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.

Chemosensory systems allow bacteria to respond and adapt to environmental conditions. Many bacteria contain more than one chemosensory system, but knowledge of their specific roles in regulating different functions remains scarce. Here, we address this issue by analyzing the function of the F6, F8, and alternative (non-motility) cellular functions (ACF) chemosensory systems of the model plant pathogen Pseudomonas syringae pv. tomato. In this work, we assign PsPto chemoreceptors to each chemosensory system, and we visualize for the first time the F6 and F8 chemosensory systems of PsPto using cryo-electron tomography. We confirm that chemotaxis and swimming motility are controlled by the F6 system, and we demonstrate how different components from the F8 and ACF systems also modulate swimming motility. We also determine how the kinase and response regulators from the F6 and F8 chemosensory systems do not work together in the regulation of biofilm, whereas both components from the ACF system contribute together to regulate these traits. Furthermore, we show how the F6, F8, and ACF kinases interact with the ACF response regulator WspR, supporting crosstalk among chemosensory systems. Finally, we reveal how all chemosensory systems play a role in regulating virulence.

This book series provides carefully selected volumes on a broad range of structural biology methods. The most important and latest techniques are wisely presented by experienced and well-known specialists in the area. Some volumes also offer insight into the activity and functioning of structurally well characterized biological systems, thereby effectively combining the knowledge of structure and function.
The volumes are aimed at all scientists in the field of structural biology. The books provide novices with the know-how and basic understanding of specific techniques required to conduct experiments successfully, and established scientists will find the series an invaluable, comprehensive reference tool to deepen and refresh their knowledge.

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.

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.

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.

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.

Pathogenic spirochetes can alter their morphologies and behaviors to infect and survive within their hosts. Previous reports demonstrate that the formation of the so-called “round bodies” and biofilms, and chemotaxis are involved in spirochete pathogenesis. Here, we report a direct link between these cellular states that involve a new class of protein sensor with hitherto unclear function. Using cryo-electron microscopy, genetics, behavioral assays, and molecular modeling, we demonstrate that spirochetes regulate these behaviors in response to the small molecule S-adenosylmethionine (SAM) via a SAM sensor that is anchored to chemotaxis arrays. Furthermore, we establish an improved model for round body formation that now includes characterizations during log phase growth.

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.

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.

The Klebsiella jumbo myophage ϕKp24 displays an unusually complex arrangement of tail fibers interactingwith 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 ofϕKp24.We determine the structure of the capsid and tail at 4.1 Å and 3.0 Å resolution. 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.

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.

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.

Animals, plants, and fungi live in a microbe-dominated world. Investigating the interactions and processes at the host-microbe interface offers insight to how bacteria influence the development, health, and disease of the host. Optimization of exist- ing imaging technologies and development of novel instrumentation will provide the tools needed to fully understand the dynamic relationship that occurs at the host-microbe interface throughout the lifetime of the host. In this review, we describe the current methods used in cryo-electron microscopy (cryo-EM) including cryo-fixation, sample processing, FIB-SEM, and cryotomography. Further, we highlight the new advances associated with these methods that open the cryo-EM discipline to large, complex multicellular samples, like symbiotic tissues. We describe the advantages and challenges associated with correlative imaging techniques and sample thinning methods like lift-out. By highlighting recent pioneering studies in the large-volume or symbiotic sample workflows, we provide insight into how symbiotic model systems will benefit from cryo- EM methods to provide artefact-free, near-native, macromolecular-scale resolution imaging at the host-microbe interface throughout the development and maintenance of symbiosis. Cryo-EM methods have brought a deep fundamental under- standing of prokaryotic biology since its conception. We propose the application of existing and novel cryo-EM techniques to symbiotic systems is the logical next step that will bring an even greater understanding how microbes interact with their host tissues.

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.

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.

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.

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.

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.

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.

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.

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.

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 remodelling.

The prokaryotic chemotaxis system is arguably the best-understood signaling pathway in biology. In all previously described species, chemoreceptors organize into a hexagonal (P6 symmetry) extended array. Here, we report an alternative symmetry (P2) of the chemotaxis apparatus that emerges from a strict linear organization of the histidine kinase CheA in Treponema denticola cells, which possesses arrays with the highest native curvature investigated thus far. Using cryo-ET, we reveal that Td chemoreceptor arrays assume an unusual arrangement of the supra-molecular protein assembly that has likely evolved to accommodate the high membrane curvature. The arrays have several atypical features, such as an extended dimerization domain of CheA and a variant CheW-CheR-like fusion protein that is critical for maintaining an ordered chemosensory apparatus. Furthermore, the previously characterized Td oxygen sensor ODP influences CheA ordering. These results suggest a greater diversity of the chemotaxis signaling system than previously thought.

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.

Motility allows many microbes to traverse their environment tofind nutrientsources or escape unfavorable environments. However, some microbes are non-motile and are restricted to their immediate conditions. Intriguingly, sporadicreports have demonstrated that many nonmotile microbes can utilize the motilitymachinery of other microbes in their vicinity. This form of transportation, calledhitchhiking, has been observed with both prokaryotic and eukaryotic microbes.Importantly, many hitchhiking microbes are pathogenic to humans or plants.Here, we discuss reports of intermicrobial hitchhiking to generate a comprehen-sive view of hitchhiking mechanisms and how such interactions may influencehuman and plant health. We hypothesize that microbial hitchhiking is ubiquitousin nature and may become the subject of an independent subfield of research inmicrobiology.

Bacteria control the length of their polysaccharides, which can control cell viability, physiology, virulence, and immune evasion. Polysaccharide chain length affects immunomodulation, but its impact on bacterial physiology and antibiotic susceptibility was unclear. We probed the consequences of truncating the mycobacterial galactan, an essential linear polysaccharide of about 30 residues. Galactan covalently bridges cell envelope layers, with the outermost cell wall linkage point occurring at residue 12. Reducing galactan chain length by approximately half compromises fitness, alters cell morphology, and increases the potency of hydrophobic antibiotics. Systematic variation of the galactan chain length revealed that it determines periplasm size. Thus, glycan chain length can directly affect cellular physiology and antibiotic activity, and mycobacterial glycans, not proteins, regulate periplasm size.

Symmetry is omnipresent in nature and we encounter symmetry routinely in our everyday life. It is also common on the microscopic level, where symmetry is often key to the proper function of core biological processes. The human brain is exquisitely well suited to recognize such symmetrical features with ease. In contrast, computational recognition of such patterns in images is still surprisingly challenging. In this paper we describe a mathematical approach to identifying smaller local symmetrical structures within larger images. Our algorithm attributes a local symmetry score to each image pixel, which subsequently allows the identification of the symmetrical centers of an object. Though there are already many methods available to detect symmetry in images, to the best of our knowledge, our algorithm is the first that is easily applicable in ImageJ/FIJI. We have created an interactive plugin in FIJI that allows the detection and thresholding of local symmetry values. The plugin combines the different reflection symmetry axis of a square to get a good coverage of reflection symmetry in all directions. To demonstrate the plugins potential, we analyzed images of bacterial chemoreceptor arrays and intracellular vesicle trafficking events, which are two prominent examples of biological systems with symmetrical patterns.

Species of the genus Shewanella are widespread in nature in various habitats, however, little is known about phages affecting Shewanella sp. Here we report the isolation of phages from diverse freshwater environments that infect and lyse strains of Shewanella oneidensis and other Shewanella sp. Sequence analysis and microscopic imaging strongly indicate that these phages form a so far unclassified genus, now named Shewanella phage Thanatos, which can be positioned within the subfamiliy of Tevenvirinae (Duplodnaviria; Heunggongvirae; Uroviricota; Caudoviricetes; Caudovirales; Myoviridae; Tevenvirinae). We characterized one member of this group in more detail using S. oneidensis MR-1 as a host. Shewanella phage Thanatos-1 possesses a prolate icosahedral capsule of about 110 nm in height and 70 nm in width and a tail of about 95 nm in length. The dsDNA genome exhibits a GC content of about 34.5 %, has a size of 160.6 kbp and encodes about 206 proteins (92 with an annotated putative function) and two tRNAs. Out of those 206, MS analyses identified about 155 phage proteins in PEG-precipitated samples of infected cells. Phage attachment likely requires the outer lipopolysaccharide of S. oneidensis, narrowing the phage’s host range. Under the applied conditions, about 20 novel phage particles per cell were produced after a latent period of approximately 40 minutes, which are stable at a pH range from 4 to 12 and resist temperatures up to 55 °C for at least 24 h. Addition of Thanatos to S. oneidensis results in partial dissolution of established biofilms, however, early exposure of planktonic cells to Thanatos significantly enhances biofilm formation. Taken together, we identified a novel genus of Myophages affecting S. oneidensis communities in different ways.

The bacterial cell wall is a multicomponent structure that provides structural support and protection. In monoderm species, the cell wall is made up predominantly of peptidoglycan, teichoic acids and capsular glycans. Filamentous monoderm Actinobacteria incorporate new cell-wall material at their tips. Here we use cryo-electron tomography to reveal the architecture of the actinobacterial cell wall of Streptomyces coelicolor. Our data shows a density difference between the apex and subapical regions. Removal of teichoic acids results in a patchy cell wall and distinct lamellae. Knock-down of tagO expression using CRISPR-dCas9 interference leads to growth retardation, presumably because build-in of teichoic acids had become rate-limiting. Absence of extracellular glycans produced by MatAB and CslA proteins results in a thinner wall lacking lamellae and patches. We propose that the Streptomyces cell wall is composed of layers of peptidoglycan and extracellular polymers that are structurally supported by teichoic acids.

Vibrio cholerae, the causative agent of the acute diarrheal disease cholera, is able to thrive in diverse habitats such as natural water bodies and inside human hosts. To ensure their survival, these bacteria rely on chemosensory pathways to sense and respond to changing environmental conditions. These pathways constitute a highly sophisticated cellular control system in Bacteria and Archaea. Reflecting the complex life cycle of V. cholerae, this organism has three different chemosensory pathways that together contain over 50 proteins expressed under different environmental conditions. Only one of them is known to control motility, while the function of the other two remains to be discovered. Here, we provide an overview of the chemosensory systems in V. cholerae and the advances towards understanding their structure and function.

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identify ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we trace the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost.

Visualizing biological structures and cellular processes in their native state is a major goal of many scientific laboratories. In the past 20 years, the technique of preserving samples by vitrification has greatly expanded, specifically for use in cryogenic electron microscopy (cryo-EM). Here, we report on improvements in the design and use of a portable manual cryogenic plunge freezer that is intended for use in laboratories that are not equipped for the cryopreservation of samples. The construction of the instrument is economical, can be produced by a local machine shop without specialized equipment, and lowers the entry barriers for newcomers with a reliable alternative to costly commercial equipment. The improved design allows for successful freezing of isolated proteins for single particle analysis as well as bacterial cells for cryo-electron tomography. With this instrument, groups will be able to prepare vitreous samples whenever and wherever necessary, which can then be imaged at local or national cryo-EM facilities.

The prokaryotic chemotaxis system is arguably the best-understood signaling pathway in biology, but most insights have been obtained from only a few model organisms. In all previously described species, chemoreceptors organize with the histidine kinase (CheA) and coupling protein (CheW) into a hexagonal (P6 symmetry) extended array that is considered universal among archaea and bacteria. Here, for the first time, we apply cryo-electron tomography to whole Treponema denticola (Td) cells to investigate the structure of a spirochete (F2) chemotaxis system. The Td chemoreceptor arrays assume a truly unusual arrangement of the supra-molecular protein assembly that has likely evolved to accommodate the high membrane curvature present in spirochetes. A two-fold (P2) symmetry of the chemotaxis apparatus in Td emerges from a strict linear organization of the kinase CheA, which generates arrays that run parallel to the cell axis. The arrays have several additional atypical features, such as an extended dimerization domain of CheA and a variant CheW-CheR-like fusion protein that is critical for maintaining an ordered, functional chemosensory apparatus in an extremely curved cell. Furthermore, the previously characterized Td oxygen sensor ODP influences array integrity and its loss substantially orders CheA. These results demonstrate the importance of examining chemotaxis structures of non-model organisms in vivo and suggest a greater diversity of this signaling system than previously thought.

The UV-inducible pili system of Sulfolobales (Ups) mediates the formation of species-specific cellular aggregates. Within these aggregates, cells exchange DNA to repair DNA double-strand breaks via homologous recombination. Substitution of the Sulfolobus acidocaldarius pilin subunits UpsA and UpsB with their homologs from Sulfolobus tokodaii showed that these subunits facilitate species-specific aggregation. A region of low conservation within the UpsA homologs is primarily important for this specificity. Aggregation assays in the presence of different sugars showed the importance of N-glycosylation in the recognition process. In addition, the N-glycan decorating the S-layer of S. tokodaii is different from the one of S. acidocaldarius. Therefore, each Sulfolobus species seems to have developed a unique UpsA binding pocket and unique N-glycan composition to ensure aggregation and, consequently, also DNA exchange with cells from only the same species, which is essential for DNA repair by homologous recombination.

Most chemotactic motile bacteria possess multiple chemotaxis signaling systems, the functions of which are not well characterized. Chemotaxis signaling is initiated by chemoreceptors that assemble as large arrays, together with chemotaxis coupling proteins (CheW) and histidine kinase proteins (CheA), which form a baseplate with the cytoplasmic tips of receptors. These cell pole-localized arrays mediate sensing, signaling, and signal amplification during chemotaxis responses. Membrane-bound chemoreceptors with different cytoplasmic domain lengths segregate into distinct arrays. Here, we show that a bacterium, Azospirillum brasilense, which utilizes two chemotaxis signaling systems controlling distinct motility parameters, coordinates its chemotactic responses through the production of two separate membrane-bound chemoreceptor arrays by mixing paralogs within chemotaxis baseplates. The polar localization of chemoreceptors of different length classes is maintained in strains that had baseplate signaling proteins from either chemotaxis system but was lost when both systems were deleted. Chemotaxis proteins (CheA and CheW) from each of the chemotaxis signaling systems (Che1 and Che4) could physically interact with one another, and chemoreceptors from both classes present in A. brasilense could interact with Che1 and Che4 proteins. The assembly of paralogs from distinct chemotaxis pathways into baseplates provides a straightforward mechanism for coordinating signaling from distinct pathways, which we predict is not unique to this system given the propensity of chemotaxis systems for horizontal gene transfer.

Chemotaxis is crucial for the survival of bacteria, and the signaling systems associated with it exhibit a high level of evolutionary conservation. The architecture of the chemosensory array and the signal transduction mechanisms have been extensively studied in Escherichia coli. More recent studies have revealed a vast diversity of the chemosensory system among bacteria. Unlike E. coli, some bacteria assemble more than one chemosensory array and respond to a broader spectrum of environmental and internal stimuli. These chemosensory arrays exhibit a great variability in terms of protein composition, cellular localization, and functional variability. Here, we present recent findings that emphasize the extent of diversity in chemosensory arrays and highlight the importance of studying chemosensory arrays in bacteria other than the common model organisms.

Bacteria sense and respond to their environment through a highly conserved assembly of transmembrane chemoreceptors (MCPs), the histidine kinase (CheA), and the coupling protein CheW, hereafter termed “the chemosensory array”. In recent years, great strides have been made in understanding the architecture of the underlying chemosensory arrays and how these assemblies engender sensitive and cooperative responses. Nonetheless, a central outstanding question surrounds how receptors modulate the activity of the chemotaxis kinase CheA, the enzymatic output of the sensory system. With a focus on recent advances, we summarize the current understanding of array structure and function to comment on the molecular mechanism by which CheA, receptors and CheW generate the high sensitivity, gain and dynamic range emblematic of bacterial chemotaxis. The complexity of the chemosensory arrays has motivated investigation with many different approaches. In particular, structural methods, genetics, cellular activity assays, nanodisc technology and cryo-electron tomography have provided advances that bridge length scales and connect molecular mechanism to cellular function. Given the high degree of component integration in the chemosensory arrays, we ultimately aim to understand how such networked molecular interactions generate a whole that is truly greater than the sum of its parts. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

Tsr, the serine chemoreceptor in Escherichia coli, transduces signals from a periplasmic ligand-binding site to its cytoplasmic tip, where it controls the activity of the CheA kinase. To function, Tsr forms trimers of homodimers (TODs), which as- sociate in vivo with the CheA kinase and CheW coupling protein. Together, these proteins assemble into extended hexagonal arrays. Here, we use cryo-electron to- mography and molecular dynamics simulation to study Tsr in the context of a near- native array, characterizing its signaling-related conformational changes at both the individual dimer and the trimer level. In particular, we show that individual Tsr dimers within a trimer exhibit asymmetric flexibilities that are a function of the sig- naling state, highlighting the effect of their different protein interactions at the re- ceptor tips. We further reveal that the dimer compactness of the Tsr trimer changes between signaling states, transitioning at the glycine hinge from a compact confor- mation in the kinase-OFF state to an expanded conformation in the kinase-ON state. Hence, our results support a crucial role for the glycine hinge: to allow the receptor flexibility necessary to achieve different signaling states while also maintaining struc- tural constraints imposed by the membrane and extended array architecture.

The Gram-negative cell envelope is a remarkable structure with core components that include an inner membrane, an outer membrane, and a peptidoglycan layer in the periplasmic space between. Multiple molecular systems function to maintain integrity of this essential barrier between the interior of the cell and its surrounding environment. We show that a conserved DUF1849-family protein, EipB, is secreted to the periplasmic space of Brucella, a monophyletic group of intracellular pathogens. In the periplasm, EipB folds into an unusual fourteen-stranded β-spiral structure that resembles the LolA and LolB lipoprotein delivery system, though the overall fold of EipB is distinct from LolA/LolB. Deletion of eipB results in defects in Brucella cell envelope integrity in vitro and in maintenance of spleen colonization in a mouse model of B. abortus infection. Transposon disruption of ttpA, which encodes a periplasmic protein containing tetratricopeptide repeats, is synthetically lethal with eipB deletion. ttpA is a reported virulence determinant in Brucella, and our studies of ttpA deletion and overexpression strains provide evidence that this gene also contributes to cell envelope function. We conclude that eipB and ttpA function in the Brucella periplasmic space to maintain cell envelope integrity, which facilitates survival in a mammalian host.

Aquatic environments are reservoirs of the human pathogen Vibrio cholerae O1, which causes the acute diarrheal disease cholera. Upon low temperature or limited nutrient availability, the cells enter a viable but non-culturable (VBNC) state. Characteristic of this state are an altered morphology, low metabolic activity, and lack of growth under standard laboratory conditions. Here, for the first time, the cellular ultrastructure of V. cholerae VBNC cells raised in natural waters was investigated using electron cryo-tomography. This was complemented by a comparison of the proteomes and the peptidoglycan composition of V. cholerae from LB overnight cultures and VBNC cells. The extensive remodeling of the VBNC cells was most obvious in the passive dehiscence of the cell envelope, resulting in improper embedment of flagella and pili. Only minor changes of the peptidoglycan and osmoregulated periplasmic glucans were observed. Active changes in VBNC cells included the production of cluster I chemosensory arrays and change of abundance of cluster II array proteins. Components involved in iron acquisition and storage, peptide import and arginine biosynthesis were overrepresented in VBNC cells, while enzymes of the central carbon metabolism were found at lower levels. Finally, several pathogenicity factors of V. cholerae were less abundant in the VBNC state, potentially limiting their infectious potential. This study gives unprecedented insight into the physiology of VBNC cells and the drastically altered presence of their metabolic and structural proteins.

Bacteria thrive in virtually all environments. Like all other living organisms, bacteria may
encounter various types of stresses, to which cells need to adapt. In this chapter, we
describe how cells cope with stressful conditions and how this may lead to dramatic
morphological changes. These changes may not only allow harmless cells to withstand
environmental insults but can also benefit pathogenic bacteria by enabling them to
escape from the immune system and the activity of antibiotics. A better understanding
of stress-induced morphogenesis will help us to develop new approaches to combat
such harmful pathogens.

Bacteria switch only intermittently to motile planktonic lifestyles under favorable conditions. Under chronic nutrient deprivation, however, bacteria orchestrate a switch to stationary phase, conserving energy by altering metabolism and stopping motility. About two-thirds of bacteria use flagella to swim, but how bacteria deactivate this large molecular machine remains unclear. Here, we describe the previously unreported ejection of polar motors by γ-proteobacteria. We show that these bacteria eject their flagella at the base of the flagellar hook when nutrients are depleted, leaving a relic of a former flagellar motor in the outer membrane. Subtomogram averages of the full motor and relic reveal that this is an active process, as a plug protein appears in the relic, likely to prevent leakage across their outer membrane; furthermore, we show that ejection is triggered only under nutritional depletion and is independent of the filament as a possible mechanosensor. We show that filament ejection is a widespread phenomenon demonstrated by the appearance of relic structures in diverse γ-proteobacteria including Plesiomonas shigelloides, Vibrio cholerae, Vibrio fischeri, Shewanella putrefaciens, and Pseudomonas aeruginosa. While the molecular details remain to be determined, our results demonstrate a novel mechanism for bacteria to halt costly motility when nutrients become scarce.

The bacterial flagellar motor, a cell-envelope-embedded macromolecular machine that functions as a cellular propeller, exhibits significant structural variability between species. Different torque-generating stator modules allow motors to operate in different pH, salt or viscosity levels. How such diversity evolved is unknown. Here we use electron cryo-tomography to determine the in situ macromolecular structures of three Gammaproteobacteria motors: Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis, providing the first views of intact motors with dual stator systems. Complementing our imaging with bioinformatics analysis, we find a correlation between the motor's stator system and its structural elaboration. Motors with a single H+-driven stator have only the core periplasmic P- and L-rings; those with dual H+-driven stators have an elaborated P-ring; and motors with Na+ or Na+/H+-driven stators have both their P- and L-rings embellished. Our results suggest an evolution of structural elaboration that may have enabled pathogenic bacteria to colonize higher-viscosity environments in animal hosts.

The chemoreceptor array, a remarkably ordered supramolecular complex, is composed of hexagonally packed trimers of receptor dimers networked by a histidine kinase and one or more coupling proteins. Even though the receptor packing is universal among chemotactic bacteria and archaea, the array architecture has been extensively studied only in selected model organisms. Here, we show that even in the complete absence of the kinase, the cluster II arrays in Vibrio cholerae retain their native spatial localization and the iconic hexagonal packing of the receptors with 12-nm spacing. Our results demonstrate that the chemotaxis array is versatile in composition, a property that allows auxiliary chemotaxis proteins such as ParP and CheV to integrate directly into the assembly. Along with its compositional variability, cluster II arrays exhibit a low degree of structural stability compared with the ultrastable arrays in Escherichia coli. We propose that the variability in chemoreceptor arrays is an important mechanism that enables the incorporation of chemotaxis proteins based on their availability.

Molecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function in B. abortus envelope stress resistance and infection. Expression of this gene, which we name eipA, is directly activated by the essential cell cycle regulator, CtrA. eipA encodes a soluble periplasmic protein that adopts an unusual eight‐stranded β‐barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the cell envelope integrity. Transposon disruption of genes required for LPS O‐polysaccharide biosynthesis is synthetically lethal with eipA deletion. This genetic connection between O‐polysaccharide and eipA is corroborated by our discovery that eipA is essential in Brucella ovis, a naturally rough species that harbors mutations in several genes required for O‐polysaccharide production. Conditional depletion of eipA expression in B. ovis results in a cell chaining phenotype, providing evidence that eipA directly or indirectly influences cell division in Brucella. We conclude that EipA is a molecular determinant of Brucella virulence that functions to maintain cell envelope integrity and influences cell division.

The intricate nature of the cellular structure in bacteria and archaea has historically been underappreciated because of their small size. However, the advent of new microscopy techniques, such as fluorescent microscopy, super resolution light microscopy, cryo-electron microscopy, electron cryotomography and correlative microscopy techniques now enables the study of intact microbial cells at unprecedented resolution. We are now able to directly observe microbial cell structures and gain insight into essential processes such as establishing the proper cell shape, cell growth, division, motility, sensing and interacting with the environment and the formation of cellular communities. In this article, we will give a brief overview into the components of bacterial and archaeal cells, and how the cells rely on these structures to thrive.

The cell wall is a shape-defining structure that envelopes almost all bacteria and protects them from environmental stresses. Bacteria can be forced to grow without a cell wall under certain conditions that interfere with cell wall synthesis, but the relevance of these wall-less cells (known as L-forms) is unclear. Here, we show that several species of filamentous actinomycetes have a natural ability to generate wall-deficient cells in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the normal mycelial mode of growth. However, prolonged exposure of S-cells to hyperosmotic stress yields variants that are able to proliferate indefinitely without their cell wall, similarly to L-forms. We propose that formation of wall-deficient cells in actinomycetes may serve as an adaptation to osmotic stress.

Most motile bacteria are able to bias their movement towards more favorable environments or to escape from obnoxious substances by a process called chemotaxis. Chemotaxis depends on a chemosensory system that is able to sense specific environmental signals and generate a behavioral response. Typically, the signal is transmitted to the bacterial flagellum, ultimately regulating the swimming behavior of individual cells. Chemotaxis is mediated by proteins that assemble into large, highly ordered arrays. It is imperative for successful chemotactic behavior and cellular competitiveness that chemosensory arrays form and localize properly within the cell. Here we review how chemotaxis arrays form and localize in Vibrio cholerae and Vibrio parahaemolyticus. We focus on how the ParC/ParP-system mediates cell cycle-dependent polar localization of chemotaxis arrays and thus ensures proper cell pole development and array inheritance upon cell division.

Cryo-electron microscopy (cryo-EM) allows the imaging of intact macromolecular complexes in the context of whole cells. The biological samples for cryo-EM are kept in a near-native state by flash freezing, without the need for any additional sample preparation or fixation steps. Since transmission electron microscopy only generates 2D projections of the samples, the specimen has to be tilted in order to recover its 3D structural information. This is done by collecting images of the sample with various tilt angles in respect to the electron beam. The acquired tilt series can then be computationally back-projected. This technique is called electron cryotomography (ECT), and has been instrumental in unraveling the architecture of chemoreceptor arrays. Here we describe the method of visualizing in vivo bacterial chemoreceptor arrays in three main steps: immobilization of bacterial cells on EM grids by plunge-freezing; 2D image acquisition in tilt series; and 3D tomogram reconstruction.

Cryo-electron microscopy (cryo-EM) enables the study of biological structures in situ in great detail and to solve protein structures at Ångstrom level resolution. Due to recent advances in instrumentation and data processing, the field of cryo-EM is a rapidly growing. Access to facilities and national centers that house the state-of-the-art microscopes is limited due to the ever-rising demand, resulting in long wait times between sample preparation and data acquisition. To improve sample storage, we have developed a cryo-storage system with an efficient, high storage capacity that enables sample storage in a highly organized manner. This system is simple to use, cost-effective and easily adaptable for any type of grid storage box and dewar and any size cryo-EM laboratory.

Chemotaxis proteins organize into large, highly ordered, chemotactic signaling arrays, which in Vibrio species are found at the cell pole. Proper localization of signaling arrays is mediated by ParP, which tethers arrays to a cell pole anchor, ParC. Here we show that ParP's C-terminus integrates into the core-unit of signaling arrays through interactions with MCP-proteins and CheA. Its intercalation within core-units stimulates array formation, whereas its N-terminal interaction domain enables polar recruitment of arrays and facilitates its own polar localization. Linkage of these domains within ParP couples array formation and localization and results in controlled array positioning at the cell pole. Notably, ParP's integration into arrays modifies its own and ParC's subcellular localization dynamics, promoting their polar retention. ParP serves as a critical nexus that regulates the localization dynamics of its network constituents and drives the localized assembly and stability of the chemotactic machinery, resulting in proper cell pole development.

Electron cryotomography (ECT) can reveal the native structure and arrangement of macromolecular complexes inside intact cells. This technique has greatly advanced our understanding of the ultrastructure of bacterial cells. Rather than undifferentiated bags of enzymes, we now view bacteria as structurally complex assemblies of macromolecular machines. To date, our group has applied ECT to nearly 90 different bacterial species, collecting more than 15,000 cryotomograms. In addition to known structures, we have observed several, to our knowledge, uncharacterized features in these tomograms. Some are completely novel structures; others expand the features or species range of known structure types. Here we present a survey of these uncharacterized bacterial structures in the hopes of accelerating their identification and study, and furthering our understanding of the structural complexity of bacterial cells.IMPORTANCE Bacteria are more structurally complex than is commonly appreciated and we present here a number of interesting structures that will initiate new lines of research investigating their identities and roles.

Most bacteria possess a peptidoglycan cell wall that determines their morphology and provides mechanical robustness during osmotic challenges. The biosynthesis of this structure is achieved by a large set of synthetic and lytic enzymes with varying substrate specificities. Although the biochemical functions of these proteins are conserved and well-investigated, the precise roles of individual factors and the regulatory mechanisms coordinating their activities in time and space remain incompletely understood. Here, we comprehensively analyze the autolytic machinery of the alphaproteobacterial model organism Caulobacter crescentus, with a specific focus on LytM-like endopeptidases, soluble lytic transglycosylases and amidases. Our data reveal a high degree of redundancy within each protein family but also specialized functions for individual family members under stress conditions. In addition, we identify two lytic transglycosylases and an amidase as new divisome components that are recruited to midcell at distinct stages of the cell cycle. The midcell localization of these proteins is affected by two LytM factors with degenerate catalytic domains, DipM and LdpF, which may serve as regulatory hubs coordinating the activities of multiple autolytic enzymes during cell constriction and fission respectively. These findings set the stage for in-depth studies of the molecular mechanisms that control peptidoglycan remodeling in C. crescentus.

Transmembrane chemotaxis receptors are found in bacteria in extended hexagonal arrays stabilized by the membrane and by cytosolic binding partners, the kinase CheA and coupling protein CheW. Models of array architecture and assembly propose receptors cluster into trimers of dimers that associate with one CheA dimer and two CheW monomers to form the minimal "core unit" necessary for signal transduction. Reconstructing in vitro chemoreceptor ternary complexes that are homogeneous and functional and exhibit native architecture remains a challenge. Here we report that His-tag-mediated receptor dimerization with divalent metals is sufficient to drive assembly of nativelike functional arrays of a receptor cytoplasmic fragment. Our results indicate receptor dimerization initiates assembly and precedes formation of ternary complexes with partial kinase activity. Restoration of maximal kinase activity coincides with a shift to larger complexes, suggesting that kinase activity depends on interactions beyond the core unit. We hypothesize that achieving maximal activity requires building core units into hexagons and/or coalescing hexagons into the extended lattice. Overall, the minimally perturbing His-tag-mediated dimerization leads to assembly of chemoreceptor arrays with native architecture and thus serves as a powerful tool for studying the assembly and mechanism of this complex and other multiprotein complexes.

Unraveling the structure and function of two-component and chemotactic signaling along with different aspects related to motility of bacteria and archaea are key research areas in modern microbiology. Escherichia coli is the traditional model organism to study chemotaxis signaling and motility. However, the recent study of a wide range of bacteria and even some archaea with different lifestyles has provided new insight into the eco-physiology of chemotaxis, which is essential for the host establishment of different pathogens or beneficial bacteria. The expanded range of model organisms has also permitted the study of chemosensory pathways unrelated to chemotaxis, multiple chemotaxis pathways within an organism, and new types of chemoreceptors. This research has greatly benefitted from technical advances in the field of cryo-microscopy that continues to reveal with increasing resolution the complexity and diversity of large protein complexes like the flagellar motor or chemoreceptor arrays. In addition, sensitive instruments now allow for an increasing number of experiments to be conducted at the single-cell level, thereby revealing information that is beginning to bridge the gap between individual cells and population behavior. Evidence has also accumulated showing that bacteria have evolved different mechanisms for surface sensing, which appears to be mediated by flagella and possibly type IV pili, and that the downstream signaling involves chemosensory pathways and two-component system based processes. Herein we summarize the recent advances and research tendencies in this field as presented at the latest Bacterial Locomotion and Signal Transduction (BLAST XIV) conference.

Archaeal swimming motility is driven by archaella: rotary motors attached to long extracellular filaments. The structure of these motors, and particularly how they are anchored in the absence of a peptidoglycan cell wall, is unknown. Here, we use electron cryotomography to visualize the archaellar basal body in vivo in Thermococcus kodakaraensis KOD1. Compared to the homologous bacterial type IV pilus (T4P), we observe structural similarities as well as several unique features. While the position of the cytoplas- mic ATPase appears conserved, it is not braced by linkages that extend upward through the cell envelope as in the T4P, but rather by cytoplasmic components that attach it to a large conical frus- tum up to 500 nm in diameter at its base. In addition to anchoring the lophotrichous bundle of archaella, the conical frustum associ- ates with chemosensory arrays and ribosome-excluding material and may function as a polar organizing center for the coccoid cells.

FtsZ, the bacterial homologue of eukaryotic tubulin, plays a central role in cell division in nearly all bacteria and many archaea. It forms filaments under the cytoplasmic membrane at the division site where, together with other proteins it recruits, it drives peptidoglycan synthesis and constricts the cell. Despite extensive study, the arrangement of FtsZ filaments and their role in division continue to be debated. Here, we apply electron cryotomography to image the native structure of intact dividing cells and show that constriction in a variety of Gram‐negative bacterial cells, including Proteus mirabilis and Caulobacter crescentus, initiates asymmetrically, accompanied by asymmetric peptidoglycan incorporation and short FtsZ‐like filament formation. These results show that a complete ring of FtsZ is not required for constriction and lead us to propose a model for FtsZ‐driven division in which short dynamic FtsZ filaments can drive initial peptidoglycan synthesis and envelope constriction at the onset of cytokinesis, later increasing in length and number to encircle the division plane and complete constriction.

Electron cryotomography (ECT) can produce three-dimensional images of biological samples such as intact cells in a near-native, frozen-hydrated state to macromolecular resolution (∼4 nm). Because one of its first and most common applications has been to bacterial chemoreceptor arrays, ECT’s contributions to this field illustrate well its past, present, and future. Al- though X-ray crystallography and nuclear magnetic resonance spectroscopy have revealed the structures of nearly all the individual components of chemoreceptor arrays, ECT has revealed the mesoscale information about how the components are arranged within cells. Receptors assemble into a universally conserved 12-nm hexagonal lattice linked by CheA/CheW rings. Membrane-bound arrays are single layered; cytoplasmic arrays are double layered. Images of in vitro reconstitutions have led to a model of how arrays assemble, and images of native arrays in different states have shown that the conformational changes associated with signal transduction are subtle, constraining models of activation and system cooperativity. Phase plates, better detectors, and more stable stages promise even higher resolution and broader application in the near future.

Nearly all motile bacterial cells use a highly sensitive and adaptable sensory system to detect changes in nutrient concentrations in the environment and guide their movements toward attractants and away from repellents. The best-studied bacterial chemoreceptor arrays are membrane-bound. Many motile bacteria contain one or more additional, sometimes purely cytoplasmic, chemoreceptor systems. Vibrio cholerae contains three chemotaxis clusters (I, II, and III). Here, using electron cryotomography, we explore V. cholerae’s cytoplasmic chemoreceptor array and establish that it is formed by proteins from cluster I. We further identify a chemoreceptor with an unusual domain architecture, DosM, which is essential for formation of the cytoplasmic arrays. DosM contains two signaling domains and spans the two-layered cytoplasmic arrays. Finally, we present evidence suggesting that this type of receptor is important for the structural stability of the cytoplasmic array.

Tenericutes are a unique class of bacteria that lack a cell wall and are typically parasites or commensals of eukaryotic hosts. Environmental 16S rDNA surveys have identified a number of tenericute clades in diverse environments, introducing the possibility that these Tenericutes may represent non-host-associated, free-living microorganisms. Metagenomic sequencing of deep-sea methane seep sediments resulted in the assembly of two genomes from a Tenericutes-affiliated clade currently known as 'NB1-n' (SILVA taxonomy) or 'RF3' (Greengenes taxonomy). Metabolic reconstruction revealed that, like cultured members of the Mollicutes, these 'NB1-n' representatives lack a tricarboxylic acid cycle and instead use anaerobic fermentation of simple sugars for substrate level phosphorylation. Notably, the genomes also contained a number of unique metabolic features including hydrogenases and a simplified electron transport chain containing an RNF complex, cytochrome bd oxidase and complex I. On the basis of the metabolic potential predicted from the annotated genomes, we devised an anaerobic enrichment media that stimulated the growth of these Tenericutes at 10 °C, resulting in a mixed culture where these organisms represented ~60% of the total cells by targeted fluorescence in situ hybridization (FISH). Visual identification by FISH confirmed these organisms were not directly associated with Eukaryotes and electron cryomicroscopy of cells in the enrichment culture confirmed an ultrastructure consistent with the defining phenotypic property of Tenericutes, with a single membrane and no cell wall. On the basis of their unique gene content, phylogenetic placement and ultrastructure, we propose these organisms represent a novel class within the Tenericutes, and suggest the names Candidatus 'Izimaplasma sp. HR1' and Candidatus 'Izimaplasma sp. HR2' for the two genome representatives.The ISME Journal advance online publication, 8 April 2016; doi:10.1038/ismej.2016.55.

We have functionally and structurally defined an essential protein phosphorelay that regulates expression of genes required for growth, division, and intracellular survival of the global zoonotic pathogen Brucella abortus. Our study delineates phosphoryl transfer through this molecular pathway, which initiates from the sensor kinase CckA and proceeds through the ChpT phosphotransferase to two regulatory substrates: CtrA and CpdR. Genetic perturbation of this system results in defects in cell growth and division site selection, and a specific viability deficit inside human phagocytic cells. Thus, proper control of B. abortus division site polarity is necessary for survival in the intracellular niche. We further define the structural foundations of signaling from the central phosphotransferase, ChpT, to its response regulator substrate, CtrA, and provide evidence that there are at least two modes of interaction between ChpT and CtrA, only one of which is competent to catalyze phosphoryltransfer. The structure and dynamics of the active site on each side of the ChpT homodimer are distinct, supporting a model in which quaternary structure of the 2:2 ChpT-CtrA complex enforces an asymmetric mechanism of phosphoryl transfer between ChpT and CtrA. Our study provides mechanistic understanding, from the cellular to the atomic scale, of a conserved transcriptional regulatory system that controls the cellular and infection biology of B. abortus. More generally, our results provide insight into the structural basis of two-component signal transduction, which is broadly conserved in bacteria, plants, and fungi.

Chemotaxis allows cells to sense and respond to their environment. In Bacteria, stimuli are detected by arrays of chemoreceptors that relay the signal to a two-component regulatory system. These arrays take the form of highly stereotyped super-lattices comprising hexagonally packed trimers-of-receptor-dimers networked by rings of histidine kinase and coupling proteins. This structure is conserved across chemotactic Bacteria, and between membrane-bound and cytoplasmic arrays, and gives rise to the highly cooperative, dynamic nature of the signalling system. The chemotaxis system, absent in eukaryotes, is also found in Archaea, where its structural details remain uncharacterized. Here we provide evidence that the chemotaxis machinery was not present in the last archaeal common ancestor, but rather was introduced in one of the waves of lateral gene transfer that occurred after the branching of Eukaryota but before the diversification of Euryarchaeota. Unlike in Bacteria, the chemotaxis system then evolved largely vertically in Archaea, with very few subsequent successful lateral gene transfer events. By electron cryotomography, we find that the structure of both membrane-bound and cytoplasmic chemoreceptor arrays is conserved between Bacteria and Archaea, suggesting the fundamental importance of this signalling architecture across diverse prokaryotic lifestyles.

Most motile bacteria sense and respond to their environment through a transmembrane chemoreceptor array whose structure and function have been well-studied, but many species also contain an additional cluster of chemoreceptors in their cytoplasm. Although the cytoplasmic cluster is essential for normal chemotaxis in some organisms, its structure and function remain unknown. Here we use electron cryotomography to image the cytoplasmic chemoreceptor cluster in Rhodobacter sphaeroides and Vibrio cholerae. We show that just like transmembrane arrays, cytoplasmic clusters contain trimers-of-receptor-dimers organized in 12-nm hexagonal arrays. In contrast to transmembrane arrays, however, cytoplasmic clusters comprise two CheA/CheW baseplates sandwiching two opposed receptor arrays. We further show that cytoplasmic fragments of normally transmembrane E. coli chemoreceptors form similar sandwiched structures in the presence of molecular crowding agents. Together these results suggest that the 12-nm hexagonal architecture is fundamentally important and that sandwiching and crowding can replace the stabilizing effect of the membrane. DOI: http://dx.doi.org/10.7554/eLife.02151.001.

Bacterial chemoreceptors cluster in highly ordered, cooperative, extended arrays with a conserved architecture, but the principles that govern array assembly remain unclear. Here we show images of cellular arrays as well as selected chemoreceptor complexes reconstituted in vitro that reveal new principles of array structure and assembly. First, in every case, receptors clustered in a trimers-of-dimers configuration, suggesting this is a highly favored fundamental building block. Second, these trimers-of-receptor dimers exhibited great versatility in the kinds of contacts they formed with each other and with other components of the signaling pathway, although only one architectural type occurred in native arrays. Third, the membrane, while it likely accelerates the formation of arrays, was neither necessary nor sufficient for lattice formation. Molecular crowding substituted for the stabilizing effect of the membrane and allowed cytoplasmic receptor fragments to form sandwiched lattices that strongly resemble the cytoplasmic chemoreceptor arrays found in some bacterial species. Finally, the effective determinant of array structure seemed to be CheA and CheW, which formed a "superlattice" of alternating CheA-filled and CheA-empty rings that linked receptor trimers-of-dimer units into their native hexagonal lattice. While concomitant overexpression of receptors, CheA, and CheW yielded arrays with native spacing, the CheA occupancy was lower and less ordered, suggesting that temporal and spatial coordination of gene expression driven by a single transcription factor may be vital for full order, or that array overgrowth may trigger a disassembly process. The results described here provide new insights into the assembly intermediates and assembly mechanism of this massive macromolecular complex.

Motile bacteria sense their physical and chemical environment through highly cooperative, ordered arrays of chemoreceptors. These signalling complexes phosphorylate a response regulator which in turn governs flagellar motor reversals, driving cells towards favourable environments. The structural changes that translate chemoeffector binding into the appropriate kinase output are not known. Here, we apply high-resolution electron cryotomography to visualize mutant chemoreceptor signalling arrays in well-defined kinase activity states. The arrays were well ordered in all signalling states, with no discernible differences in receptor conformation at 2-3 nm resolution. Differences were observed, however, in a keel-like density that we identify here as CheA kinase domains P1 and P2, the phosphorylation site domain and the binding domain for response regulator target proteins. Mutant receptor arrays with high kinase activities all exhibited small keels and high proteolysis susceptibility, indicative of mobile P1 and P2 domains. In contrast, arrays in kinase-off signalling states exhibited a range of keel sizes. These findings confirm that chemoreceptor arrays do not undergo large structural changes during signalling, and suggest instead that kinase activity is modulated at least in part by changes in the mobility of key domains.

The apparent handedness of an EM-tomography reconstruction depends on a number of conventions and can be confused in many ways. As the number of different hardware and software combinations being used for electron tomography continue to climb, and the reconstructions being produced reach higher and higher resolutions, the need to verify the hand of the results has increased. Here we enumerate various steps in a typical tomography experiment that affect handedness and show that DNA origami gold nanoparticle helices can be used as convenient and fail-safe handedness standards.

In eukaryotes, the differentiation of cellular extensions such as cilia or neuronal axons depends on the partitioning of proteins to distinct plasma membrane domains by specialized diffusion barriers. However, examples of this compartmentalization strategy are still missing for prokaryotes, although complex cellular architectures are also widespread among this group of organisms. This study reveals the existence of a protein-mediated membrane diffusion barrier in the stalked bacterium Caulobacter crescentus. We show that the Caulobacter cell envelope is compartmentalized by macromolecular complexes that prevent the exchange of both membrane and soluble proteins between the polar stalk extension and the cell body. The barrier structures span the cross-sectional area of the stalk and comprise at least four proteins that assemble in a cell-cycle-dependent manner. Their presence is critical for cellular fitness because they minimize the effective cell volume, allowing faster adaptation to environmental changes that require de novo synthesis of envelope proteins.

Chemoreceptor arrays are supramolecular transmembrane machines of unknown structure that allow bacteria to sense their surroundings and respond by chemotaxis. We have combined X-ray crystallography of purified proteins with electron cryotomography of native arrays inside cells to reveal the arrangement of the component transmembrane receptors, histidine kinases (CheA) and CheW coupling proteins. Trimers of receptor dimers lie at the vertices of a hexagonal lattice in a "two-facing-two" configuration surrounding a ring of alternating CheA regulatory domains (P5) and CheW couplers. Whereas the CheA kinase domains (P4) project downward below the ring, the CheA dimerization domains (P3) link neighboring rings to form an extended, stable array. This highly interconnected protein architecture underlies the remarkable sensitivity and cooperative nature of transmembrane signaling in bacterial chemotaxis.

Bacterial chemoreceptors cluster into exquisitively sensitive, tunable, highly ordered, polar arrays. While these arrays serve as paradigms of cell signalling in general, it remains unclear what conformational changes transduce signals from the periplasmic tips, where attractants and repellents bind, to the cytoplasmic signalling domains. Conflicting reports support and contest the hypothesis that activation causes large changes in the packing arrangement of the arrays, up to and including their complete disassembly. Using electron cryotomography, here we show that in Caulobacter crescentus, chemoreceptor arrays in cells grown in different media and immediately after exposure to the attractant galactose all exhibit the same 12 nm hexagonal packing arrangement, array size and other structural parameters. ΔcheB and ΔcheR mutants mimicking attractant- or repellent-bound states prior to adaptation also show the same lattice structure. We conclude that signal transduction and amplification must be accomplished through only small, nanoscale conformational changes.

The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species.

How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (> 80 nm) helical filaments exist near or along either surface of the inner membrane. We also use correlated cryo-fluorescent light microscopy (cryo-fLM) and electron cryo-tomography (ECT) to identify cytoplasmic bundles of MreB, showing that MreB filaments are detectable by ECT. In light of these results, the structure and function of MreB must be reconsidered: instead of acting as a large, rigid scaffold that localizes cell-wall synthetic machinery, moving MreB complexes may apply tension to growing peptidoglycan strands to ensure their orderly, linear insertion.

In bacteria, cytokinesis is dependent on lytic enzymes that facilitate remodelling of the cell wall during constriction. In this work, we identify a thus far uncharacterized periplasmic protein, DipM, that is required for cell division and polarity in Caulobacter crescentus. DipM is composed of four peptidoglycan binding (LysM) domains and a C-terminal lysostaphin-like (LytM) peptidase domain. It binds to isolated murein sacculi in vitro, and is recruited to the site of constriction through interaction with the cell division protein FtsN. Mutational analyses showed that the LysM domains are necessary and sufficient for localization of DipM, while its peptidase domain is essential for function. Consistent with a role in cell wall hydrolysis, DipM was found to interact with purified murein sacculi in vitro and to induce cell lysis upon overproduction. Its inactivation causes severe defects in outer membrane invagination, resulting in a significant delay between cytoplasmic compartmentalization and final separation of the daughter cells. Overall, these findings indicate that DipM is a periplasmic component of the C. crescentus divisome that facilitates remodelling of the peptidoglycan layer and, thus, coordinated constriction of the cell envelope during the division process.

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy. Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (approximately 4nm).(1, 2) In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (< -180 degrees C). For slender cells (up to approximately 500 nm in thickness(3)), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.(1, 2) In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.

Bacterial cell morphogenesis requires coordination among multiple cellular systems, including the bacterial cytoskeleton and the cell wall. In the vibrioid bacterium Caulobacter crescentus, the intermediate filament-like protein crescentin forms a cell envelope-associated cytoskeletal structure that controls cell wall growth to generate cell curvature. We undertook a genetic screen to find other cellular components important for cell curvature. Here we report that deletion of a gene (wbqL) involved in the lipopolysaccharide (LPS) biosynthesis pathway abolishes cell curvature. Loss of WbqL function leads to the accumulation of an aberrant O-polysaccharide species and to the release of the S layer in the culture medium. Epistasis and microscopy experiments show that neither S-layer nor O-polysaccharide production is required for curved cell morphology per se but that production of the altered O-polysaccharide species abolishes cell curvature by apparently interfering with the ability of the crescentin structure to associate with the cell envelope. Our data suggest that perturbations in a cellular pathway that is itself fully dispensable for cell curvature can cause a disruption of cell morphogenesis, highlighting the delicate harmony among unrelated cellular systems. Using the wbqL mutant, we also show that the normal assembly and growth properties of the crescentin structure are independent of its association with the cell envelope. However, this envelope association is important for facilitating the local disruption of the stable crescentin structure at the division site during cytokinesis.

The cytoskeleton has a key function in the temporal and spatial organization of both prokaryotic and eukaryotic cells. Here, we report the identification of a new class of polymer-forming proteins, termed bactofilins, that are widely conserved among bacteria. In Caulobacter crescentus, two bactofilin paralogues cooperate to form a sheet-like structure lining the cytoplasmic membrane in proximity of the stalked cell pole. These assemblies mediate polar localization of a peptidoglycan synthase involved in stalk morphogenesis, thus complementing the function of the actin-like cytoskeleton and the cell division machinery in the regulation of cell wall biogenesis. In other bacteria, bactofilins can establish rod-shaped filaments or associate with the cell division apparatus, indicating considerable structural and functional flexibility. Bactofilins polymerize spontaneously in the absence of additional cofactors in vitro, forming stable ribbon- or rod-like filament bundles. Our results suggest that these structures have evolved as an alternative to intermediate filaments, serving as versatile molecular scaffolds in a variety of cellular pathways.

Chemoreceptors are key components of the high-performance signal transduction system that controls bacterial chemotaxis. Chemoreceptors are typically localized in a cluster at the cell pole, where interactions among the receptors in the cluster are thought to contribute to the high sensitivity, wide dynamic range, and precise adaptation of the signaling system. Previous structural and genomic studies have produced conflicting models, however, for the arrangement of the chemoreceptors in the clusters. Using whole-cell electron cryo-tomography, here we show that chemoreceptors of different classes and in many different species representing several major bacterial phyla are all arranged into a highly conserved, 12-nm hexagonal array consistent with the proposed "trimer of dimers" organization. The various observed lengths of the receptors confirm current models for the methylation, flexible bundle, signaling, and linker sub-domains in vivo. Our results suggest that the basic mechanism and function of receptor clustering is universal among bacterial species and was thus conserved during evolution.

A new method for recording both fluorescence and cryo-EM images of small bacterial cells was developed and used to identify chemoreceptor arrays in cryotomograms of intact Caulobacter crescentus cells. We show that in wild-type cells preserved in a near-native state, the chemoreceptors are hexagonally packed with a lattice spacing of 12 nm, just a few tens of nanometers away from the flagellar motor that they control. The arrays were always found on the convex side of the cell, further demonstrating that Caulobacter cells maintain dorsal/ventral as well as anterior/posterior asymmetry. Placing the known crystal structure of a trimer of receptor dimers at each vertex of the lattice accounts well for the density and agrees with other constraints. Based on this model for the arrangement of receptors, there are between one and two thousand receptors per array.

Cell polarization is an integral part of many unrelated bacterial processes. How intrinsic cell polarization is achieved is poorly understood. Here, we provide evidence that Caulobacter crescentus uses a multimeric pole-organizing factor (PopZ) that serves as a hub to concurrently achieve several polarizing functions. During chromosome segregation, polar PopZ captures the ParB*ori complex and thereby anchors sister chromosomes at opposite poles. This step is essential for stabilizing bipolar gradients of a cell division inhibitor and setting up division near midcell. PopZ also affects polar stalk morphogenesis and mediates the polar localization of the morphogenetic and cell cycle signaling proteins CckA and DivJ. Polar accumulation of PopZ, which is central to its polarizing activity, can be achieved independently of division and does not appear to be dictated by the pole curvature. Instead, evidence suggests that localization of PopZ largely relies on PopZ multimerization in chromosome-free regions, consistent with a self-organizing mechanism.

A newly characterized archaeal rudivirus Stygiolobus rod-shaped virus (SRV), which infects a hyperthermophilic Stygiolobus species, was isolated from a hot spring in the Azores, Portugal. Its virions are rod-shaped, 702 (+/- 50) by 22 (+/- 3) nm in size, and nonenveloped and carry three tail fibers at each terminus. The linear double-stranded DNA genome contains 28,096 bp and an inverted terminal repeat of 1,030 bp. The SRV shows morphological and genomic similarities to the other characterized rudiviruses Sulfolobus rod-shaped virus 1 (SIRV1), SIRV2, and Acidianus rod-shaped virus 1, isolated from hot acidic springs of Iceland and Italy. The single major rudiviral structural protein is shown to generate long tubular structures in vitro of similar dimensions to those of the virion, and we estimate that the virion constitutes a single, superhelical, double-stranded DNA embedded into such a protein structure. Three additional minor conserved structural proteins are also identified. Ubiquitous rudiviral proteins with assigned functions include glycosyl transferases and a S-adenosylmethionine-dependent methyltransferase, as well as a Holliday junction resolvase, a transcriptionally coupled helicase and nuclease implicated in DNA replication. Analysis of matches between known crenarchaeal chromosomal CRISPR spacer sequences, implicated in a viral defense system, and rudiviral genomes revealed that about 10% of the 3,042 unique acidothermophile spacers yield significant matches to rudiviral genomes, with a bias to highly conserved protein genes, consistent with the widespread presence of rudiviruses in hot acidophilic environments. We propose that the 12-bp indels which are commonly found in conserved rudiviral protein genes may be generated as a reaction to the presence of the host CRISPR defense system.

Two Gram-positive, coccoid, non-spore-forming bacteria (strains 3-M5-R-4(T) and 3-M5-R-7), cells of which formed diploid, tetrad and cluster arrangements, were isolated from air of the Regenerative Enclosed Life Support Module Simulator system. On the basis of 16S rRNA gene sequence similarity, these strains were shown to belong to the family Intrasporangiaceae and were related to members of the genus Tetrasphaera, with similarities to the seven known species of the genus Tetrasphaera of 96.71-97.76 %. The fatty acid profile supported affiliation of these novel isolates to the genus Tetrasphaera, although larger amounts of octadecanoic acid (C(18 : 0)) and cis-9-octadecenoic acid (C(18 : 1)) were observed in the isolates, thus enabling them to be differentiated from other Tetrasphaera species. In addition, DNA-DNA hybridization studies indicated that these strains belonged to a novel species that could be readily distinguished from its nearest neighbour, Tetrasphaera japonica DSM 13192(T), which had less than 20 % DNA-DNA relatedness. Physiological and biochemical tests showed few phenotypic differences, but genotypic analysis enabled these gelatin-liquefying strains to be differentiated from the seven Tetrasphaera species. The strains described in this study therefore represent a novel species, for which the name Tetrasphaera remsis sp. nov. is proposed; the type strain is 3-M5-R-4(T) (=ATCC BAA-1496(T) =CIP 109413(T)).

Electron cryotomography is an emerging technology that enables thin samples, including small intact prokaryotic cells, to be imaged in three dimensions in a near-native 'frozen-hydrated' state to a resolution sufficient to recognize very large macromolecular complexes in situ. Following years of visionary technology development by a few key pioneers, several laboratories are now using the technique to produce biological results of major significance in the field of prokaryotic ultrastructure. Recent discoveries have included the surprising generality and complexity of the cytoskeleton, the connectivity of key membrane compartments, the location and architecture of large macromolecular machines such as the ribosome and flagellar motors, and the structure of some extraordinary external appendages. Through further technology development, identification of the most revealing model systems and sustained effort, electron cryotomography is poised to help resolve many fundamentally important questions about prokaryotic ultrastructure.

Electron cryotomography is the highest-resolution structural technique currently available that can be applied to unique objects such as flexible large protein complexes, irregular viruses, organelles and small cells. Specimens are preserved in a near-native, 'frozen-hydrated' state by vitrification. The thickness of the vitreous ice must be optimized for each specimen, and gold fiducials are typically added to facilitate image alignment. Here, we describe in detail our protocols for electron cryotomography sample preparation including (i) introduction of fiducial markers into the sample and (ii) sample vitrification. Because we almost exclusively use an automated, climate-controlled plunge-freezing device (the FEI Vitrobot) to vitrify our samples, we discuss its operation and parameters in detail. A session in which eight grids are prepared takes 1.5-2 h.

While the absence of any cytoskeleton was once recognized as a distinguishing feature of prokaryotes, it is now clear that a number of different bacterial proteins do form filaments in vivo. Despite the critical roles these proteins play in cell shape, genome segregation and cell division, molecular mechanisms have remained obscure in part for lack of electron microscopy-resolution images where these filaments can be seen acting within their cellular context. Here, electron cryotomography was used to image the widely studied model prokaryote Caulobacter crescentus in an intact, near-native state, producing three-dimensional reconstructions of these cells with unprecedented clarity and fidelity. We observed many instances of large filament bundles in various locations throughout the cell and at different stages of the cell cycle. The bundles appear to fall into four major classes based on shape and location, referred to here as 'inner curvature', 'cytoplasmic', 'polar' and 'ring-like'. In an attempt to identify at least some of the filaments, we imaged cells where crescentin and MreB filaments would not be present. The inner curvature and cytoplasmic bundles persisted, which together with their localization patterns, suggest that they are composed of as-yet unidentified cytoskeletal proteins. Thus bacterial filaments are frequently found as bundles, and their variety and abundance is greater than previously suspected.

Proteinaceous, hair-like appendages known as fimbriae or pili commonly extend from the surface of prokaryotic cells and serve important functions such as cell adhesion, biofilm formation, motility and DNA transfer. Here we show that a novel group of archaea from cold, sulphidic springs has developed cell surface appendages of an unexpectedly high complexity with a well-defined base-to-top organization. It represents a new class of filamentous cell appendages, for which the term 'hamus' is proposed. Each archaeal cell is surrounded by a halo of about 100 hami, which mediate strong adhesion of the cells to surfaces of different chemical composition. The hami are mainly composed of 120 kDa subunits and remained stable in a broad temperature and pH range (0-70 degrees C; 0.5-11.5). Electron microscopy and cryo-electron tomography revealed that the hamus filament possesses a helical basic structure. At periodic distances, three prickles emanate from the filament, giving it the character of industrially produced barbwire. At its distal end the hami carry a tripartite, barbed grappling hook (60 nm in diameter). The architecture of this molecular hook is reminiscent of man-made fishhooks, grapples and anchors. It appears that nature has developed a perfect mechanical nano-tool in the course of biological evolution, which also might prove useful in the field of nanobiotechnology.