleavage specificity. We found that exchanging loop 2 of MazF‐ec with loop 2 regions from either MazF‐mx or MazF‐mt3 created a new cleavage sequence at (A/U)(A/U)AA and C in addition to the original cleavage site, A and CA, whereas exchanging loop 1 did not alter cleavage specificity. Intriguingly, exchange of loop 2 with 8 or 12 consecutive Gly residues also resulted in a new RNA cleavage site at (A/U)(A/U)AA and C. The present study suggests a method for expanding the RNA cleavage repertoire of mRNA interferases, which is crucial for potential use in the regulation of specific gene expression and for biotechnological applications. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

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.     

55.2, a Phage T4 ORFan Gene,Encodes an Inhibitor of Escherichia coli Topoisomerase I and Increases Phage Fitness

Topoisomerases are enzymes that alter the topological properties of DNA. Phage T4 encodes its own topoisomerase but it can also utilize host-encoded topoisomerases. Here we characterized 55.2, a phage T4 predicted ORF of unknown function. High levels of expression of the cloned 55.2 gene are toxic in E. coli. This toxicity is suppressed either by increased topoisomerase I expression or by partial inactivation of the ATPase subunit of the DNA gyrase. Interestingly, very low-level expression of 55.2, which is non-lethal to wild type E. coli, prevents the growth of a deletion mutant of the topoisomerase I (topA) gene. In vitro, gp55.2 binds DNA and blocks specifically the relaxation of negatively supercoiled DNA by topoisomerase I. In vivo, expression of gp55.2 at low non-toxic levels alters the steady state DNA supercoiling of a reporter plasmid. Although 55.2 is not an essential gene, competition experiments indicate that it is required for optimal phage growth. We propose that the role of gp55.2 is to subtly modulate host topoisomerase I activity during infection to insure optimal T4 phage yield.     

Structural insights into the inhibition mechanism of bacterial toxin LsoA by bacteriophage antitoxin Dmd

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.     

Identification,functional characterization,assembly and structure of ToxIN type III toxin–antitoxin complex from E. coli

Toxin–antitoxin (TA) systems are proposed to play crucial roles in bacterial growth under stress conditions such as phage infection. The type III TA systems consist of a protein toxin whose activity is inhibited by a noncoding RNA antitoxin. The toxin is an endoribonuclease, while the antitoxin consists of multiple repeats of RNA. The toxin assembles with the individual antitoxin repeats into a cyclic complex in which the antitoxin forms a pseudoknot structure. While structure and functions of some type III TA systems are characterized, the complex assembly process is not well understood. Using bioinformatics analysis, we have identified type III TA systems belonging to the ToxIN family across different Escherichia coli strains and found them to be clustered into at least five distinct clusters. Furthermore, we report a 2.097 Å resolution crystal structure of the first E. coli ToxIN complex that revealed the overall assembly of the protein-RNA complex. Isothermal titration calorimetry experiments showed that toxin forms a high-affinity complex with antitoxin RNA resulting from two independent (5′ and 3′ sides of RNA) RNA binding sites on the protein. These results further our understanding of the assembly of type III TA complexes in bacteria.     

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.     

Functional mapping of community‐acquired respiratory distress syndrome (CARDS) toxin of Mycoplasma pneumoniae defines regions with ADP‐ribosyltransferase,vacuolating and receptor‐binding activities

Community‐acquired respiratory distress syndrome (CARDS) toxin from Mycoplasma pneumoniae is a 591‐amino‐acid virulence factor with ADP‐ribosyltransferase (ADPRT) and vacuolating activities. It is expressed at low levels during in vitro growth and at high levels during colonization of the lung. Exposure of experimental animals to purified recombinant CARDS toxin alone is sufficient to recapitulate the cytopathology and inflammatory responses associated with M. pneumoniae infection in humans and animals. Here, by molecular modelling, serial truncations and site‐directed mutagenesis, we show that the N‐terminal region is essential for ADP‐ribosylating activity. Also, by systematic truncation and limited proteolysis experiments we identified a portion of the C‐terminal region that mediates toxin binding to mammalian cell surfaces and subsequent internalization. In addition, the C‐terminal region alone induces vacuolization in a manner similar to full‐length toxin. Together, these data suggest that CARDS toxin has a unique architecture with functionally separable N‐terminal and C‐terminal domains.     

Prophage encoding toxin/antitoxin system PfiT/PfiA inhibits Pf4 production in Pseudomonas aeruginosa

Pf prophages are ssDNA filamentous prophages that are prevalent among various Pseudomonas aeruginosa strains. The genomes of Pf prophages contain not only core genes encoding functions involved in phage replication, structure and assembly but also accessory genes. By studying the accessory genes in the Pf4 prophage in P. aeruginosa PAO1, we provided experimental evidence to demonstrate that PA0729 and the upstream ORF Rorf0727 near the right attachment site of Pf4 form a type II toxin/antitoxin (TA) pair. Importantly, we found that the deletion of the toxin gene PA0729 greatly increased Pf4 phage production. We thus suggest the toxin PA0729 be named PfiT for Pf 4 i nhibition t oxin and Rorf0727 be named PfiA for Pf iT a ntitoxin. The PfiT toxin directly binds to PfiA and functions as a corepressor of PfiA for the TA operon. The PfiAT complex exhibited autoregulation by binding to a palindrome (5′-AATTC N₅GTTAA -3′) overlapping the -35 region of the TA operon. The deletion of pfiT disrupted TA autoregulation and activated pfiA expression. Additionally, the deletion of pfiT also activated the expression of the replication initiation factor gene PA0727. Moreover, the Pf4 phage released from the pfiT deletion mutant overcame the immunity provided by the phage repressor Pf4r. Therefore, this study reveals that the TA systems in Pf prophages can regulate phage production and phage immunity, providing new insights into the function of TAs in mobile genetic elements.     

Structure of the sirtuin‐linked macrodomain SAV0325 from Staphylococcus aureus

Cells use the post‐translational modification ADP‐ribosylation to control a host of biological activities. In some pathogenic bacteria, an operon‐encoded mono‐ADP‐ribosylation cycle mediates response to host‐induced oxidative stress. In this system, reversible mono ADP‐ribosylation of a lipoylated target protein represses oxidative stress response. An NAD⁺‐dependent sirtuin catalyzes the single ADP‐ribose (ADPr) addition, while a linked macrodomain‐containing protein removes the ADPr. Here we report the crystal structure of the sitruin‐linked macrodomain protein from Staphylococcus aureus, SauMacro (also known as SAV0325) to 1.75‐Å resolution. The monomeric SauMacro bears a previously unidentified Zn²⁺‐binding site that putatively aids in substrate recognition and catalysis. An amino‐terminal three‐helix bundle motif unique to this class of macrodomain proteins provides a structural scaffold for the Zn²⁺ site. Structural features of the enzyme further indicate a cleft proximal to the Zn²⁺ binding site appears well suited for ADPr binding, while a deep hydrophobic channel in the protein core is suitable for binding the lipoate of the lipoylated protein target.     

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.