CslA and GlxA from Streptomyces lividans form a functional cellulose synthase complex.

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. IMPORTANCE: Cellulose stands out as the most abundant polysaccharide on Earth. While the synthesis of this polysaccharide has been extensively studied in plants and Gram-negative bacteria, the mechanisms in Gram-positive bacteria have remained largely unknown. Our research unveils a novel cellulose synthase complex formed by the interaction between the cellulose synthase-like protein CslA and the radical copper oxidase GlxA from Streptomyces lividans, a soil-dwelling Gram-positive bacterium. This discovery provides molecular insights into the distinctive cellulose biosynthesis machinery. Beyond expanding our understanding of cellulose biosynthesis, this study also opens avenues for exploring biotechnological applications and ecological roles of cellulose in Gram-positive bacteria, thereby contributing to the broader field of microbial cellulose biosynthesis and biofilm research.

Phage fibers and spikes: a nanoscale Swiss army knife for host infection.

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 cryo-electron 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.

Genome sequence and characterization of Streptomyces phages Vanseggelen and Verabelle, representing two new species within the genus Camvirus.

Despite the rising interest in bacteriophages, little is known about their infection cycle and lifestyle in a multicellular host. Even in the model system Streptomyces, only a small number of phages have been sequenced and well characterized so far. Here, we report the complete characterization and genome sequences of Streptomyces phages Vanseggelen and Verabelle isolated using Streptomyces coelicolor as a host. A wide range of Streptomyces strains could be infected by both phages, but neither of the two phages was able to infect members of the closely related sister genus Kitasatospora. The phages Vanseggelen and Verabelle have a double-stranded DNA genome with lengths of 48,720 and 48,126 bp, respectively. Both phage genomes contain 72 putative genes, and the presence of an integrase encoding protein indicates a lysogenic lifestyle. Characterization of the phages revealed their stability over a wide range of temperatures (30-45 °C) and pH values (4-10). In conclusion, Streptomyces phage Vanseggelen and Streptomyces phage Verabelle are newly isolated phages that can be classified as new species in the genus Camvirus, within the subfamily Arquattrovirinae.

Characterization of Bacteriophage cd2, a Siphophage Infecting Carnobacterium divergens and a Representative Species of a New Genus of Phage.

Carnobacterium divergens is frequently isolated from natural environments and is a predominant species found in refrigerated foods, particularly meat, seafood, and dairy. While there is substantial interest in using C. divergens as biopreservatives and/or probiotics, some strains are known to be fish pathogens, and the uncontrolled growth of C. divergens has been associated with food spoilage. Bacteriophages offer a selective approach to identify and control the growth of bacteria; however, to date, few phages targeting C. divergens have been reported. In this study, we characterize bacteriophage cd2, which we recently isolated from minced beef. A detailed host range study reveals that phage cd2 infects certain phylogenetic groups of C. divergens. This phage has a latent period of 60 min and a burst size of ~28 PFU/infected cell. The phage was found to be acid and heat sensitive, with a complete loss of phage activity when stored at pH 2 or heated to 60°C. Electron microscopy shows that phage cd2 is a siphophage, and while it shares the B3 morphotype with a unique cluster of Listeria and Enterococcus phages, a comparison of genomes reveals that phage cd2 comprises a new genus of phage, which we have termed as Carnodivirus. IMPORTANCE Currently, very little is known about phages that infect carnobacteria, an important genus of lactic acid bacteria with both beneficial and detrimental effects in the food and aquaculture industries. This report provides a detailed characterization of phage cd2, a novel siphophage that targets Carnobacterium divergens, and sets the groundwork for understanding the biology of these phages and their potential use in the detection and biocontrol of C. divergens isolates.

Thermophilic Dehalococcoidia with unusual traits shed light on an unexpected past.

Although the phylum Chloroflexota is ubiquitous, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic bacteria from hot spring sediments belonging to the genus Tepidiforma and class Dehalococcoidia within the phylum Chloroflexota. A combination of cryo-electron tomography, exometabolomics, and cultivation experiments using stable isotopes of carbon revealed three unusual traits: flagellar motility, a peptidoglycan-containing cell envelope, and heterotrophic activity on aromatics and plant-associated compounds. Outside of this genus, flagellar motility has not been observed in Chloroflexota, and peptidoglycan-containing cell envelopes have not been described in Dehalococcoidia. Although these traits are unusual among cultivated Chloroflexota and Dehalococcoidia, ancestral character state reconstructions showed flagellar motility and peptidoglycan-containing cell envelopes were ancestral within the Dehalococcoidia, and subsequently lost prior to a major adaptive radiation of Dehalococcoidia into marine environments. However, despite the predominantly vertical evolutionary histories of flagellar motility and peptidoglycan biosynthesis, the evolution of enzymes for degradation of aromatics and plant-associated compounds was predominantly horizontal and complex. Together, the presence of these unusual traits in Dehalococcoidia and their evolutionary histories raise new questions about the timing and selective forces driving their successful niche expansion into global oceans.

A Flexible and Efficient Microfluidics Platform for the Characterization and Isolation of Novel Bacteriophages.

Bacteriophages are viruses that infect bacteria. This property makes them highly suitable for varied uses in industry or in the development of the treatment of bacterial infections. However, the conventional methods that are used to isolate and analyze these bacteriophages from the environment are generally cumbersome and time consuming. Here, we adapted a high-throughput microfluidic setup for long-term analysis of bacteriophage-bacteria interaction and demonstrate isolation of phages from environmental samples. IMPORTANCE Bacteriophages are gaining increased attention for their potential application as agents to combat antibiotic-resistant infections. However, isolation and characterization of new phages are time consuming and limited by currently used methods. The microfluidics platform presented here allows the isolation and long-term analysis of phages and their effect on host cells with fluorescent light microscopy imaging. Furthermore, this new workflow allows high-throughput characterization of environmental samples for the identification of phages alongside gaining detailed insight into the host response. Taken together, this microfluidics platform will be a valuable tool for phage research, enabling faster and more efficient screening and characterization of host-phage interactions.

High-resolution reconstruction of a Jumbo-bacteriophage infecting capsulated bacteria using hyperbranched tail fibers.

The Klebsiella jumbo myophage ϕKp24 displays an unusually complex arrangement of tail fibers interacting with a host cell. In this study, we combine cryo-electron microscopy methods, protein structure prediction methods, molecular simulations, microbiological and machine learning approaches to explore the capsid, tail, and tail fibers of ϕKp24. We determine the structure of the capsid and tail at 4.1 Šand 3.0 Šresolution. We observe the tail fibers are branched and rearranged dramatically upon cell surface attachment. This complex configuration involves fourteen putative tail fibers with depolymerase activity that provide ϕKp24 with the ability to infect a broad panel of capsular polysaccharide (CPS) types of Klebsiella pneumoniae. Our study provides structural and functional insight into how ϕKp24 adapts to the variable surfaces of capsulated bacterial pathogens, which is useful for the development of phage therapy approaches against pan-drug resistant K. pneumoniae strains.

Genome sequence and characterization of Streptomyces phage Pablito, representing a new species within the genus Janusvirus.

Streptomycetes are ubiquitous soil bacteria. Here we report the complete and annotated genome sequence and characterization of Streptomyces phage Pablito, isolated from a soil sample in Haarlem, the Netherlands using Streptomyces lividans as host. This phage was able to infect a diverse range of Streptomyces strains, but none belonging to the sister genus Kitasatospora. Phage Pablito has double-stranded DNA with a genome length of 49,581 base pairs encoding 76 putative proteins, of which 26 could be predicted. The presence of a serine integrase protein indicated the lysogenic nature of phage Pablito. The phage remained stable over a wide range of temperatures (25-45 °C) and at pH ≥ 7.0, but lost infectivity at temperatures above 55 °C or when the pH dropped below 6.0. This newly isolated phage is closely related to Streptomyces phage Janus and Hank144 and considered a new species classified in the genus Janusvirus, within the subfamily Arquattrovirinae.

Discovery of a Novel Inner Membrane-Associated Bacterial Structure Related to the Flagellar Type III Secretion System.

The bacterial flagellar type III secretion system (fT3SS) is a suite of membrane-embedded and cytoplasmic proteins responsible for building the flagellar motility machinery. Homologous nonflagellar (NF-T3SS) proteins form the injectisome machinery that bacteria use to deliver effector proteins into eukaryotic cells, and other family members were recently reported to be involved in the formation of membrane nanotubes. Here, we describe a novel, evolutionarily widespread, hat-shaped structure embedded in the inner membranes of bacteria, of yet-unidentified function, that is present in species containing fT3SS. Mutant analysis suggests a relationship between this novel structure and the fT3SS, but not the NF-T3SS. While the function of this novel structure remains unknown, we hypothesize that either some of the fT3SS proteins assemble within the hat-like structure, perhaps including the fT3SS core complex, or that fT3SS components regulate other proteins that form part of this novel structure. IMPORTANCE The type III secretion system (T3SS) is a fascinating suite of proteins involved in building diverse macromolecular systems, including the bacterial flagellar motility machine, the injectisome machinery that bacteria use to inject effector proteins into host cells, and probably membrane nanotubes which connect bacterial cells. Here, we accidentally discovered a novel inner membrane-associated complex related to the flagellar T3SS. Examining our lab database, which is comprised of more than 40,000 cryo-tomograms of dozens of species, we discovered that this novel structure is both ubiquitous and ancient, being present in highly divergent classes of bacteria. Discovering a novel, widespread structure related to what are among the best-studied molecular machines in bacteria will open new venues for research aiming at understanding the function and evolution of T3SS proteins.

Reversible bacteriophage resistance by shedding the bacterial cell wall.

Phages are highly abundant in the environment and pose a major threat for bacteria. Therefore, bacteria have evolved sophisticated defence systems to withstand phage attacks. Here, we describe a previously unknown mechanism by which mono- and diderm bacteria survive infection with diverse lytic phages. Phage exposure leads to a rapid and near-complete conversion of walled cells to a cell-wall-deficient state, which remains viable in osmoprotective conditions and can revert to the walled state. While shedding the cell wall dramatically reduces the number of progeny phages produced by the host, it does not always preclude phage infection. Altogether, these results show that the formation of cell-wall-deficient cells prevents complete eradication of the bacterial population and suggest that cell wall deficiency may potentially limit the efficacy of phage therapy, especially in highly osmotic environments or when used together with antibiotics that target the cell wall.

The VarA-CsrA regulatory pathway influences cell shape in Vibrio cholerae.

Despite extensive studies on the curve-shaped bacterium Vibrio cholerae, the causative agent of the diarrheal disease cholera, its virulence-associated regulatory two-component signal transduction system VarS/VarA is not well understood. This pathway, which mainly signals through the downstream protein CsrA, is highly conserved among gamma-proteobacteria, indicating there is likely a broader function of this system beyond virulence regulation. In this study, we investigated the VarA-CsrA signaling pathway and discovered a previously unrecognized link to the shape of the bacterium. We observed that varA-deficient V. cholerae cells showed an abnormal spherical morphology during late-stage growth. Through peptidoglycan (PG) composition analyses, we discovered that these mutant bacteria contained an increased content of disaccharide dipeptides and reduced peptide crosslinks, consistent with the atypical cellular shape. The spherical shape correlated with the CsrA-dependent overproduction of aspartate ammonia lyase (AspA) in varA mutant cells, which likely depleted the cellular aspartate pool; therefore, the synthesis of the PG precursor amino acid meso-diaminopimelic acid was impaired. Importantly, this phenotype, and the overall cell rounding, could be prevented by means of cell wall recycling. Collectively, our data provide new insights into how V. cholerae use the VarA-CsrA signaling system to adjust its morphology upon unidentified external cues in its environment.

How advances in cryo-electron tomography have contributed to our current view of bacterial cell biology.

Advancements in the field of cryo-electron tomography have greatly contributed to our current understanding of prokaryotic cell organization and revealed intracellular structures with remarkable architecture. In this review, we present some of the prominent advancements in cryo-electron tomography, illustrated by a subset of structural examples to demonstrate the power of the technique. More specifically, we focus on technical advances in automation of data collection and processing, sample thinning approaches, correlative cryo-light and electron microscopy, and sub-tomogram averaging methods. In turn, each of these advances enabled new insights into bacterial cell architecture, cell cycle progression, and the structure and function of molecular machines. Taken together, these significant advances within the cryo-electron tomography workflow have led to a greater understanding of prokaryotic biology. The advances made the technique available to a wider audience and more biological questions and provide the basis for continued advances in the near future.

UVC inactivation of pathogenic samples suitable for cryo-EM analysis.

Cryo-electron microscopy has become an essential tool to understand structure and function of biological samples. Especially for pathogens, such as disease-causing bacteria and viruses, insights gained by cryo-EM can aid in developing cures. However, due to the biosafety restrictions of pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the ultrastructure of the sample. Alternatively, researchers use in vitro or ex vivo models, which are non-pathogenic but lack the complexity of the pathogen of interest. Here we show that ultraviolet-C (UVC) radiation applied at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of Vibrio cholerae and the bacterial virus, the ICP1 bacteriophage. We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography followed by sub-tomogram averaging, and single particle analysis (SPA). Additionally, we applied the UVC irradiation to the protein apoferritin (ApoF), which is a widely used test sample for high-resolution SPA studies. The UVC-treated ApoF sample resulted in a 2.1 Å structure indistinguishable from an untreated published map. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.

Cell wall deficiency as an escape mechanism from phage infection.

The cell wall plays a central role in protecting bacteria from some environmental stresses, but not against all. In fact, in some cases, an elaborate cell envelope may even render the cell more vulnerable. For example, it contains molecules or complexes that bacteriophages recognize as the first step of host invasion, such as proteins and sugars, or cell appendages such as pili or flagella. In order to counteract phages, bacteria have evolved multiple escape mechanisms, such as restriction-modification, abortive infection, CRISPR/Cas systems or phage inhibitors. In this perspective review, we present the hypothesis that bacteria may have additional means to escape phage attack. Some bacteria are known to be able to shed their cell wall in response to environmental stresses, yielding cells that transiently lack a cell wall. In this wall-less state, the bacteria may be temporarily protected against phages, since they lack the essential entities that are necessary for phage binding and infection. Given that cell wall deficiency can be triggered by clinically administered antibiotics, phage escape could be an unwanted consequence that limits the use of phage therapy for treating stubborn infections.

In situ imaging of bacterial outer membrane projections and associated protein complexes using electron cryo-tomography.

The ability to produce outer membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among diderm bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of ~90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified outer MEs and MVs in 13 diderm bacterial species and classified several major ultrastructures: (1) tubes with a uniform diameter (with or without an internal scaffold), (2) tubes with irregular diameter, (3) tubes with a vesicular dilation at their tip, (4) pearling tubes, (5) connected chains of vesicles (with or without neck-like connectors), (6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.

Loss of the Bacterial Flagellar Motor Switch Complex upon Cell Lysis.

The bacterial flagellar motor is a complex macromolecular machine whose function and self-assembly present a fascinating puzzle for structural biologists. Here, we report that in diverse bacterial species, cell lysis leads to loss of the cytoplasmic switch complex and associated ATPase before other components of the motor. This loss may be prevented by the formation of a cytoplasmic vesicle around the complex. These observations suggest a relatively loose association of the switch complex with the rest of the flagellar machinery. IMPORTANCE We show in eight different bacterial species (belonging to different phyla) that the flagellar motor loses its cytoplasmic switch complex upon cell lysis, while the rest of the flagellum remains attached to the cell body. This suggests an evolutionary conserved weak interaction between the switch complex and the rest of the flagellum which is important to understand how the motor evolved. In addition, this information is crucial for mimicking such nanomachines in the laboratory.

Microbial hitchhiking: how Streptomyces spores are transported by motile soil bacteria.

Streptomycetes are sessile bacteria that produce metabolites that impact the behavior of microbial communities. Emerging studies have demonstrated that Streptomyces spores are distributed through various mechanisms, but it remains unclear how spores are transported to their preferred microenvironments, such as plant roots. Here, we show that Streptomyces spores are capable of utilizing the motility machinery of other soil bacteria. Motility assays and microscopy studies reveal that Streptomyces spores are transported to plant tissues by interacting directly with the flagella of both gram-positive and gram-negative bacteria. Genetics experiments demonstrate that this form of motility is facilitated by structural proteins on the spore coat. These results demonstrate that nonmotile bacteria are capable of utilizing the motility machinery of other microbes to complete necessary stages of their lifecycle.

Formation of wall-less cells in Kitasatospora viridifaciens requires cytoskeletal protein FilP in oxygen-limiting conditions.

The cell wall is considered an essential component for bacterial survival, providing structural support, and protection from environmental insults. Under normal growth conditions, filamentous actinobacteria insert new cell wall material at the hyphal tips regulated by the coordinated activity of cytoskeletal proteins and cell wall biosynthetic enzymes. Despite the importance of the cell wall, some filamentous actinobacteria can produce wall-deficient S-cells upon prolonged exposure to hyperosmotic stress. Here, we performed cryo-electron tomography and live cell imaging to further characterize S-cell extrusion in Kitasatospora viridifaciens. We show that exposure to hyperosmotic stress leads to DNA compaction, membrane and S-cell extrusion, and thinning of the cell wall at hyphal tips. Additionally, we find that the extrusion of S-cells is abolished in a cytoskeletal mutant strain that lacks the intermediate filament-like protein FilP. Furthermore, micro-aerobic culturing promotes the formation of S-cells in the wild type, but the limited oxygen still impedes S-cell formation in the ΔfilP mutant. These results demonstrate that S-cell formation is stimulated by oxygen-limiting conditions and dependent on functional cytoskeleton remodeling.

Engineered chemotaxis core signaling units indicate a constrained kinase-off state.

Bacterial chemoreceptors, the histidine kinase CheA, and the coupling protein CheW form transmembrane molecular arrays with remarkable sensing properties. The receptors inhibit or stimulate CheA kinase activity depending on the presence of attractants or repellants, respectively. We engineered chemoreceptor cytoplasmic regions to assume a trimer of receptor dimers configuration that formed well-defined complexes with CheA and CheW and promoted a CheA kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar electron-spin resonance spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryo-electron microscopy. The kinase-off state was especially stable, had relatively low domain mobility, and associated the histidine substrate and docking domains with the kinase core, thus preventing catalytic activity. Together, these data provide an experimentally restrained model for the inhibited state of the core signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.