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Pseudomonas species are ubiquitous in the environment and serve as valuable source of enzymes and secondary metabolites for industrial applications. Pseudomonas aeruginosa secretes metalloproteases, such as elastase LasB and produces bioactive small molecules, including pyocyanin, rhamnolipids, and pyoverdine, with potential biotechnological applications. However, the interest in P. aeruginosa for industrial use has been limited due to the virulence-associated Type III Secretion System (T3SS), a key factor in host-pathogen interactions. In this study, we genotypically and phenotypically characterized a collection of P. aeruginosa strains naturally lacking T3SS-encoding genes. Phylogenetic analysis revealed that these strains belong to two distinct clades. Several strains exhibited low or no cytotoxicity on epithelial cell lines and were avirulent in the Galleria infection model. The level of LasB and the three metabolites-pyocyanin, rhamnolipids, and pyoverdine-varied independently of virulence profiles. Notably, we identified avirulent strains capable of producing at least two secondary metabolites, including mono-rhamnolipids, highlighting their potential for biotechnological applications.

Bacteriophages are a promising tool for treating bacterial infections, given the rise and spread of antibiotic resistances. However, phage-resistant bacteria can emerge during treatment, jeopardizing the success of therapy. In vitro studies with model organisms have provided valuable insights into the mechanisms by which phage resistance can evolve. However, the relevance of these findings often remains unclear. Here, we investigate the selection of phage-resistant variants and the cost of phage resistance in vitro and in the murine gut using a clinically relevant Escherichia coli K1 strain and a strain-specific phage cocktail. By performing experimental evolution studies in both settings, we obtained different phage-resistant E. coli mutants. Genome resequencing identified lipopolysaccharide (LPS) and the K1 capsule as bacterial surface structures altered in phage-resistant mutants. Targeted deletions of waaO, encoding an ɑ-1,3 glucosyltransferase, involved in the synthesis of the R core of LPS, a gene encoding a predicted O-antigen ligase and emrR involved in capsule gene regulation were generated and confirmed their role in phage-resistance. Escherichia coli mutants deficient in LPS or capsule showed a growth advantage in vitro when exposed to phages but LPS-deficient mutants exhibited severely attenuated growth in the murine gut, even in the presence of phages. Our observations add to the evidence that bacteria in the intestinal environment face a high cost of phage resistance conferred by cell surface alteration, which is not apparent in nutrient-rich culture media. Therefore, it is crucial to carefully consider the context in which phage cocktails are tested, particularly when studying phage efficacy and evolution of phage resistance.

Clustered Regularly Interspersed Short Palindromic Repeats and CRISPR-associated genes (CRISPR-Cas) is a bacterial immune system also famous for its use in genome editing. The diversity of known systems could be significantly increased by metagenomic data. Here we present the Metagenomic CRISPR Array Analysis Tool (MCAAT), a highly sensitive algorithm for finding CRISPR arrays in unassembled metagenomic data. It takes advantage of the properties of CRISPR arrays that form multicycles in de Bruijn graphs. We show that MCAAT reliably predicts CRISPR arrays in bacterial genome sequences and that its assembly-free graph-based strategy outperforms assembly-based workflows and other assembly-free methods on synthetic and real metagenomes. Our new approach will help to increase the diversity of known CRISPR-Cas systems and enable studies of spacer evolution within metagenomic data sets.

DNA repair processes are the foundation for genome integrity and survival, especially in extreme environments where DNA damage occurs more frequently and where archaea are found. Nevertheless, first-hand experimental information on repair pathways in archaea is scarce, and assignment of repair proteins is currently largely based on homology. We showed previously that DNA lesions induced by clustered regularly interspaced short palindromic repeats Cas (CRISPR-Cas) self-targeting are repaired by microhomology-mediated end joining (MMEJ). To identify proteins involved in the archaeal MMEJ pathway, we used deletion strains devoid of proteins assigned to the key steps of MMEJ, to examine changes in the repair outcome. In addition, we used aphidicolin to inhibit the activity of the essential PolB1 protein. For the first time, we were thereby able to experimentally identify proteins involved in this repair pathway in the euryarchaeal model organism Haloferax volcanii. This study confirms that Mre11, Rad50, Fen1, PolB1, LigA, and LigN take part in MMEJ, as previously inferred. In addition, we show that Cas1 and Hel308a are also involved in the MMEJ pathway.

CRISPR-Cas9 systems are widely used for bacterial genome editing, yet their heterologous expression has been associated with cytotoxicity. The Cas9 nuclease from Streptococcus pyogenes (SpyCas9) has been one common source, with reports of cytotoxicity with the nuclease alone or in combination with a single-guide RNA observed in some bacteria. However, the potential cytotoxic effects of other components of the CRISPR-Cas9 system remain unknown. Here, we report that expression of the short isoform of the trans-activating CRISPR RNA (tracr-S) from the S. pyogenes CRISPR-Cas locus is cytotoxic in Lacticaseibacillus paracasei, even in the absence of SpyCas9. Deleting a putative transcription regulator in L. paracasei alleviates tracr-S cytotoxicity and leads to expression of the long isoform of the trans-activating CRISPR RNA (tracr-L). Furthermore, cytotoxicity was specific to the tracr-S sequence and was linked to direct interactions with host RNAs. This work thus reveals that additional CRISPR components beyond Cas9 can interfere with the use of heterologous CRISPR-Cas systems in bacteria, with potential implications for the evolution of CRISPR immunity.

This meeting report summarizes the scientific activities of the ninth annual conference of Phages.fr, organized by the French Phages network. This year, the conference took place from 12 to 14 November 2024, in Sète, in the south of France. The conference hosted 136 participants from both the public and private sectors, representing 63 French groups and 16 international ones from Austria, Belgium, Finland, Germany, Guinea, Sweden, the UK, and the USA. The meeting brought together both young and senior scientists, offering them the opportunity to share their findings and ideas across four main topics: Ecology and Evolution, Phage-Host Interaction, Structure and Assembly, and Applications in Therapy and Biotechnology. For the first time, Phages.fr also offered a special session dedicated to the social and human sciences applied to microbiology. Over the 3 days, a total of 62 presentations were given (20 oral presentations and 42 posters), and five invited speakers delivered exceptional lectures introducing each session. The ninth annual symposium concluded with a public lecture titled "Viruses of Bacteria: New Allies in Human and Agricultural Health." The lecture aimed to raise public awareness about the therapeutic potential of phages in combating harmful bacteria that affect human and plant health, as well as their role in food safety.

Actinomycetes are important producers of valuable natural products that are applied in medicine or industry. The enzymes necessary for the synthesis of those compounds are encoded in biosynthetic gene clusters (BGCs) in the genome. However, the discovery of new natural products or the improvement of production levels can be hindered by difficulties in genetic manipulation, since standard methods often do not or not efficiently work in actinomycetes. One possible explanation for this could be the presence of nucleic acid defense systems such as CRISPR-Cas. Even though there is a lot of research published about CRISPR-Cas systems in general, the knowledge about the function of CRISPR-Cas in actinomycetes is very limited. Based on sequence data it is known that CRISPR-Cas systems occur in around half of all sequenced actinobacterial genomes. Moreover, in silico analyses of those systems have led to the discovery of new subtypes. The few examples of experimental evidence of CRISPR-Cas activity in vivo or in vitro, however, point to some special features, regarding crRNA maturation or life-cycle dependent CRISPR-Cas activity. This short review draws attention to this neglected research area and highlights the available data about CRISPR-Cas in actinomycetes.

Bacteriophage endolysins targeting Gram-positive bacteria typically feature a modular architecture of one or more enzymatically active domains (EADs) and cell wall binding domains (CBDs). Several endolysins also feature internal translational start sites (iTSSs) that produce short variant (SV) isoforms alongside the full-length (FL) endolysin. While the lytic activity of endolysins and their isoforms has been extensively studied as exogenous agents, the purpose behind producing the SV isoform during the phage infection cycle remains to be explored. In this study, we used staphylococcal phage φ2638A as a model to determine the interplay between its FL endolysin, Ply2638A, and its SV isoform during phage infection. X-ray crystallography structures and AlphaFold-generated models enabled elucidation of individual functions of the M23 endopeptidase, central amidase, and SH3b domains of Ply2638A. Production of the SV isoform (amidase and SH3b) was confirmed during phage infection and shown to form a heterodimer complex with Ply2638A via interamidase domain interactions. Using genetically engineered phage variants, we show that production of both isoforms provides an advantage during phage infection as phages producing only one isoform presented delayed progeny phage release as well as impaired lytic activity, which was partly restored through complementation of the missing isoform protein. Interestingly, when applied as an antimicrobial against Staphylococcus aureus in culture, the activity of Ply2638A remained constant regardless of SV isoform complementation. We propose that the SV isoform enhances the efficiency of cell lysis and progeny release at the end of the lytic cycle, providing a functional explanation for iTSSs conservation across diverse phage genomes.

The tricarboxylic acid (TCA) cycle is well known as a crucial pathway in central metabolism in many organisms. A less known analogous pathway is the methylcitrate cycle (MCC). It is present in various fungi such as Aspergillus species and bacteria such as Escherichia coli, with some of them being pathogenic. The MCC catalyzes an alpha-oxidation of propionyl-CoA to pyruvate and is of interest in view of biotechnology and pharmacology. To elucidate the potential interaction of the MCC with other central metabolic pathways, we investigated the MCC by Elementary-flux-mode analysis. We first established a reaction network model, using information from both the KEGG database and literature. This reaction network contains enzymes of the MCC as well as of the TCA cycle, glyoxylate shunt, and carbon source-utilizing pathways, such as amino acid degradation. The network was then used to calculate the elementary flux modes (EFMs) by using the simulation software Metatool 4.3. We identified 76 EFMs, with 39 of them containing the MCC. In this way, some previously known pathways were confirmed theoretically and, additionally, some new EFMs were discovered. Among these, a different, but shorter version of the MCC was identified. The EFMs were systematically analyzed with respect to their ATP yield and the robustness of the network was computed. Predictions on the impact of enzyme deletion or inhibition on the network were made. From these analyses and based on the absence of the MCC in humans, we conclude that the methylcitrate synthase represents a promising drug target against various human pathogens.