microLife最新文献

Many bacteria and archaea use CRISPR-Cas systems, which provide RNA-based, adaptive, and inheritable immune defenses against invading viruses and other foreign genetic elements. The proper processing of CRISPR guide RNAs (crRNAs) is a crucial step in the maturation of the defense complexes and is frequently performed by specialized ribonucleases encoded by cas genes. However, some systems employ enzymes associated with degradosome or housekeeping functions, such as RNase III or the endoribonuclease RNase E. Here, the endo- and 5´-exoribonuclease RNase J was identified as an additional enzyme involved in crRNA maturation, acting jointly with RNase E in the crRNA maturation of a type III-Bv CRISPR-Cas system, and possibly together with a further RNase in the cyanobacterium Synechocystis sp. PCC 6803. Co-IP experiments revealed a small set of proteins that were co-enriched with RNase J, among them the exoribonuclease polyribonucleotide nucleotidyltransferase (PNPase). Despite a measured, strong 3' exonucleolytic activity of the recombinant enzyme, PNPase was not confirmed to contribute to crRNA maturation. However, the co-IP results indicate that PNPase in Synechocystis is an enzyme that can recruit either RNase E or RNase J, together with additional proteins.

Bdellovibrio bacteriovorus is an obligate predatory bacterium that invades the periplasm of diderm prey bacteria, where it elongates and produces multiple daughter cells through nonbinary division. Investigating the molecular determinants of this lifecycle is challenging because deleting genes required for predation also impairs survival. Furthermore, the scarcity of robust conditional gene expression systems has restricted functional studies in this bacterium. Here, we address these limitations by expanding the genetic toolbox for B. bacteriovorus. First, we analysed the relative strength of a series of promoters, providing new resources to fine-tune gene expression. We then established an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible expression system that can be activated during both the attack and growth phases of the predator. Finally, we designed a CRISPR interference (CRISPRi) module for IPTG-inducible gene knockdown, enabling rapid and targeted depletion. As a proof of principle, CRISPRi-mediated silencing of the cell curvature gene bd1075 reproduced the straight phenotype of the deletion mutant. Likewise, depletion of the tubulin homologue FtsZ-which we showed is essential for B. bacteriovorus survival-blocked cell division within the first replicative cycle, yielding filamentous progeny still able of exiting the prey cell. This highlights the intriguing potential of uncoupling key cell cycle and predatory processes. Overall, these tools significantly broaden the scope of genetic manipulation in B. bacteriovorus and open new avenues for in-depth investigation of its noncanonical biology.

Clustered regularly interspaced palindromic repeats (CRISPR)-associated transposons (CAST) consist of an integration between certain class 1 or class 2 CRISPR-Cas systems and Tn7-like transposons. Class 2 type V-K CAST systems are restricted to cyanobacteria. Here, we identified a unique subgroup of type V-K systems through phylogenetic analysis, classified as V-K_V2. Subgroup V-K_V2 CAST systems are characterized by an alternative tracrRNA, the exclusive use of Arc_2-type transcriptional regulators, and distinct differences in the length of protein domains in TnsB and TnsC. Although the occurrence of V-K_V2 CAST systems is restricted to Nostocales cyanobacteria, it shows signs of horizontal gene transfer, indicating its capability for genetic mobility. The predicted V-K_V2 tracrRNA secondary structure has been integrated into an updated version of the CRISPRtracrRNA program available on GitHub under https://github.com/BackofenLab/CRISPRtracrRNA/releases/tag/2.0.

The 2024 International Union of Microbiological Societies Congress was held in Florence, the city of Renaissance. The theme was to increase the awareness of the power of microbial life, recognizing that it can lead the transformation towards a sustainable planet. The meeting gathered over 1400 experts from more than 90 countries and focused on the transformative potential of microbiology in addressing global challenges and aligning microbial science with the Sustainable Development Goals. Six roundtable discussions explored the pivotal role of microbiology in mitigating climate change, preparing for pandemics, producing sustainable energy, promoting a One Health approach, understanding microbiome dynamics, and developing data infrastructure. The discussions revealed that microbes are still overlooked agents in sustainable solutions. Expert panellists at the roundtables discussed microbial innovations in hydrogen and biofuel production, conversion of greenhouse gases, biomanufacturing, and soil restoration, the role of microbiome in immune health, the importance of cross-kingdom interactions, and the integration of food, environmental, and microbiomes under the One Health framework. Panels stressed the need for equitable access to vaccines, diagnostics, and data sharing, especially in the face of antimicrobial resistance. The importance of global collaboration, data repositories, and regulatory alignment, was repeatedly emphasized. The congress invited calls for the formation of an international microbiology coalition, need for interdisciplinary partnerships, increased investment in microbial technologies, updating of regulatory frameworks, and integration of microbiome science into public health and environmental policy. Microorganisms are the oldest architects of nature, able to build a sustainable future for the planet.

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.