Architecture and activation mechanism of the bacterial PARIS defence system.

Bacteria and their viruses (bacteriophages or phages) are engaged in an intense evolutionary arms race1-5. While the mechanisms of many bacterial antiphage defence systems are known1, how these systems avoid toxicity outside infection yet activate quickly after infection is less well understood. Here we show that the bacterial phage anti-restriction-induced system (PARIS) operates as a toxin-antitoxin system, in which the antitoxin AriA sequesters and inactivates the toxin AriB until triggered by the T7 phage counterdefence protein Ocr. Using cryo-electron microscopy, we show that AriA is related to SMC-family ATPases but assembles into a distinctive homohexameric complex through two oligomerization interfaces. In uninfected cells, the AriA hexamer binds to up to three monomers of AriB, maintaining them in an inactive state. After Ocr binding, the AriA hexamer undergoes a structural rearrangement, releasing AriB and allowing it to dimerize and activate. AriB is a toprim/OLD-family nuclease, the activation of which arrests cell growth and inhibits phage propagation by globally inhibiting protein translation through specific cleavage of a lysine tRNA. Collectively, our findings reveal the intricate molecular mechanisms of a bacterial defence system triggered by a phage counterdefence protein, and highlight how an SMC-family ATPase has been adapted as a bacterial infection sensor.

High-Resolution Bacterial Cytological Profiling Reveals Intrapopulation Morphological Variations upon Antibiotic Exposure.

Phenotypic heterogeneity is crucial to bacterial survival and could provide insights into the mechanism of action (MOA) of antibiotics, especially those with polypharmacological actions. Although phenotypic changes among individual cells could be detected by existing profiling methods, due to the data complexity, only population average data were commonly used, thereby overlooking the heterogeneity. In this study, we developed a high-resolution bacterial cytological profiling method that can capture morphological variations of bacteria upon antibiotic treatment. With an unprecedented single-cell resolution, this method classifies morphological changes of individual cells into known MOAs with an overall accuracy above 90%. We next showed that combinations of two antibiotics induce altered cell morphologies that are either unique or similar to that of an antibiotic in the combinations. With these combinatorial profiles, this method successfully revealed multiple cytological changes caused by a natural product-derived compound that, by itself, is inactive against Acinetobacter baumannii but synergistically exerts its multiple antibacterial activities in the presence of colistin. The findings have paved the way for future single-cell profiling in bacteria and have highlighted previously underappreciated intrapopulation variations caused by antibiotic perturbation.

Machine learning from Pseudomonas aeruginosa transcriptomes identifies independently modulated sets of genes associated with known transcriptional regulators.

The transcriptional regulatory network (TRN) of Pseudomonas aeruginosa coordinates cellular processes in response to stimuli. We used 364 transcriptomes (281 publicly available + 83 in-house generated) to reconstruct the TRN of P. aeruginosa using independent component analysis. We identified 104 independently modulated sets of genes (iModulons) among which 81 reflect the effects of known transcriptional regulators. We identified iModulons that (i) play an important role in defining the genomic boundaries of biosynthetic gene clusters (BGCs), (ii) show increased expression of the BGCs and associated secretion systems in nutrient conditions that are important in cystic fibrosis, (iii) show the presence of a novel ribosomally synthesized and post-translationally modified peptide (RiPP) BGC which might have a role in P. aeruginosa virulence, (iv) exhibit interplay of amino acid metabolism regulation and central metabolism across different carbon sources and (v) clustered according to their activity changes to define iron and sulfur stimulons. Finally, we compared the identified iModulons of P. aeruginosa with those previously described in Escherichia coli to observe conserved regulons across two Gram-negative species. This comprehensive TRN framework encompasses the majority of the transcriptional regulatory machinery in P. aeruginosa, and thus should prove foundational for future research into its physiological functions.

HORMA Domain Proteins and a Trip13-like ATPase Regulate Bacterial cGAS-like Enzymes to Mediate Bacteriophage Immunity.

Bacteria are continually challenged by foreign invaders, including bacteriophages, and have evolved a variety of defenses against these invaders. Here, we describe the structural and biochemical mechanisms of a bacteriophage immunity pathway found in a broad array of bacteria, including E. coli and Pseudomonas aeruginosa. This pathway uses eukaryotic-like HORMA domain proteins that recognize specific peptides, then bind and activate a cGAS/DncV-like nucleotidyltransferase (CD-NTase) to generate a cyclic triadenylate (cAAA) second messenger; cAAA in turn activates an endonuclease effector, NucC. Signaling is attenuated by a homolog of the AAA+ ATPase Pch2/TRIP13, which binds and disassembles the active HORMA-CD-NTase complex. When expressed in non-pathogenic E. coli, this pathway confers immunity against bacteriophage λ through an abortive infection mechanism. Our findings reveal the molecular mechanisms of a bacterial defense pathway integrating a cGAS-like nucleotidyltransferase with HORMA domain proteins for threat sensing through protein detection and negative regulation by a Trip13 ATPase.

Arylthiazole antibiotics targeting intracellular methicillin-resistant Staphylococcus aureus (MRSA) that interfere with bacterial cell wall synthesis.

The promising antibacterial potency of arylthiazole antibiotics is offset by their limited activity against intracellular bacteria (namely methicillin-resistant Staphylococcus aureus (MRSA)), similar to many clinically-approved antibiotics. The failure to target these hidden pathogens is due to the compounds' lack of proper characteristics to accumulate intracellularly. Fine tuning of the size and polar-surface-area of the linking heteroaromatic ring provided a new series of 5-thiazolylarylthiazoles with balanced properties that allow them to sufficiently cross and accumulate inside macrophages infected with MRSA. The most promising compound 4i exhibited rapid bactericidal activity, good metabolic stability and produced over 80% reduction of intracellular MRSA in infected macrophages.

Imaging mass spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of Bacillus subtilis.

During bacterial cannibalism, a differentiated subpopulation harvests nutrients from their genetically identical siblings to allow continued growth in nutrient-limited conditions. Hypothesis-driven imaging mass spectrometry (IMS) was used to identify metabolites active in a Bacillus subtilis cannibalism system in which sporulating cells lyse nonsporulating siblings. Two candidate molecules with sequences matching the products of skfA and sdpC, genes for the proposed cannibalistic factors sporulation killing factor (SKF) and sporulation delaying protein (SDP), respectively, were identified and the structures of the final products elucidated. SKF is a cyclic 26-amino acid (aa) peptide that is posttranslationally modified with one disulfide and one cysteine thioether bridged to the α-position of a methionine, a posttranslational modification not previously described in biology. SDP is a 42-residue peptide with one disulfide bridge. In spot test assays on solid medium, overproduced SKF and SDP enact a cannibalistic killing effect with SDP having higher potency. However, only purified SDP affected B. subtilis cells in liquid media in fluorescence microscopy and growth assays. Specifically, SDP treatment delayed growth in a concentration-dependent manner, caused increases in cell permeability, and ultimately caused cell lysis accompanied by the production of membrane tubules and spheres. Similarly, SDP but not SKF was able to inhibit the growth of the pathogens Staphylococcus aureus and Staphylococcus epidermidis with comparable IC(50) to vancomycin. This investigation, with the identification of SKF and SDP structures, highlights the strength of IMS in investigations of metabolic exchange of microbial colonies and also demonstrates IMS as a promising approach to discover novel biologically active molecules.

ParE toxin encoded by the broad-host-range plasmid RK2 is an inhibitor of Escherichia coli gyrase.

Broad-host-range plasmid RK2 encodes a post-segregational killing system, parDE, which contributes to the stable maintenance of this plasmid in Escherichia coli and many distantly related bacteria. The ParE protein is a toxin that inhibits cell growth, causes cell filamentation and eventually cell death. The ParD protein is a specific ParE antitoxin. In this work, the in vitro activities of these two proteins were examined. The ParE protein was found to inhibit DNA synthesis using an E. coli oriC supercoiled template and a replication-proficient E. coli extract. Moreover, ParE inhibited the early stages of both chromosomal and plasmid DNA replication, as measured by the DnaB helicase- and gyrase-dependent formation of FI*, a highly unwound form of supercoiled DNA. The presence of ParD prevented these inhibitory activities of ParE. We also observed that the addition of ParE to supercoiled DNA plus gyrase alone resulted in the formation of a cleavable gyrase-DNA complex that was converted to a linear DNA form upon addition of sodium dodecyl sulphate (SDS). Adding ParD before or after the addition of ParE prevented the formation of this cleavable complex. These results demonstrate that the target of ParE toxin activity in vitro is E. coli gyrase.