microLife最新文献

Prokaryotic microorganisms coexist with mobile genetic elements (MGEs), which can be both genetic threats and evolutionary catalysts. In Haloferax lucentense, a halophilic archaeon, we have recently identified an unusual genomic arrangement: a complete type I-B CRISPR-Cas system encoded on a megaplasmid and an incomplete type I-B system within an integrated provirus in the main chromosome. The provirus-encoded system lacks the adaptation genes (cas1, cas2, and cas4), suggesting its potential reliance on the megaplasmid-encoded CRISPR-Cas module for the acquisition of new spacers. This arrangement suggests a potential instance of "adaptive outsourcing," where a provirus might leverage a co-resident MGE for a key function. Through comparative genomics, we show that similar proviral CRISPR-Cas systems are found in distantly related haloarchaea (e.g. Natrinema and Halobacterium), indicating probable virus-mediated horizontal transfer and suggesting they may function as mobile defense modules. Phylogenetic analysis highlights distinct evolutionary origins of the two systems: the plasmid system clusters with other Haloferax CRISPR-Cas systems, while the proviral system clusters with those from other genera, consistent with horizontal acquisition. Interestingly, spacer analysis reveals that the proviral systems predominantly target viral sequences, while the plasmid system appears to target both plasmids and viral sequences, a distribution mirroring broader trends observed in other plasmid- and chromosome-encoded CRISPR systems. This observed targeting preference suggests a potential for complementarity that could support a model of cooperative immunity, where each system may protect its genetic "owner" from competition and, indirectly, the host.

[This corrects the article DOI: 10.1093/femsml/uqaf019.].

Scientific publishing faces a credibility crisis driven to a very large extent by predatory journals, paper mills, and exploitative open-access (OA) practices. Structural pressures-publish-or-perish culture, mandatory OA policies, and author publication charges-driven business models-fuel the proliferation of low-quality or fraudulent research, now exacerbated by artificial intelligence-generated content. This opinion, which aligns with a growing clamour from the research community-calls for an international journal accreditation system, guided by a transparent code of conduct and enforced by funding agencies, to restore integrity, prioritize quality over quantity for professional progression, and safeguard trust in scientific communication.

Prokaryotic organisms execute multiple stress response mechanisms in order to cope with rapidly changing environments. Some mechanisms respond to specific cues, such as the OxyR-dependent response to hydrogen peroxide or the SOS-response that is induced upon DNA-damage. These specific responses complement general mechanisms that respond to multiple and diverse stressors. One example is nucleoid condensation, which is a rapid and effective mechanism for genome protection and observed in response to various stresses, including entry into stationary phase. Recently, the upregulation of small membrane proteins (SMPs) in response to stress was observed, but details on how this emerging class of proteins modulate the stress response is largely unknown. Here, we demonstrate that the production of two SMPs, YohP and YncL, cause nucleoid condensation in Escherichia coli. Nucleoid condensation is the result of YohP-/YncL-induced sublethal membrane depolarization, which induces the phage-shock response and leads to a reduction of global protein synthesis. YohP production also prevents the oligomerization of the antimicrobial peptide magainin-2 in the E. coli membrane and reduces the metabolic activity of E. coli cells. Thus, the synthesis of YohP and likely of other SMPs potentially protects bacterial cells against some unfavorable conditions by shifting them into a metabolically silent state.

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.