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Phage therapy offers a promising strategy against bacterial pathogens in medicine and agriculture, but the rise of phage-resistant bacteria presents a significant challenge to its sustainability. Here, we used an environmental model bacterium, Pseudomonas protegens CHA0, to investigate phage resistance mechanisms in laboratory conditions through genomic analysis of four phage-resistant variants (C2, C4, C17, C18). Whole-genome sequencing revealed frequent deletions, insertions, and single nucleotide substitutions, particularly in genes encoding enzymes involved in cell surface modifications. The T428P mutation in AlgC, a phosphoglucomutase, and the P229T substitution in YkcC, a glycosyltransferase, each conferred resistance by altering phage receptor accessibility while preserving bacterial fitness. These findings emphasize that subtle mutations in surface-modifying enzymes enable P. protegens to evolve resistance to bacteriophages without compromising their ecological performance.

The Helicobacter pylori cag pathogenicity island (cagPAI) encodes a complex virulence-associated type IV secretion system (CagT4SS). Recently, structural detail on the CagT4SS has been substantially improved by Cryo-EM. However, important structural and functional information is still missing. In the present study, we followed the hypothesis that H. pylori T4SS external proteins may form a surface-exposed assembly, together with non-CagT4SS proteins, which may be essential for T4SS function. Using interaction screens followed by biochemical and functional characterization, we have enhanced the knowledge about functional protein-protein interactions of the CagT4SS extracellular proteins. This comprises newly identified interactions of CagT4SS surface proteins, including the VirB2 homolog CagC, the VirB5 homolog CagL and CagN, with outer membrane proteins HopQ and HopZ. We have further quantitated direct, pH dependent, interactions of T4SS surface proteins with HopZ and HopQ, with host cell factors CEACAM and integrin, and self-interactions of both HopZ and HopQ. Utilizing chromosomal tag insertions in H. pylori, we detected surface-exposed colocalization of HopQ with T4SS components in the absence or, for HopQ, also in the presence of human gastric epithelial cells. Functionally antagonistic roles of HopQ and HopZ were uncovered in early proinflammatory human epithelial cell activation by the T4SS. In summary, we identified a network of interactions between H. pylori outer membrane proteins and CagT4SS surface proteins that are functionally relevant for T4SS-dependent transport processes. This study provides a valuable resource guiding future studies to refine structure and mechanistic roles of the surface-exposed portions of the CagT4SS.

Solo-Cas4 homologs are Cas4-family proteins found outside of canonical CRISPR-Cas operons. Here, we present the biochemical characterization of Solo-Cas4 from Methanosarcina mazei Gö1. We found significantly upregulated solo-cas4 transcript levels during stationary phase, while remaining constant under oxygen exposure, temperature shifts, high salt conditions or virus challenge. Heterologously expressed as a SUMO-fusion, the purified tag-free protein displays an absorption peak at 420 nm, indicative of a [4Fe-4S]-cluster​. Size-exclusion-chromatography revealed that Solo-Cas4 forms a higher oligomeric complex, with an apparent molecular mass of 318 kDa. In vitro nuclease activity assays demonstrated that Solo-Cas4 cleaves metal-dependent linear dsDNA, with highest cleavage activity in the presence of Mn²⁺, followed by Mg²⁺, while Ca²⁺ and Cu²⁺ result in negligible cleavage. Isoleucine169 was identified to be crucial for catalysis, mutating it to alanine completely abolished nuclease activity​. Mutating any of the four conserved cysteines-proposed to coordinate the [4Fe-4S]-cluster did not affect nuclease activity; however, it abolishes metal cluster binding. Supercoiled circular dsDNA was preferentially nicked by Solo-Cas4 in the presence of Mg²⁺, whereas Mn²⁺ also led to linearization followed by complete degradation. Besides, ssDNA was cleaved by Solo-Cas4 but with lower activity. In agreement, Microscale thermophoresis analysis revealed strong dsDNA binding with highest affinity to supercoiled circular DNA, and weak ssDNA binding. Overall, these findings indicate that M. mazei Solo-Cas4 is a high oligomeric Cas4-family nuclease that preferentially targets supercoiled dsDNA and is upregulated during stationary growth.

Filamentous hyphae are the main invasive morphotype of the opportunistic fungal pathogen Candida albicans. However, yeast cells seem better suited for dissemination through the bloodstream during the progression of life-threatening systemic infections. While yeast cells are present together with hyphae in the intestine during commensal colonization, how yeast cells ultimately reach the blood following translocation of invasive hyphae is unknown. In this study we investigated potential mechanisms proposed for how yeast cells may enter the blood using an in vitro model of translocation based on intestinal epithelial cells (IECs). Our data show that yeast cells can passively translocate with invasive hyphae, though this requires host-cell damage facilitated by the peptide toxin candidalysin, encoded by ECE1. Independent of fungal-mediated damage, chemical disruption of the IEC layer by the mycotoxin patulin was sufficient to foster efficient translocation of C. albicans yeast cells alone. This was dependent on a significant loss of barrier integrity rather than host-cell damage itself. The same phenomenon was observed for oral clinical isolates, which more readily grow as yeast and pseudohyphal cells as compared to the standard SC5314 strain. The transition of hypha-to-yeast growth was also associated with translocation across IECs by increased expression of the yeast-essential gene PES1. This is the first study to directly investigate the mechanisms by which C. albicans yeast cells can translocate across IECs and to describe the fungal factors that contribute to this process.

Bacteria define their heritable cell shape using membrane integral glycosyltransferases (GTases) of the shape, elongation, division, and sporulation protein family and monofunctional D, D-transpeptidases of the class B penicillin-binding protein family (bPBP). Current models support bPBPs pairing with cognate GTases to drive cell elongation, cell division, or spore formation. Recent studies in Salmonella enterica and Clostridioides difficile however support different models with more than one bPBP interacting with a particular GTase. Here, we mined databases to assess how this plasticity in interacting proteins is represented across the domain Bacteria. Like Salmonella, many bacteria of Enterobacterales encode alternative bPBPs while having a single set of morphogenetic GTases. When extended to the domain Bacteria, the analysis uncovered bPBPs lacking the pedestal domain required to interact with the GTase and GTases with β-sheet-rich regions facing outward from the membrane. We also identified large size chimeric bPBPs fused to a GTase (FtsW/RodA/SpoVE) domain as putative 'bifunctional' class B peptidoglycan synthases. Alteration of the bPBP:GTase 1:1 ratio appears as common feature, in some cases with unbalanced proliferation of both partners or with absence of one canonical bPBP (MrdA or FtsI). Bacteria were also found with some morphogenetic functions counter-selected involving pseudogenization in highly conserved loci like ftsI, mrdA, mreC, or spoVE. Most of these bacteria encode non-canonical bPBPs bearing a PBP-A dimerisation domain instead of the canonical pedestal domain. Altogether, our findings challenge classical morphogenetic models and predict in many bacteria significant flexibility in how bPBPs and GTases combine to define cell shape.

The domain of unknown function 1127 (DUF1127) is widely distributed among bacteria, often in proteins shorter than 50 amino acids. In the plant pathogen Agrobacterium tumefaciens, the absence of three small DUF1127 proteins leads to a range of phenotypic changes. In this study, we investigated the role of these small DUFs in phosphate acquisition. Upregulation of phosphate transport systems in the triple mutant resulted in increased phosphate uptake, polyphosphate accumulation, and growth defects. Using Far-Western dot blots, pulldown experiments, and the bacterial two-hybrid system, we identified a direct interaction between the small DUFs and the sensor kinase PhoR, which regulates phosphate metabolism together with the response regulator PhoB. Complementation studies revealed that DUF1127 proteins from Sinorhizobium meliloti, Rhodobacter sphaeroides, and Escherichia coli could restore the phenotypes in the A. tumefaciens triple mutant. Notably, an E. coli mutant lacking YjiS, the sole DUF1127 protein in this species, showed upregulated expression of phosphate uptake genes and accelerated phosphate uptake. Furthermore, we provide evidence for an interaction between YjiS and E. coli PhoR, suggesting that DUF1127-containing proteins may share a conserved regulatory function across different bacterial species. These findings provide new insights into the function of small DUF1127 proteins, demonstrating that they can act through protein-protein interactions.

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.