RelA-SpoT Homolog toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis.

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.

A hyperpromiscuous antitoxin protein domain for the neutralization of diverse toxin domains.

Toxin-antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids as well as temperate bacteriophages. They act as regulatory switches, with the toxin limiting the growth of bacteria and archaea by compromising diverse essential cellular targets and the antitoxin counteracting the toxic effect. To uncover previously uncharted TA diversity across microbes and bacteriophages, we analyzed the conservation of genomic neighborhoods using our computational tool FlaGs (for flanking genes), which allows high-throughput detection of TA-like operons. Focusing on the widespread but poorly experimentally characterized antitoxin domain DUF4065, our in silico analyses indicated that DUF4065-containing proteins serve as broadly distributed antitoxin components in putative TA-like operons with dozens of different toxic domains with multiple different folds. Given the versatility of DUF4065, we have named the domain Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralized TA pairs. While the majority of validated PanA-neutralized toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises transcription and translation as well as inducing RelA-dependent accumulation of the nucleotide alarmone (p)ppGpp. We find that Panacea-containing antitoxins form a complex with their diverse cognate toxins, characteristic of the direct neutralization mechanisms employed by Type II TA systems. Finally, through directed evolution, we have selected PanA variants that can neutralize noncognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain.

Structural basis for HflXr-mediated antibiotic resistance in Listeria monocytogenes.

HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.

Photorhabdus antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of 23S ribosomal RNA.

Bacteria have evolved sophisticated mechanisms to deliver potent toxins into bacterial competitors or into eukaryotic cells in order to destroy rivals and gain access to a specific niche or to hijack essential metabolic or signaling pathways in the host. Delivered effectors carry various activities such as nucleases, phospholipases, peptidoglycan hydrolases, enzymes that deplete the pools of NADH or ATP, compromise the cell division machinery, or the host cell cytoskeleton. Effectors categorized in the family of polymorphic toxins have a modular structure, in which the toxin domain is fused to additional elements acting as cargo to adapt the effector to a specific secretion machinery. Here we show that Photorhabdus laumondii, an entomopathogen species, delivers a polymorphic antibacterial toxin via a type VI secretion system. This toxin inhibits protein synthesis in a NAD+-dependent manner. Using a biotinylated derivative of NAD, we demonstrate that translation is inhibited through ADP-ribosylation of the ribosomal 23S RNA. Mapping of the modification further showed that the adduct locates on helix 44 of the thiostrepton loop located in the GTPase-associated center and decreases the GTPase activity of the EF-G elongation factor.

A role for the Saccharomyces cerevisiae ABCF protein New1 in translation termination/recycling.

Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3'-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling.

HicA toxin of Escherichia coli derepresses hicAB transcription to selectively produce HicB antitoxin.

Antitoxins encoded by type II toxin - antitoxin (TA) modules neutralize cognate toxins by direct protein - protein contact and in addition, regulate TA operon transcription by binding to operators in the promoter regions. On top of the simple negative feed-back regulation, canonical type II TA operons are regulated by a mechanism called 'Conditional Cooperativity'(CC). In CC, the cellular toxin:antitoxin (T:A) ratio controls the transcription-rate such that low T:A ratios favour repression and high T:A ratios favour de-repression of TA operon transcription. Here a new molecular mechanism that secures selective synthesis of antitoxin in the presence of excess toxin was unravelled. The hicAB locus of E. coli K-12 encodes HicA mRNase and HicB antitoxin. It was shown that hicAB is transcribed by two promoters, an upstream one that is activated by CRP-cAMP and competence factor Sxy and a downstream one that is autorepressed solely by HicB. Excess HicA destabilizes the HicB•operator complex in vitro and consistently, activates hicAB transcription in vivo. Remarkably, the hicAB transcript synthesized from the HicB-controlled promoter produces HicB but not HicA. Thus, the HicA-mediated derepression of hicAB transcription provides a mechanism that conditionally and selectively stimulates synthesis of HicB antitoxin under conditions of excess HicA toxin.

Chp1 is a dedicated chaperone at the ribosome that safeguards eEF1A biogenesis.

Cotranslational protein folding depends on general chaperones that engage highly diverse nascent chains at the ribosomes. Here we discover a dedicated ribosome-associated chaperone, Chp1, that rewires the cotranslational folding machinery to assist in the challenging biogenesis of abundantly expressed eukaryotic translation elongation factor 1A (eEF1A). Our results indicate that during eEF1A synthesis, Chp1 is recruited to the ribosome with the help of the nascent polypeptide-associated complex (NAC), where it safeguards eEF1A biogenesis. Aberrant eEF1A production in the absence of Chp1 triggers instant proteolysis, widespread protein aggregation, activation of Hsf1 stress transcription and compromises cellular fitness. The expression of pathogenic eEF1A2 variants linked to epileptic-dyskinetic encephalopathy is protected by Chp1. Thus, eEF1A is a difficult-to-fold protein that necessitates a biogenesis pathway starting with dedicated folding factor Chp1 at the ribosome to protect the eukaryotic cell from proteostasis collapse.

The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArg and tRNALys isoacceptors in the P site.

The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.

Synthetic oxepanoprolinamide iboxamycin is active against Listeria monocytogenes despite the intrinsic resistance mediated by VgaL/Lmo0919 ABCF ATPase.

Background: Listeriosis is a food-borne disease caused by the Gram-positive Bacillota (Firmicute) bacterium Listeria monocytogenes. Clinical L. monocytogenes isolates are often resistant to clinically used lincosamide clindamycin, thus excluding clindamycin as a viable treatment option. Objectives: We have established newly developed lincosamide iboxamycin as a potential novel antilisterial agent. Methods: We determined MICs of the lincosamides lincomycin, clindamycin and iboxamycin for L. monocytogenes, Enterococcus faecalis and Bacillus subtilis strains expressing synergetic antibiotic resistance determinants: ABCF ATPases that directly displace antibiotics from the ribosome and Cfr, a 23S rRNA methyltransferase that compromises antibiotic binding. For L. monocytogenes strains, either expressing VgaL/Lmo0919 or lacking the resistance factor, we performed time-kill kinetics and post-antibiotic effect assays. Results: We show that the synthetic lincosamide iboxamycin is highly active against L. monocytogenes and can overcome the intrinsic lincosamide resistance mediated by VgaL/Lmo0919 ABCF ATPase. While iboxamycin is not bactericidal against L. monocytogenes, it displays a pronounced post-antibiotic effect, which is a valuable pharmacokinetic feature. We demonstrate that VmlR ABCF of B. subtilis grants significant (33-fold increase in MIC) protection from iboxamycin, while LsaA ABCF of E. faecalis grants an 8-fold protective effect. Furthermore, the VmlR-mediated iboxamycin resistance is cooperative with that mediated by the Cfr, resulting in up to a 512-fold increase in MIC. Conclusions: While iboxamycin is a promising new antilisterial agent, our findings suggest that emergence and spread of ABCF ARE variants capable of defeating next-generation lincosamides in the clinic is possible and should be closely monitored.

Structural basis for PoxtA-mediated resistance to phenicol and oxazolidinone antibiotics.

PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF). They confer resistance to oxazolidinone and phenicol antibiotics, such as linezolid and chloramphenicol, which stall translating ribosomes when certain amino acids are present at a defined position in the nascent polypeptide chain. These proteins are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria, and are thought to confer resistance by binding to the ribosome and dislodging the bound antibiotic. However, the mechanistic basis of this resistance remains unclear. Here we refine the PoxtA spectrum of action, demonstrate alleviation of linezolid-induced context-dependent translational stalling, and present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome. PoxtA perturbs the CCA-end of the P-site tRNA, causing it to shift by ∼4 Šout of the ribosome, corresponding to a register shift of approximately one amino acid for an attached nascent polypeptide chain. We postulate that the perturbation of the P-site tRNA by PoxtA thereby alters the conformation of the attached nascent chain to disrupt the drug binding site.

Mastering inflectional suffixes: a longitudinal study of beginning writers' spellings*.

This study tracked the order in which ten beginning spellers (M age=5 ; 05; SD=0·21 years) mastered the correct spellings of common inflectional suffixes in English. Spellings from children's journals from kindergarten and grade 1 were coded. An inflectional suffix was judged to be mastered when children spelled it accurately in 90 percent of the contexts in which it was grammatically required, a criterion used to study the order of acquisition of grammatical morphemes in oral language. The results indicated that the order in which children learned to spell inflectional suffixes correctly is similar to the order in which they learn to use them in oral language, before school age. Discrepancies between the order of mastery for inflectional suffixes in written and oral language are discussed in terms of English spelling conventions, which introduce variables into the spelling of inflected words that are not present in oral language.

Structural basis of ABCF-mediated resistance to pleuromutilin, lincosamide, and streptogramin A antibiotics in Gram-positive pathogens.

Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target. One class of such proteins are the antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F-subtype (ARE-ABCFs), which are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition from antibiotics that target the large ribosomal subunit. Here, we present single-particle cryo-EM structures of ARE-ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaALC and Listeria monocytogenes VgaL. Supported by extensive mutagenesis analysis, these structures enable a general model for antibiotic resistance mediated by these ARE-ABCFs to be proposed. In this model, ABCF binding to the antibiotic-stalled ribosome mediates antibiotic release via mechanistically diverse long-range conformational relays that converge on a few conserved ribosomal RNA nucleotides located at the peptidyltransferase center. These insights are important for the future development of antibiotics that overcome such target protection resistance mechanisms.

In Vitro Studies of Persister Cells.

Many bacterial pathogens can permanently colonize their host and establish either chronic or recurrent infections that the immune system and antimicrobial therapies fail to eradicate. Antibiotic persisters (persister cells) are believed to be among the factors that make these infections challenging. Persisters are subpopulations of bacteria which survive treatment with bactericidal antibiotics in otherwise antibiotic-sensitive cultures and were extensively studied in a hope to discover the mechanisms that cause treatment failures in chronically infected patients; however, most of these studies were conducted in the test tube. Research into antibiotic persistence has uncovered large intrapopulation heterogeneity of bacterial growth and regrowth but has not identified essential, dedicated molecular mechanisms of antibiotic persistence. Diverse factors and stresses that inhibit bacterial growth reduce killing of the bulk population and may also increase the persister subpopulation, implying that an array of mechanisms are present. Hopefully, further studies under conditions that simulate the key aspects of persistent infections will lead to identifying target mechanisms for effective therapeutic solutions.

Intramolecular Interactions Dominate the Autoregulation of Escherichia coli Stringent Factor RelA.

Amino acid starvation in Escherichia coli activates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands - such as "starved" ribosomal complexes, deacylated tRNA and pppGpp - and by individual RelA domains. The auto-regulation of RelA is proposed to act either in cis (inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) or in trans (CTD-mediated dimerization leading to enzyme inhibition). In this report, we probed the regulatory roles of the individual domains of E. coli RelA and our results are not indicative of RelA dimerization being the key regulatory mechanism. First, at growth-permitting levels, ectopic expression of RelA CTD does not interfere with activation of native RelA, indicating lack of regulation via inhibitory complex formation in the cell. Second, in our biochemical assays, increasing RelA concentration does not decrease the enzyme activity, as would be expected in the case of efficient auto-inhibition via dimerization. Third, while high-level CTD expression efficiently inhibits the growth, the effect is independent of native RelA and is mediated by direct inhibition of protein synthesis, likely via direct interaction with the ribosomal A-site. Finally, deletion of the RRM domain of the CTD region leads to growth inhibition mediated by accumulation of (p)ppGpp, suggesting de-regulation of the synthetic activity in this mutant.

The E. coli HicB Antitoxin Contains a Structurally Stable Helix-Turn-Helix DNA Binding Domain.

The E. coli hicAB type II toxin-antitoxin locus is unusual by being controlled by two promoters and by having the toxin encoded upstream of the antitoxin. HicA toxins contain a double-stranded RNA-binding fold and cleaves both mRNA and tmRNA in vivo, while HicB antitoxins contain a partial RNase H fold and either a helix-turn-helix (HTH) or ribbon-helix-helix domain. It is not known how an HTH DNA-binding domain affects higher-order structure for the HicAB modules. Here, we present crystal structures of the isolated E. coli HicB antitoxin and full-length HicAB complex showing that HicB forms a stable DNA-binding module and interacts in a canonical way with HicA despite the presence of an HTH-type DNA-binding domain. No major structural rearrangements take place upon binding of the toxin. Both structures expose well-ordered DNA-binding motifs allowing a model for DNA binding by the antitoxin to be generated.

Serine-Threonine Kinases Encoded by Split hipA Homologs Inhibit Tryptophanyl-tRNA Synthetase.

Type II toxin-antitoxin (TA) modules encode a stable toxin that inhibits cell growth and an unstable protein antitoxin that neutralizes the toxin by direct protein-protein contact. hipBA of Escherichia coli strain K-12 codes for HipA, a serine-threonine kinase that phosphorylates and inhibits glutamyl-tRNA synthetase. Induction of hipA inhibits charging of glutamyl-tRNA that, in turn, inhibits translation and induces RelA-dependent (p)ppGpp synthesis and multidrug tolerance. Here, we describe the discovery of a three-component TA gene family that encodes toxin HipT, which exhibits sequence similarity with the C-terminal part of HipA. A genetic screening revealed that trpS in high copy numbers suppresses HipT-mediated growth inhibition. We show that HipT of E. coli O127 is a kinase that phosphorylates tryptophanyl-tRNA synthetase in vitro at a conserved serine residue. Consistently, induction of hipT inhibits cell growth and stimulates production of (p)ppGpp. The gene immediately upstream from hipT, called hipS, encodes a small protein that exhibits sequence similarity with the N terminus of HipA. HipT kinase was neutralized by cognate HipS in vivo, whereas the third component, HipB, encoded by the first gene of the operon, did not counteract HipT kinase activity. However, HipB augmented the ability of HipS to neutralize HipT. Analysis of two additional hipBST-homologous modules showed that, indeed, HipS functions as an antitoxin in these cases also. Thus, hipBST constitutes a novel family of tricomponent TA modules where hipA has been split into two genes, hipS and hipT, that function as a novel type of TA pair.IMPORTANCE Bacterial toxin-antitoxin (TA) modules confer multidrug tolerance (persistence) that may contribute to the recalcitrance of chronic and recurrent infections. The first high-persister gene identified was hipA of Escherichia coli strain K-12, which encodes a kinase that inhibits glutamyl-tRNA synthetase. The hipA gene encodes the toxin of the hipBA TA module, while hipB encodes an antitoxin that counteracts HipA. Here, we describe a novel, widespread TA gene family, hipBST, that encodes HipT, which exhibits sequence similarity with the C terminus of HipA. HipT is a kinase that phosphorylates tryptophanyl-tRNA synthetase and thereby inhibits translation and induces the stringent response. Thus, this new TA gene family may contribute to the survival and spread of bacterial pathogens.

Behavioral Sleep Problems and their Potential Impact on Developing Executive Function in Children.

Bedtime resistance and night waking are common sleep problems throughout childhood, especially in the early years. These sleep problems may lead to difficulties in neurobehavioral functioning, but most research into childhood sleep problems has not emphasized the importance of the developmental context in which disruptions in neurobehavioral and daytime functioning occur. We review the development of sleep as well as executive functioning (EF) in childhood and suggest that EF may be particularly vulnerable to the effects of these common childhood sleep problems because of its prolonged course of maturation. Behavioral problems associated with common sleep problems suggest poor self-regulation in the context of sleep loss, and developing EF skills play important roles in self-regulation. A research agenda that considers a developmental approach to sleep and sleep problems in the context of childhood EF performance is outlined to promote future research in this area. CITATION: Turnbull K; Reid GJ; Morton JB. Behavioral sleep problems and their potential impact on developing executive function in children. SLEEP 2013;36(7):1077-1084.

Molecular diversity of methanogens in fecal samples from Bactrian camels (Camelus bactrianus) at two zoos.

Animals are dependent on mutualistic microbial communities that reside in their gastrointestinal track for essential physiological functions such as nutrition and pathogen resistance. The composition of microbial communities in an animal is influenced by various factors, including species, diet and geographical location. In this preliminary study, the population structure of fecal methanogens in Bactrian camels (Camelus bactrianus) from two zoos was studied using separate 16S rRNA gene libraries for each zoo. While methanogen sequences belonging to the genus Methanobrevibacter were dominant in both libraries, they showed significant differences in diversity (p=0.05) and structure (p<0.0001). Population structure analysis revealed that only two operational taxonomic units (OTUs) were shared between libraries, while two OTUs were unique to the Southwick Zoo library and seven OTUs were unique to the Potter Park Zoo library. These preliminary results highlight how methanogen population structures can vary greatly between animals of the same species maintained in captivity at different locations.