Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen .

The human pathogen causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including . OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance.

Maintenance of the Shigella sonnei Virulence Plasmid Is Dependent on Its Repertoire and Amino Acid Sequence of Toxin-Antitoxin Systems.

Shigella sonnei is a major cause of bacillary dysentery and an increasing concern due to the spread of multidrug resistance. S. sonnei harbors pINV, an ∼210 kb plasmid that encodes a type III secretion system (T3SS), which is essential for virulence.

Proteome Dynamics during Antibiotic Persistence and Resuscitation.

During antibiotic persistence, bacterial cells become transiently tolerant to antibiotics by restraining their growth and metabolic activity. Detailed molecular characterization of antibiotic persistence is hindered by low count of persisting cells and the need for their isolation. Here, we used sustained addition of stable isotope-labeled lysine to selectively label the proteome during -induced persistence and -induced resuscitation of Escherichia coli cells in minimal medium after antibiotic treatment.

PasT of Escherichia coli sustains antibiotic tolerance and aerobic respiration as a bacterial homolog of mitochondrial Coq10.

Antibiotic-tolerant persisters are often implicated in treatment failure of chronic and relapsing bacterial infections, but the underlying molecular mechanisms have remained elusive. Controversies revolve around the relative contribution of specific genetic switches called toxin-antitoxin (TA) modules and global modulation of cellular core functions such as slow growth. Previous studies on uropathogenic Escherichia coli observed impaired persister formation for mutants lacking the pasTI locus that had been proposed to encode a TA module.

Inhibition of Type III CRISPR-Cas Immunity by an Archaeal Virus-Encoded Anti-CRISPR Protein.

Bacteria and archaea possess a striking diversity of CRISPR-Cas systems divided into six types, posing a significant barrier to viral infection. As part of the virus-host arms race, viruses encode protein inhibitors of type I, II, and V CRISPR-Cas systems, but whether there are natural inhibitors of the other, mechanistically distinct CRISPR-Cas types is unknown. Here, we present the discovery of a type III CRISPR-Cas inhibitor, AcrIIIB1, encoded by the Sulfolobus virus SIRV2.

CRP Interacts Specifically With Sxy to Activate Transcription in .

Horizontal gene transfer through natural competence is an important driving force of bacterial evolution and antibiotic resistance development. In several Gram-negative pathogens natural competence is regulated by the concerted action of cAMP receptor protein (CRP) and the transcriptional co-regulator Sxy through a subset of CRP-binding sites (CRP-S sites) at genes encoding competence factors. Despite the wealth of knowledge on CRP's structure and function it is not known how CRP and Sxy act together to activate transcription.

Intramolecular Interactions Dominate the Autoregulation of Stringent Factor RelA.

Amino acid starvation in 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 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.

SosA inhibits cell division in Staphylococcus aureus in response to DNA damage.

Inhibition of cell division is critical for viability under DNA-damaging conditions. DNA damage induces the SOS response that in bacteria inhibits cell division while repairs are being made. In coccoids, such as the human pathogen, Staphylococcus aureus, this process remains poorly studied.

Serine-Threonine Kinases Encoded by Split 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. of strain K-12 codes for HipA, a serine-threonine kinase that phosphorylates and inhibits glutamyl-tRNA synthetase. Induction of inhibits charging of glutamyl-tRNA that, in turn, inhibits translation and induces RelA-dependent (p)ppGpp synthesis and multidrug tolerance.

Birth and Resuscitation of (p)ppGpp Induced Antibiotic Tolerant Persister Cells.

Transient antibiotic treatment typically eradicates most sensitive bacteria except a few survivors called persisters. The second messenger (p)ppGpp plays a key role in persister formation in Escherichia coli populations but the underlying mechanisms have remained elusive. In this study we induced (p)ppGpp synthesis by modulating tRNA charging and then directly observed the stochastic appearance, antibiotic tolerance, and resuscitation of persister cells using live microscopy.

(p)ppGpp-mediated stress response induced by defects in outer membrane biogenesis and ATP production promotes survival in Escherichia coli.

Cellular growth requires a high level of coordination to ensure that all processes run in concert. The role of the nucleotide alarmone (p)ppGpp has been extensively studied in response to external stresses, such as amino acid starvation, in Escherichia coli, but much less is known about the involvement of (p)ppGpp in response to perturbations in intracellular processes. We therefore employed CRISPRi to transcriptionally repress essential genes involved in 14 vital processes and investigated whether a (p)ppGpp-mediated response would be induced.

Transcriptome-Wide Analysis of Protein-RNA and RNA-RNA Interactions in Pathogenic Bacteria.

Regulatory RNAs and RNA-binding proteins (RBPs) play critical roles in virulence gene expression in pathogenic bacteria. A wealth of regulatory RNAs have been identified in bacterial pathogens using RNA-seq and recent technical advances are uncovering their mRNA targets. UV-crosslinking is a powerful tool for identifying protein binding sites throughout the transcriptome providing base-pair resolution of sites in vivo.

The Interplay Between Immune Response and Bacterial Infection in COPD: Focus Upon Non-typeable .

Chronic obstructive pulmonary disease (COPD) is a debilitating respiratory disease and one of the leading causes of morbidity and mortality worldwide. It is characterized by persistent respiratory symptoms and airflow limitation due to abnormalities in the lower airway following consistent exposure to noxious particles or gases. Acute exacerbations of COPD (AECOPD) are characterized by increased cough, purulent sputum production, and dyspnea.

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.

The kinases HipA and HipA7 phosphorylate different substrate pools in to promote multidrug tolerance.

The bacterial serine-threonine protein kinase HipA promotes multidrug tolerance by phosphorylating the glutamate-tRNA ligase (GltX), leading to a halt in translation, inhibition of growth, and induction of a physiologically dormant state (persistence). The HipA variant HipA7 substantially increases persistence despite being less efficient at inhibiting cell growth. We postulated that this phenotypic difference was caused by differences in the substrates targeted by both kinases.

Publisher Correction: Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity.

In the original version of this Article, molecular weight markers in Figs 1c, 2c,d and 4d were displaced during the production process, so that they were not correctly aligned with the corresponding bands. In addition, in Fig. 4c, molecular masses given for three different elution volumes were displaced so that they appeared to the left of the correct positions.

Update on non-typeable Haemophilus influenzae-mediated disease and vaccine development.

Introduction: Non-typeable Haemophilus influenzae (NTHi) has attracted more interest in recent years due to an increased prevalence of infections caused by the pathogen. This upsurge is at least partly ascribed to the introduction of the pneumococcal conjugated vaccines that has resulted in an aetiological shift in NTHi's favor with respect to upper respiratory tract infections. Moreover, an increased antimicrobial resistance has been associated with the pathogen, a fact that further strengthens the case for novel vaccine development.

Activation of the Stringent Response by Loading of RelA-tRNA Complexes at the Ribosomal A-Site.

RelA/SpoT homologs (RSHs) are ubiquitous bacterial enzymes that synthesize and hydrolyze (p)ppGpp in response to environmental challenges. Bacteria cannot survive in hosts and produce infection without activating the (p)ppGpp-mediated stringent response, but it is not yet understood how the enzymatic activities of RSHs are controlled. Using UV crosslinking and deep sequencing, we show that Escherichia coli RelA ((p)ppGpp synthetase I) interacts with uncharged tRNA without being activated.

Quorum Sensing-Regulated Phenol-Soluble Modulins Limit Persister Cell Populations in .

Incomplete killing of bacterial pathogens by antibiotics is an underlying cause of treatment failure and accompanying complications. Among those avoiding chemotherapy are persisters being individual cells in a population that for extended periods of time survive high antibiotic concentrations proposedly by being in a quiescent state refractory to antibiotic killing. While investigating the human pathogen and the influence of growth phase on persister formation, we noted that spent supernatants of stationary phase cultures of or , but not of distantly related bacteria, significantly reduced the persister cell frequency upon ciprofloxacin challenge when added to exponentially growing and stationary phase cells.

Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity.

Viruses employ a range of strategies to counteract the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), including mutational escape and physical blocking of enzymatic function using anti-CRISPR proteins (Acrs). Acrs have been found in many bacteriophages but so far not in archaeal viruses, despite the near ubiquity of CRISPR-Cas systems in archaea. Here, we report the functional and structural characterization of two archaeal Acrs from the lytic rudiviruses, SIRV2 and SIRV3.

Toxins, Targets, and Triggers: An Overview of Toxin-Antitoxin Biology.

Bacterial toxin-antitoxin (TA) modules are abundant genetic elements that encode a toxin protein capable of inhibiting cell growth and an antitoxin that counteracts the toxin. The majority of toxins are enzymes that interfere with translation or DNA replication, but a wide variety of molecular activities and cellular targets have been described. Antitoxins are proteins or RNAs that often control their cognate toxins through direct interactions and, in conjunction with other signaling elements, through transcriptional and translational regulation of TA module expression.

Structural basis for (p)ppGpp synthesis by the small alarmone synthetase RelP.

The stringent response is a global reprogramming of bacterial physiology that renders cells more tolerant to antibiotics and induces virulence gene expression in pathogens in response to stress. This process is driven by accumulation of the intracellular alarmone guanosine-5'-di(tri)phosphate-3'-diphosphate ((p)ppGpp), which is produced by enzymes of the RelA SpoT homologue (RSH) family. The Gram-positive Firmicute pathogen, , encodes three RSH enzymes: a multidomain RSH (Rel) that senses amino acid starvation on the ribosome and two small alarmone synthetase (SAS) enzymes, RelQ (SAS1) and RelP (SAS2).

Structural conservation of the PIN domain active site across all domains of life.

The PIN (PilT N-terminus) domain is a compact RNA-binding protein domain present in all domains of life. This 120-residue domain consists of a central and parallel β sheet surrounded by α helices, which together organize 4-5 acidic residues in an active site that binds one or more divalent metal ions and in many cases has endoribonuclease activity. In bacteria and archaea, the PIN domain is primarily associated with toxin-antitoxin loci, consisting of a toxin (the PIN domain nuclease) and an antitoxin that inhibits the function of the toxin under normal growth conditions.

Higher-Order Structure in Bacterial VapBC Toxin-Antitoxin Complexes.

Toxin-antitoxin systems are widespread in the bacterial kingdom, including in pathogenic species, where they allow rapid adaptation to changing environmental conditions through selective inhibition of key cellular processes, such as DNA replication or protein translation. Under normal growth conditions, type II toxins are inhibited through tight protein-protein interaction with a cognate antitoxin protein. This toxin-antitoxin complex associates into a higher-order macromolecular structure, typically heterotetrameric or heterooctameric, exposing two DNA binding domains on the antitoxin that allow auto-regulation of transcription by direct binding to promoter DNA.