Type IV secretion systems (T4SS) are versatile machines with variable functions including DNA uptake and release, protein translocation, and DNA conjugation. However, the diversity, distribution, and functional roles of the T4SS in the Ralstonia genus remain poorly understood. The Ralstonia solanacearum species complex (RSSC) comprises three species of plant-pathogenic bacteria that cause bacterial wilt disease. The Ralstonia genus also includes non-RSSC species that are primarily environmental bacteria and rare opportunistic human pathogens. This study compared the diversity and phylogenetic distribution of T4SSs in the RSSC phytopathogens vs. non-RSSC environmentals. Phylogenetic analysis of VirB4 sequences and synteny analysis revealed 16 distinct T4SS clusters in Ralstonia, with 10 clusters found in RSSC phytopathogen genomes, 12 in non-RSSC environmental genomes, and 6 clusters in both groups. Collectively, these gene clusters were more prevalent in non-RSSC environmental genomes. The presence of type IV coupling protein and relaxase genes suggests that at least 14 of these T4SS gene clusters are putative DNA-conjugation systems. The clusters were encoded on accessory plasmids of various sizes or as integrative and conjugative elements on the chromosome or megaplasmid. The putative regions of transfer for T4SS gene clusters in the RSSC phytopathogen genomes often contained type III effectors, type VI secretion toxin/antitoxin clusters, and haemagglutinin gene clusters. In contrast, the non-RSSC environmentals were enriched in heavy metal metabolism and resistance genes. One of the 16 T4SS clusters, cluster i, exhibited evidence of specialization for the RSSC phytopathogens. These findings shed light on the eco-evolutionary differences within the genus Ralstonia.

Unspecific Peroxygenases (UPOs) are enzymes with significant potential as catalysts in synthetic chemistry as they catalyse selective oxygenation reactions on organic substrates at the expense of only hydrogen peroxide as the external oxidant. The demand for UPOs has stimulated considerable research into efficient heterologous expression systems for their production, which have included optimisation of the signal peptide (SP) included upstream of the UPO gene. In this study we report a comparison of the production of the prototypical and widely-used UPO variant from the fungus Agrocybe aegerita (rAaeUPO-PaDa-I-H) using both its native SP and variant SPs evolved for improved heterologous expression in yeast hosts. The results show that improvements in protein production identified using variant SPs in S. cerevisiae are not necessarily extended to the yeast Komagataella phaffii. Indeed in K. phaffii we observed a five to sevenfold improvement in the activity of crude secretates produced using the native fungal SP of AaeUPO, compared with SPs that were evolved for improved expression and screened in S. cerevisiae. These findings suggest that superior yields of this widely-used UPO can be obtained from scaled production in K. phaffii by using the native SP from the source fungus.

The escalating volume of plastic debris in the aquatic environment has created a novel ecological niche for microorganisms. This habitat, known as the plastisphere, hosts ecologically diverse microbial communities, yet its degree of divergence in composition and function from natural substrates remains a key topic of debate. In this Review, we provide a comprehensive overview of the aquatic plastisphere, highlighting its community composition, assembly dynamics and key functional traits, such as degradation pathways and nutrient cycling. We explore how this novel environment acts as a critical hub for the persistence and dissemination of human pathogens and antimicrobial resistance. We discuss how dynamic environmental factors, together with complex inter-kingdom metabolic interactions, can reshape community composition, accelerate the evolution of resistant pathotypes and influence pathogen virulence. We conclude by emphasizing that climate-induced changes in temperature, ultraviolet light radiation, salinity and hydrodynamic patterns are likely to further influence the microbial composition and phenotypic expression of pathogens within the aquatic plastisphere. Finally, we underscore the need for a One Health approach to study the plastisphere and advocate moving the field from descriptive ecology towards a mechanistic, policy-relevant framework to address the growing public health threat amid accelerating plastic pollution and climate-driven shifts. In this Review, Ormsby and Quilliam discuss how plastic pollutants create a critical, novel habitat for complex microbial communities, highlighting the community composition, assembly dynamics and key functional traits of the plastisphere. They explore the dual public health risks of pathogen dissemination and enrichment of antimicrobial-resistance determinants.

CRISPR RNAs (crRNAs) guide recognition and targeting of intracellular invaders as part of adaptive immunity by CRISPR-Cas systems. crRNAs are transcribed from CRISPR arrays of conserved repeats interlaced with invader-derived spacers. While crRNA production is essential for immunity, its optimization for defense remains poorly understood. Here, we show that, in diverse RNA-targeting type VI CRISPR-Cas systems, the leader RNA encoded upstream of the CRISPR array prevents formation of an invader-independent extraneous crRNA (ecrRNA) by blocking processing of the first repeat. Using the VI-B2 system from Porphyromonas gingivalis as a model, we demonstrate that the leader RNA and first repeat form a conserved inhibitory hairpin that precludes binding and processing by the system’s Cas13b nuclease. Disrupting this hairpin enables ecrRNA production, which in turn can deplete invader-derived crRNAs and reduce Cas13b-mediated phage defense. Structure prediction indicates that these leader-repeat hairpins are widespread across diverse type VI subtypes, highlighting a conserved regulatory mechanism. Our findings reveal how a prevalent branch of CRISPR-Cas systems suppresses ecrRNA formation to promote RNA-guided immunity.

Chemotaxis receptor complexes sense chemical gradient in the cellular environment to direct swimming towards favorable environments. The core signaling units of these complexes are made up of two trimers-of-dimers of chemoreceptors, two CheW and a CheA dimer, which further assemble into large hexagonal signaling arrays. Structural and biochemical studies have provided important information on the architecture and interfaces of these complexes. However, the signaling pathway of these complexes that controls the kinase is not fully understood. In this review we highlight the highest resolution models of this system and examine the current consensus on the protein-protein interfaces based on models and interface experiments. We also highlight differences observed between signaling states for the individual proteins and the protein interfaces that are proposed to be part of the signaling mechanism. Overall, we conclude that there is strong structural consensus for the protein interfaces but, despite some intriguing results, more information is needed to understand how the interfaces change between signaling states and the role they play in signaling. An animated Interactive 3D Complement (I3DC) is available in Proteopedia https://proteopedia.org/w/Journal:FEMS_Microbiology_Reviews:1.

APOBEC3A catalyzes cytosine-to-uracil deamination in single-stranded DNA and RNA. Physiologically, APOBEC3A functions in innate immunity and aberrant deamination is associated with cytosine mutations in enzymatically preferred YTCW substrate motifs in multiple cancers. Much less is known about the potential contribution of APOBEC3A-catalyzed RNA editing to virus and cancer evolution. Here, we present HAMMER (hairpin-based APOBEC3A-mediated messenger RNA editing reporter), a rapid luminescence-based cellular assay for measuring RNA editing by APOBEC3A. HAMMER reports APOBEC3A activity as a reduction in the ratio of firefly to renilla luciferase activity. Briefly, tandem renilla and firefly luciferase open reading frames are separated by an optimal APOBEC3A hairpin substrate, in which C-to-U editing of a CGA motif yields a UGA stop codon thus preventing translation of the downstream firefly luciferase reporter, without impacting the upstream renilla reporter. HAMMER activation is dose-responsive, catalytic activity-dependent, and specific to human APOBEC3A. A panel of herpesviral ribonucleotide reductase constructs was used to show that direct inhibition of APOBEC3A results in a dose-responsive recovery of firefly luciferase expression. HAMMER is therefore a scalable and easy-to-use method for quantifying cellular APOBEC3A RNA editing activity and characterizing inhibitors.