Cryo-electron tomography (cryo-ET) has revolutionized visualization of bacterial nanomachines by revealing molecular structures in situ under near-native conditions. This review highlights recent advances extending cryo-ET from isolated cells to host-microbe interactions within intact tissues. We discuss technical solutions for large-volume imaging such as cryo-focused ion beam milling, correlative light and electron microscopy, and serial lift-out workflows that preserve native structural context. Computational developments in AI-driven denoising, segmentation, and sub-tomogram averaging enhance interpretability of low-dose tomograms. Using examples from predatory bacteria, intracellular pathogens, and squid symbionts, we demonstrate how cryo-ET elucidates mechanistic details of microbial interactions.

Environmental changes, whether due to climate change or human influences, compromise the resilience of plants to biotic and abiotic stresses, such as pathogens, drought and heat. Plant microbiota are known to promote plant resilience. To be able to harness the power of the plant microbiome we need to identify microbiota with health-promoting properties. Recent studies have demonstrated that the bacterium Chitinophaga pinensis enhances plant health and increases resistance to fungal infections. Here, we show that C. pinensis exhibits an unusually high morphological plasticity, switching between a filamentous and a spherical cell state, each of which is characterized by a distinct transcriptional profile. Despite these transcriptional differences, spherical cells remained metabolically active and replicating, while lacking structural characteristics typically associated with dormant states. Furthermore, the spherical cell morphology of C. pinensis facilitates hitchhiking behaviour and motility via surfactin cheating, potentially influencing its dispersal and interactions within the plant microbiome. To investigate the structural dynamics and transcriptional adaptation of this plant endophyte, we applied a combination of microscopy and culture-based techniques. Taken together, our study provides new insights into the morphological flexibility and transcriptional regulation of the plant-beneficial C. pinensis.

Bacteria produce natural products to adapt to their environments, with phage interactions as major ecological and evolutionary drivers. While some natural products protect bacteria from phages, the prevalence and diversity of chemical anti-phage defenses remain largely unexplored. Here, we identify a widespread family of lanthipeptide biosynthetic gene clusters (BGCs) in Actinobacteria, which we term lanthivirin BGCs, that confer anti-phage activity. Lanthivirin BGCs often colocalize with other defense systems in genomic defense islands. We demonstrate anti-phage activity both in a native Streptomyces context and through heterologous expression of six distinct lanthivirin systems. Mutational analyses of the biosynthetic machinery and core peptide show that lanthivirin maturation is required for anti-phage activity. Genetic and biochemical analyses during infection support a phage-protein-dependent inhibition of phage DNA replication, with no host toxicity. Our findings highlight the untapped potential of BGCs as sources of anti-phage natural products, offering a new avenue for discovering antiviral compounds.

Most bacterial phyla have few or no pure cultures, including Atribacterota, comprised of ubiquitous anaerobes. Here, we report genome-guided enrichment and isolation of two Atribacterota species representing a new family, Caldatribacterium saccharofermentans from a hot spring, and Caldatribacterium inferamans from a deep aquifer. Both were co-enriched with sulfate-reducing bacteria and initially resisted isolation, which we link to inadvertent removal of precipitated folic acid by filter-sterilization of unbuffered Wolin's vitamin solution. We then predict folate auxotrophy across the Atribacterota and ~29% of all bacteria, with extensive auxotrophy in 27% of phyla. Since ≥604 of 791 ( ≥ 76%) media with folic acid additions in the MediaDive database use unbuffered vitamin solutions in which folic acid is likely removed during filter-sterilization, we propose that folate auxotrophy limits culturability in defined media en masse. We also uncover unusual features of Caldatribacterium, including three lipid membrane-like layers (LMLs), with the inner LML surrounding the nucleoid, and a high percentage of secreted proteins, supporting a unique cell biology of Atribacterota.

Outbreaks of cholera pose a major threat to human health. Currently, antibiotics are the most effective treatment against the causative agent, the bacterium Vibrio cholerae. However, the use of antibiotics eventually leads to the emergence of resistant strains, which necessitates the need for alternative approaches. The use of bacteriophages to target the infection by antibiotic-resistant bacteria is one promising alternative. While clearance of Vibrio cholerae with the use of phages has been performed on several animal models, none of these models are naturalistic hosts of V. cholerae. Therefore, we set out to investigate the interaction between V. cholerae and bacteriophage ICP1 both in vitro and in vivo in a naturalistic host, the zebrafish model, Danio rerio. To study the interplay between host, bacteria, and phages, we used a combination of light and ultrastructural imaging techniques, including confocal fluorescence microscopy, serial block face scanning electron microscopy (EM) imaging, and cryogenic EM, which allowed us to investigate both the colonization process by V. cholerae and clearance by the ICP1 bacteriophage. In addition, we determined the effects of the microbiome on this treatment by using germ-free, conventionalized, and monoassociated zebrafish larvae as a host. Independent of the presence and composition of microbiomes used here, V. cholerae efficiently colonized the larval intestine. Finally, we demonstrate significant in vivo clearance of V. cholerae N16961-dsRED by ICP1, underscoring the role of phage-bacteria dynamics in shaping pathogen colonization within the zebrafish larval host.IMPORTANCECholera remains a life-threatening disease that causes recurring outbreaks and significant mortality, particularly in developing and conflict-affected regions. As antimicrobial resistance continues to rise, there is an urgent need to better understand the ecological and microbial dynamics that govern Vibrio cholerae colonization and persistence. This research investigates how V. cholerae interacts with bacteriophages, the host environment, and the resident microbiota within a natural vertebrate host, offering new insights into the factors that influence pathogen clearance and shaping of the gut ecosystem during infection. The powerful combination of serial block-face scanning and cryogenic electron microscopy, fluorescence microscopy, and traditional colony/plaque counting methods revealed previously unobserved aspects of the interplay between host, pathogen, phages, and selected microsymbionts, highlighting phage-driven clearance of V. cholerae during colonization.

AbstractInflammatory bowel diseases (IBDs) are a major public and veterinary health concern, but the causes are poorly understood. In this review, we discuss the potential of mycobacteria as causative factors for such diseases. We focus on similarities between the most common human IBD, Crohn's disease, and a common IBD in cattle, Johne's disease. Both are multifactorial diseases, leading to a chronic hyperinflammatory immune response of the intestines. However, the underlying genetic and environmental factors, such as variations in the intestinal microbiome, are still poorly understood. While Johne's disease has been shown to be caused by Mycobacterium avium subsp. paratuberculosis, the likeliness of mycobacteria as a causative factor of Crohn's disease is still heavily debated. In this review, we summarize the advances in research that could be used to further investigate the role of mycobacteria in intestinal diseases and to give better estimations about which mycobacterial species are most probably hazards for health. New advances in molecular, genetic and imaging methods give hope for better diagnostics, disease prevention, and development of new therapeutics. In addition, these techniques offer researchers a toolbox and general model systems for better understanding the mechanisms of intestinal diseases caused by mycobacteria and the role of the microbiome in the control of disease progression.

Understanding the structural and functional mechanisms of bacteriophage 7-7-1, the flagellotropic phage infecting Agrobacterium sp. H13-3, offers promising insights into phage-host interactions. Using single particle analysis (SPA) cryo-electron microscopy (cryo-EM), we determined the capsid and tail structure, and built atomic models of capsid hexamers, pentamers and tail. Combined with cryo-electron tomography (cryo-ET) and machine learning methodologies, our findings indicate that phage 7-7-1 uses capsid fibers to establish initial contact with the host flagellum, followed by subsequent attachment to cell surface receptors. Proteinase K treatment confirmed the time-dependent degradation of capsid fibers. The study also demonstrated that capsid fibers are flexible and can interact with other phages and host flagella, suggesting a cooperative infection strategy. These results provide crucial structural insights and may open avenues for developing phage-based therapeutics against resistant bacterial pathogens. ### Competing Interest Statement The authors have declared no competing interest.

The cell wall represents an essential structure conserved among most bacteria, playing a crucial role in growth and development. While extensively studied model bacteria have provided insights into cell wall synthesis coordination, the mechanism governing polar growth in actinobacteria remains enigmatic. Here we identify the stomatin-like protein StlP as a pivotal factor for orchestrating polar growth in filamentous actinobacteria under hyperosmotic stress. StlP facilitates the establishment of a membrane microdomain with increased membrane fluidity, a process crucial for maintaining proper growth. The absence of StlP leads to branching of filaments, aberrant cell wall synthesis, thinning of the cell wall, and the extrusion of cell wall-deficient cells at hyphal tips. StlP interacts with key components of the apical glycan synthesis machinery, providing protection to filaments during apical growth. Introduction of StlP in actinobacteria lacking this protein enhances polar growth and resilience under hyperosmotic stress, accompanied by the formation of a membrane microdomain. Our findings imply that stomatin-like proteins, exemplified by StlP, confer a competitive advantage to actinobacteria encountering hyperosmotic stress. Given the widespread conservation of StlP in filamentous actinobacteria, our results propose that the mediation of polar growth through membrane microdomain formation is a conserved phenomenon in these bacteria.

The morphogenetic protein DivIVA exhibits diverse functions across bacterial phyla. In Bacillota, DivIVA is primarily involved in cell division, whereas in Actinomycetota, it plays a central role in coordinating polar growth. Due to its essentiality in Actinomycetota, gaining insight into its structural functions is challenging. We studied truncated DivIVA proteins using a unique divIVA deletion mutant in cell wall-deficient Kitasatospora viridifaciens L-forms. DivIVA comprises an N-terminal domain consisting of a coiled-coil segment bearing a membrane-targeting structure at the N-terminal end, followed by an intercoil region, a larger coiled-coil and a C-terminal domain. Deleting either the intercoil or C-terminal region affected branching. We also created a minimized variant in which both were deleted simultaneously, retaining the N-terminal domain and the second coiled-coil. Expression of this variant caused severe growth defects. Cells showed increased hyphal width, thicker cell walls and frequent tip bursting. Finally, we successfully introduced chimeric DivIVA from the unicellular actinobacterium Mycolicibacterium smegmatis with the membrane targeting domain of K. viridifaciens DivIVA, demonstrating functional conservation within the phylum. By contrast, chimeric DivIVA proteins from Bacillus subtilis could not support growth, underscoring that polar growth is encoded in Actinomycetota-specific amino acid motifs in the first and second coiled-coils. These findings enhance our understanding of the structure-function relationship for DivIVA and present new opportunities to study polar growth.

The morphogenetic protein DivIVA exhibits diverse functions across bacterial phyla. In Bacillota, DivIVA is primarily involved in cell division, whereas in Actinomycetota, it plays a central role in coordinating polar growth. Due to its essential nature, gaining insight into DivIVA function is challenging. Here we report on the functionality of truncated DivIVA proteins, using a unique divIVA deletion mutant created in cell wall-deficient Kitasatospora viridifaciens L-forms. DivIVA comprises an N-terminal domain, two coiled-coil regions separated by an intercoil linker, and a C-terminal domain. Deleting either the intercoil or the C-terminal region impacted branching dynamics. We also created a minimized variant wherein both were deleted simultaneously, containing the N-terminus and fused coiled-coils, resembling DivIVA from unicellular bacteria. Expression of this minimized variant resulted in severe growth defects. Cells exhibited a strong increase in hyphal width and cell wall thickness, accompanied by frequent tip bursting. Finally, we successfully introduced chimeric DivIVA from the unicellular actinobacterium Mycolicibacterium smegmatis with an N-terminal domain of Kitasatospora viridifaciens , demonstrating functional conservation within the phylum. In contrast, a chimeric DivIVA from Bacillus subtilis could not support growth, underscoring that polar growth is encoded within Actinomycetota-specific amino acid motifs encoded in the first and second coiled-coil. These findings enhance our understanding of the structure-function relationship for DivIVA and present new opportunities to study polar growth. Impact DivIVA is essential for polar growth in Actinomycetota. In Streptomycetaceae, this membrane-binding protein localizes at growing hyphal tips and along lateral hyphal walls where new branches emerge. Due to its essentiality, the structural relationship of DivIVA between unicellular and multicellular species remains elusive. Using a Kitasatospora viridifaciens L-form divIVA deletion mutant, we expressed truncated DivIVA variants to identify essential regions. Deleting two large unstructured domains produced a minimized variant containing the N-terminus and fused coiled-coils, resembling DivIVA from unicellular bacteria. This strongly impacted morphogenesis, increasing hyphal width and cell wall thickness, and leading to hyphal tip bursting. Finally, we successfully substituted DivIVA of K. viridifaciens with that from Mycolicibacterium smegmatis . Furthermore, Bacillus subtilis DivIVA could not facilitate reversion, showing that polar growth depends on amino acid motifs unique to Actinomycetota. These findings enhance our understanding of the structure-function relationship of DivIVA and offer new opportunities to study polar growth. ### Competing Interest Statement The authors have declared no competing interest. Dutch Research Council, VI.C.192.002 Swedish Research Council, 2019-04643

The field of cryo-EM offers the possibility to gain high-resolution structural information of biomolecules in their native state. Advances in sample thinning of cryo-EM samples allows the study of proteins inside intact cells using tomography, opening the door for ‘visual proteomics’. However, thicker samples such as tissues or entire organisms are still largely unsuitable for cryo-electron tomography (cryo-ET). Therefore, significant efforts are directed toward developing and improving preparation methods to enable cryo-ET of such complex samples. We focused on the binary association between the Hawaiian bobtail squid Euprymna scolopes and the luminous bacteria Vibrio fischeri . The Squid–Vibrio system has long been studied to understand host-symbiont interactions. Our goal is to study the bacterial-host interface using cryo-ET, at a resolution previously unattainable by conventional EM methods. Here, we present a multi-modal preparation and correlative imaging workflow—including cryo- fluorescence microscopy, microCT, freeze-substitution electron tomography (FS-ET), and serial blockface SEM—to localize and prepare specific regions of the dissected symbiotic light organs for cryo-ET. This approach enabled us to directly visualize symbiotic V. fischeri within the internal host crypts at macromolecular resolution, revealing spatial organization, physical contact, and putative exchange interfaces between host and microbe. Our findings provide structural insights into a foundational model of host–microbe symbiosis and demonstrate the feasibility of cryo-ET for investigating intact tissues at the nanoscale. ### Competing Interest Statement The authors have declared no competing interest. Gordon and Betty Moore Foundation, Symbiosis Model Systems, Grant #9328 Max Planck Society, https://ror.org/01hhn8329 European Union's Horizon Europe Projects, IMAGINE (GA#101094250), ‘SymPore’ (951292) M J Murdock Charitable Trust, 1-339-02548

The phylum Atribacterota is ubiquitous in anoxic environments where it plays important roles in syntrophic carbon and hydrogen metabolism; however, only two species have been isolated. Here, we report the isolation of two additional species representing a new family, Caldatribacterium saccharofermentans from a hot spring and Caldatribacterium inferamans from a deep fractured-rock aquifer. Both were co-enriched on carbohydrates with sulfate-reducing bacteria (SRB). Despite unsuccessful attempts to isolate them on the defined enrichment medium, we finally isolated both species by adding yeast extract to the medium. We show that folate was the key factor provided by the SRBs and the yeast extract and that folate provided in vitamin solutions was inadvertently removed by filter sterilization according to standard media preparation protocols. We further confirm the absence of folate biosynthesis pathways across the phylum and suggest that folate precipitation during media preparation limits cultivation of Atribacterota in toto. The two Caldatribacterium species share unusual features with other Atribacterota , including three lipid membrane-like layers (LMLs), with the inner LML that appears to surround a nucleoid, and a high percentage of transmembrane proteins. This study expands the culturability of Atribacterota and identifies conserved features, including sugar fermentation, unusual cell ultrastructure, and vitamin dependencies necessary for their cultivation.

The bacterial chemotaxis system is one of the best-understood cellular pathways and serves as the model for signal transduction systems. Most chemotaxis research has been conducted with transmembrane chemotaxis systems from Escherichia coli and has established paradigms of the system that were thought to be universal. However, emerging research has revealed that many bacteria possess alternative features of their chemotaxis system, demonstrating that these systems are likely more complex than previously assumed. Here, we compare the canonical chemotaxis system of E. coli with systems that diverge in supramolecular architecture, sensory mechanisms, and protein composition. The alternative features have likely evolved to accommodate chemical specificities of natural niches and cell morphologies. Collectively, these studies demonstrate that bacterial chemotaxis systems are a rapidly expanding field that offers many new opportunities to explore this exceedingly diverse system.

Bacteriophages are being rediscovered as potent agents for medical and industrial applications. However, finding a suitable phage relies on numerous factors, including host specificity, burst size, and infection cycle. The host range of a phage is, besides phage defense systems, initially determined by the recognition and attachment of receptor-binding proteins (RBPs) to the target receptors of susceptible bacteria. RBPs include tail (or occasionally head) fibers and tailspikes. Owing to the potential flexibility and heterogeneity of these structures, they are often overlooked during structural studies. Recent advances in cryoelectron microscopy studies and computational approaches have begun to unravel their structural and fundamental mechanisms during phage infection. In this review, we discuss the current state of research on different phage tail and head fibers, spike models, and molecular mechanisms. These details may facilitate the manipulation of phage-host specificity, which in turn will have important implications for science and society.

The genus Mycobacterium includes species such as Mycobacterium tuberculosis, which can cause deadly human diseases. These bacteria have a protective cell envelope that can be remodeled to facilitate their survival in challenging conditions. Understanding how such conditions affect membrane remodeling can facilitate antibiotic discovery and treatment. To this end, we describe an optimized fluorogenic probe, N-QTF, that reports on mycolyltransferase activity, which is vital for cell division and remodeling. N-QTF is a glycolipid probe that can reveal dynamic changes in the mycobacterial cell envelope in both fast- and slow-growing mycobacterial species. Using this probe to monitor the consequences of antibiotic treatment uncovered distinct cellular phenotypes. Even antibiotics that do not directly inhibit cell envelope biosynthesis cause conspicuous phenotypes. For instance, mycobacteria exposed to the RNA polymerase inhibitor rifampicin release fluorescent extracellular vesicles (EVs). While all mycobacteria release EVs, fluorescent EVs were detected only in the presence of RIF, indicating that exposure to the drug alters EV content. Macrophages exposed to the EVs derived from RIF-treated cells released lower levels of cytokines, suggesting the EVs moderate immune responses. These data suggest that antibiotics can alter EV content to impact immunity. Our ability to see such changes in EV constituents directly results from exploiting these chemical probes.

Filamentous growth of streptomycetes coincides with the synthesis and deposition of an uncharacterized protective glucan at hyphal tips. Synthesis of this glucan depends on the integral membrane protein CslA and the radical copper oxidase GlxA, which are part of a presumably large multiprotein complex operating at growing tips. Here, we show that CslA and GlxA interact by forming a protein complex that is sufficient to synthesize cellulose in vitro. Mass spectrometry analysis revealed that the purified complex produces cellulose chains with a degree of polymerization of at least 80 residues. Truncation analyses demonstrated that the removal of a significant extracellular segment of GlxA had no impact on complex formation, but significantly diminished activity of CslA. Altogether, our work demonstrates that CslA and GlxA form the active core of the cellulose synthase complex and provide molecular insights into a unique cellulose biosynthesis system that is conserved in streptomycetes.