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Streptococcus agalactiae (Group B Streptococcus, GBS) is primarily known as a major neonatal pathogen. In adults, these bacteria often colonize the gastrointestinal and urogenital tracts. Treatment of infections using antibiotics is often complicated by recurrences caused by multi-resistant streptococci. Endolysin EN534 from prophage A2 of human isolate Streptococcus agalactiae KMB-534 has a modular structure consisting of two terminal catalytic domains, amidase_3 and CHAP, and one central binding domain, LysM. The EN534 gene was cloned into an expression vector, and the corresponding recombinant protein EN534-C was expressed in Escherichia coli in a soluble form and isolated by affinity chromatography. The lytic activity of this endolysin was tested on cell wall substrates from different GBS serotypes, B. subtilis, L. jensenii, and E. coli. The enzyme lysed streptococci, but not beneficial vaginal lactobacilli. The isolated protein is stable in a temperature range of 20-37 °C. Calcium ions enhanced the activity of the enzyme in the pH range from 5.0 to 8.0. The exolytic activity of EN534-C was observed by time-lapse fluorescence microscopy on a S. agalactiae CCM 6187 substrate. Recombinant endolysin EN534-C may have the potential to become an antimicrobial agent for the treatment of S. agalactiae infections.

An overview of a unique life science facility at the European XFEL is provided, and its importance in sample preparation, characterization and analysis prior to measurements at the different European XFEL instruments is discussed. The capabilities that the facility can provide for the research community are highlighted, alongside examples of recent successful studies., The science of X-ray free-electron lasers (XFELs) critically depends on the performance of the X-ray laser and on the quality of the samples placed into the X-ray beam. The stability of biological samples is limited and key biomolecular transformations occur on short timescales. Experiments in biology require a support laboratory in the immediate vicinity of the beamlines. The XBI BioLab of the European XFEL (XBI denotes XFEL Biology Infrastructure) is an integrated user facility connected to the beamlines for supporting a wide range of biological experiments. The laboratory was financed and built by a collaboration between the European XFEL and the XBI User Consortium, whose members come from Finland, Germany, the Slovak Republic, Sweden and the USA, with observers from Denmark and the Russian Federation. Arranged around a central wet laboratory, the XBI BioLab provides facilities for sample preparation and scoring, laboratories for growing prokaryotic and eukaryotic cells, a Bio Safety Level 2 laboratory, sample purification and characterization facilities, a crystallization laboratory, an anaerobic laboratory, an aerosol laboratory, a vacuum laboratory for injector tests, and laboratories for optical microscopy, atomic force microscopy and electron microscopy. Here, an overview of the XBI facility is given and some of the results of the first user experiments are highlighted.

Graphical abstract, Here we use singe-molecule optical proteomics and computational analysis of live cell bacterial images, using millisecond super-resolved tracking and quantification of fluorescently labelled protein SpoIIE in single live Bacillus subtilis bacteria to understand its crucial role in cell development. Asymmetric cell division during sporulation in Bacillus subtilis presents a model system for studying cell development. SpoIIE is a key integral membrane protein phosphatase that couples morphological development to differential gene expression. However, the basic mechanisms behind its operation remain unclear due to limitations of traditional tools and technologies. We instead used advanced single-molecule imaging of fluorescently tagged SpoIIE in real time on living cells to reveal vital changes to the patterns of expression, localization, mobility and stoichiometry as cells undergo asymmetric cell division then engulfment of the smaller forespore by the larger mother cell. We find, unexpectedly, that SpoIIE forms tetramers capable of cell- and stage-dependent clustering, its copy number rising to ~ 700 molecules as sporulation progresses. We observed that slow moving SpoIIE clusters initially located at septa are released as mobile clusters at the forespore pole as phosphatase activity is manifested and compartment-specific RNA polymerase sigma factor, σF, becomes active. Our findings reveal that information captured in its quaternary organization enables one protein to perform multiple functions, extending an important paradigm for regulatory proteins in cells. Our findings more generally demonstrate the utility of rapid live cell single-molecule optical proteomics for enabling mechanistic insight into the complex processes of cell development during the cell cycle.

Chromium of anthropogenic origin contaminates the environment worldwide. The toxicity of chromium, a group I human carcinogen, is greatest when it is in a hexavalent oxidation state, Cr(VI). Cr(VI) is actively transported into the cell, triggering oxidative damage intracellularly. Due to the abundance of unspecific intracellular reductants, any microbial species is capable of bio-transformation of toxic Cr(VI) to innocuous Cr(III), however, this process is often lethal. Only some bacterial species are capable of sustaining the vegetative growth in the presence of a high concentration of Cr(VI) and thus operate as self-sustainable bioremediation agents. One of the successful microbial Cr(VI) detoxification strategies is the activation of chromate efflux pumps. This work describes transplantation of the chromate efflux pump from the potentially pathogenic but highly Cr resistant

Bacillus subtilis is a rod-shaped bacterium which divides precisely at mid-cell during vegetative growth. Unlike Escherichia coli, another model organism used for studying cell division, B. subtilis can also divide asymmetrically during sporulation, the simplest cell differentiation process. The asymmetrically positioned sporulation septum serves as a morphological foundation for establishing differential gene expression in the smaller forespore and larger mother cell. Both vegetative and sporulation septation events are fine-tuned with cell cycle, and placement of both septa are highly precise. We understand in some detail how this is achieved during vegetative growth but have limited information about how the asymmetric septation site is determined during sporulation.

Bacillus subtilis endospores are exceptionally resistant cells encircled by two protective layers: a petidoglycan layer, termed the cortex, and the spore coat, a proteinaceous layer. The formation of both structures depends upon the proper assembly of a basement coat layer, which is composed of two proteins, SpoIVA and SpoVM. The present work examines the interactions of SpoIVA and SpoVM with coat proteins recruited to the spore surface during the early stages of coat assembly. We showed that the alanine racemase YncD associates with two morphogenetic proteins, SpoIVA and CotE. Mutant spores lacking the yncD gene were less resistant against wet heat and germinated to a greater extent than wild-type spores in the presence of micromolar concentrations of l-alanine. In seeking a link between the coat and cortex formation, we investigated the interactions between SpoVM and SpoIVA and the proteins essential for cortex synthesis and found that SpoVM interacts with a penicillin-binding protein, SpoVD, and we also demonstrated that SpoVM is crucial for the proper localization of SpoVD. This study shows that direct contacts between coat morphogenetic proteins with a complex of cortex-synthesizing proteins could be one of the tools by which bacteria couple cortex and coat formation.

Communications Physics 3(1), 97 (2020). doi:10.1038/s42005-020-0362-y, The emergence of high repetition-rate X-ray free-electron lasers (XFELs) powered by superconducting accelerator technology enables the measurement of significantly more experimental data per day than was previously possible. The European XFEL is expected to provide 27,000 pulses per second, over two orders of magnitude more than any other XFEL. The increased pulse rate is a key enabling factor for single-particle X-ray diffractive imaging, which relies on averaging the weak diffraction signal from single biological particles. Taking full advantage of this new capability requires that all experimental steps, from sample preparation and delivery to the acquisition of diffraction patterns, are compatible with the increased pulse repetition rate. Here, we show that single-particle imaging can be performed using X-ray pulses at megahertz repetition rates. The results obtained pave the way towards exploiting high repetition-rate X-ray free-electron lasers for single-particle imaging at their full repetition rate., Published by Springer Nature, London

This study revealed a novel, potentially conserved mechanism involved in the biosynthesis of microbial natural products, exemplified by the mitochondrial active depsipeptide cereulide. Similar to other bioactive substances, such as the last-resort antibiotics vancomycin and daptomycin, the antitumor drug cryptophycin or the cholesterol-lowering agent lovastatin, cereulide is synthesized nonribosomally by multienzyme machinery, requiring the concerted actions of multiple proteins to ensure correct product assembly. Given the importance of microbial secondary metabolites in human and veterinary medicine, it is critical to understand how these processes are orchestrated within the host cells. By revealing that tethering of a biosynthetic enzyme to the cell membrane by an ABC transporter is essential for nonribosomal peptide production, our study provides novel insights into synthesis of microbial secondary metabolites, which could contribute to isolation of novel compounds from cryptic secondary metabolite clusters or improve the yield of produced pharmaceuticals., Nonribosomal peptide synthetases (NRPSs) and polyketide synthetases (PKSs) play a pivotal role in the production of bioactive natural products, such as antibiotics and cytotoxins. Despite biomedical and pharmaceutical importance, the molecular mechanisms and architectures of these multimodular enzyme complexes are not fully understood. Here, we report on an ABC transporter that forms a vital part of the nonribosomal peptide biosynthetic machinery. Emetic Bacillus cereus produces the highly potent, mitochondrial active nonribosomal depsipeptide cereulide, synthesized by the NRPS Ces. The ces gene locus includes, next to the structural cesAB genes, a putative ABC transporter, designated cesCD. Our study demonstrates that tethering of CesAB synthetase to the cell membrane by CesCD is critical for peptide assembly. In vivo studies revealed that CesAB colocalizes with CesCD on the cell membrane, suggesting direct involvement of this ABC transporter in the biosynthesis of a nonribosomal peptide. Mutation of cesCD, disrupting the assembly of the CesCD complex, resulted in decreased interaction with CesAB and, as a consequence, negatively affected cereulide biosynthesis. Specific domains within CesAB synthetase interacting with CesC were identified. Furthermore, we demonstrated that the structurally similar BerAB transporter from Bacillus thuringiensis complements CesCD function in cereulide biosynthesis, suggesting that the direct involvement of ABC transporter in secondary metabolite biosynthesis could be a widespread mechanism. In summary, our study revealed a novel, noncanonical function for ABC transporter, which is essential for megaenzyme functionality of NRPS. The new insights into natural product biosynthesis gained may facilitate the discovery of new metabolites with bioactive potential.

Peptidoglycan is generally considered one of the main determinants of cell shape in bacteria. In rod-shaped bacteria, cell elongation requires peptidoglycan synthesis to lengthen the cell wall. In addition, peptidoglycan is synthesized at the division septum during cell division. Sporulation of Bacillus subtilis begins with an asymmetric cell division. Formation of the sporulation septum requires almost the same set of proteins as the vegetative septum, however, these two septa are significantly different. In addition to their differences in localization, the sporulation septum is thinner and it contains SpoIIE, a crucial sporulation specific protein. Here we show that peptidoglycan biosynthesis is linked to the cell division machinery during sporulation septum formation. We detected a direct interaction between SpoIIE and GpsB and found that both proteins co-localize during the early stages of asymmetric septum formation. We propose that SpoIIE is part of a multi-protein complex which includes GpsB, other division proteins and peptidoglycan synthesis proteins, and could provide a link between the peptidoglycan synthesis machinery and the complex morphological changes required for forespore formation during B. subtilis sporulation.

DivIVA is a protein initially identified as a spatial regulator of cell division in the model organism Bacillus subtilis, but its homologues are present in many other Gram-positive bacteria, including Clostridia species. Besides its role as topological regulator of the Min system during bacterial cell division, DivIVA is involved in chromosome segregation during sporulation, genetic competence, and cell wall synthesis. DivIVA localizes to regions of high membrane curvature, such as the cell poles and cell division site, where it recruits distinct binding partners. Previously, it was suggested that negative curvature sensing is the main mechanism by which DivIVA binds to these specific regions. Here, we show that Clostridioides difficile DivIVA binds preferably to membranes containing negatively charged phospholipids, especially cardiolipin. Strikingly, we observed that upon binding, DivIVA modifies the lipid distribution and induces changes to lipid bilayers containing cardiolipin. Our observations indicate that DivIVA might play a more complex and so far unknown active role during the formation of the cell division septal membrane.

© 2019, Springer Nature B.V. Investigation of bacterial chromate tolerance has mostly focused on strains originating from polluted sites. In the present study, we isolated 33 chromate tolerant strains from diverse environments harbouring varying concentrations of chromium (Cr). All of these strains were able to grow on minimal media with at least 2 mM hexavalent chromium (Cr(VI)) and their classification revealed that they belonged to 12 different species and 8 genera, with a majority (n = 20) being affiliated to the Bacillus cereus group. Selected B. cereus group strains were further characterised for their chromate tolerance level and the ability to remove toxic Cr(VI) from solution. A similar level of chromate tolerance was observed in isolates originating from environments harbouring high or low Cr. Reference B. cereus strains exhibited the same Cr(VI) tolerance which indicates that a high chromate tolerance could be an intrinsic group characteristic. Cr(VI) removal varied from 22.9% (strain PCr2a) to 98.5% (strain NCr4). Strains NCr1a and PCr12 exhibited the ability to grow to the greatest extent in Cr(VI) containing media (maximum growth of 65.3% and 64.9% relative to that in the absence of Cr(VI), respectively) accompanied with high chromate removal activity (73.7% and 74.4%, respectively), making them prime candidates for the investigation of chromate tolerance mechanisms in Gram-positive bacteria and Cr(VI) bioremediation applications.

Bacillus subtilis spore coat is a bacterial proteinaceous structure with amazing characteristics of self-organization, unique resiliency, toughness and flexibility in the same time. The spore coat represents a complex multilayered protein structure which is composed of over 80 coat proteins. Some of these proteins form two dimensional crystal structures who’s low resolution ternary structure as was determined by electron microscopy. However, there are no 3D structure of these proteins known, due to a problem of preparing 3D crystals which could be analyzed by synchrotron X-ray sources. In the present study, Grazing-Incidence Wide-Angle X-ray Scattering (GIWAXS) was applied to investigate a diffraction pattern of CotY 2D crystals formed on Langmuir monolayer films. We observed two distinct diffraction rings and their position corresponds to a structure with the lattice spacing of 10.6 A and 5.0 A, respectively. Obtaining diffractions of 2D crystals pave the way to determination of 3D structure of coat proteins by using strong X-ray sources.

The spore of Bacillus subtilis, a dormant type of cell, is surrounded by a complex multilayered protein structure known as the coat. It is composed of over 70 proteins and essential for the spore to withstand extreme environmental conditions and allow germination under favorable conditions. However, understanding how the properties of the coat arise from the interactions among all these proteins is an important challenge. Moreover, many specific protein-protein interactions among the coat proteins are crucial for coat assembly. In this study, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) was applied to investigate the interaction as a dynamic process between two morphogenetic coat proteins, CotE and CotZ. The unbinding force and kinetic parameters characterizing the interaction between CotE and CotZ were obtained. It is found that there is a strong affinity between CotE and CotZ. Furthermore, the assembly behaviors of CotE and CotZ, individually or in combination, were studied by AFM at solid-liquid interfaces. Our results revealed that CotE-CotZ assembly is dependent on their molar ratios and the interaction between CotE and CotZ involves in the CotE-CotZ assembly.

Bacterial nanotubes are membranous structures that have been reported to function as conduits between cells to exchange DNA, proteins, and nutrients. Here, we investigate the morphology and formation of bacterial nanotubes using Bacillus subtilis. We show that nanotube formation is associated with stress conditions, and is highly sensitive to the cells' genetic background, growth phase, and sample preparation methods. Remarkably, nanotubes appear to be extruded exclusively from dying cells, likely as a result of biophysical forces. Their emergence is extremely fast, occurring within seconds by cannibalizing the cell membrane. Subsequent experiments reveal that cell-to-cell transfer of non-conjugative plasmids depends strictly on the competence system of the cell, and not on nanotube formation. Our study thus supports the notion that bacterial nanotubes are a post mortem phenomenon involved in cell disintegration, and are unlikely to be involved in cytoplasmic content exchange between live cells.

Spore formation following asymmetric cell division in Bacillus subtilis offers a model system to study development, morphogenesis and signal transduction in more complex organisms. Extensive biochemical and genetic details of its sporulation factors are known, however, the molecular mechanisms by which asymmetry is generated remain unclear. A crucial membrane phosphatase, SpoIIE, couples gene regulation to morphology changes, but how it performs different functions dependent on cell stage is unknown. We addressed this puzzle using high-speed single-molecule fluorescence microscopy on live B. subtilis expressing genomically encoded SpoIIE fluorescent protein fusions during sporulation. Copy number analysis indicated a few tens of SpoIIE at sporulation onset increasing to 400-600 molecules per cell following asymmetric cell division with up to 30% greater proportion in the forespore, corresponding to a concentration enhancement in the smaller forespore sufficient for differential dephosphorylation of an anti-sigma factor antagonist and activation of the forespore specific transcription factor, σF. Step-wise photobleach analysis indicates that SpoIIE forms tetramers capable of reversible oligomerisation to form clusters correlated with stage-specific functions. Specifically, low mobility SpoIIE clusters which initially localize to the asymmetric septum are released as mobile SpoIIE clusters around the forespore when phosphatase activity is manifested. SpoIIE is subsequently recaptured at the septum in a SpoIIQ-dependent manner. After mother cell engulfment of the forespore, SpoIIE is released as a mix of higher mobility clusters and tetramers. Our findings suggest that additional information captured in the changing state of multimerization and mobility enable one protein to perform different roles at different cell stages.Significance/impactCertain bacteria undergo sporulation involving cells dividing asymmetrically. A crucial protein SpoIIE facilitates this morphological asymmetry and directly links it to asymmetry in gene expression. Here, we used specialized light microscopy, capable of observing single molecules, plus biophysics, genetics and biochemical tools, to monitor SpoIIE in single living bacteria in real time, allowing us to count how many molecules are present in different cell regions, and how mobile they are. We find that SpoIIE clusters and moves depending on development stages, indicating that it has different roles depending on other binding proteins and their cellular locations. Our results suggest that changes in molecular stoichiometry and mobility may be used as switches in more complex cell processes.

Vegetative cell division in Bacillus subtilis takes place precisely at the middle of the cell to ensure that two viable daughter cells are formed. The first event in cell division is the positioning of the FtsZ Z-ring at the correct site. This is controlled by the coordinated action of both negative and positive regulators. The existence of positive regulators has been inferred, but none have presently been identified in B. subtilis. Noc and the Min system belong to negative regulators; Noc prevents division from occurring over the chromosomes, and the Min system inhibits cell division at the poles. Here we report that the morphogenic protein, RodZ, an essential cell shape determinant, is also required for proper septum positioning during vegetative growth. In rodZ mutant cells, the vegetative septum is positioned off center, giving rise to small, round, DNA-containing cells. Searching for the molecular mechanism giving rise to this phenotype led us to discover that RodZ directly interacts with MinJ. We hypothesize that RodZ may aid the Min system in preventing non-medial vegetative division.

Spores formed by Bacillus subtilis are surrounded by a protective and multilayered shell, termed the coat, which grants the spores resistance to various environmental stresses and facilitates spore germination. The spore coat consists of more than seventy different proteins, arranged into at least four distinct structural layers: the undercoat, inner coat, outer coat and crust. However, how these proteins, especially the morphogenetic proteins, interact to establish the organized, functional coat layers remains poorly understood. CotY and CotZ as the components of the crust, play a morphogenetic role in the crust assembly around the spore. In this study, the single molecule force spectroscopy was used to investigate the interaction and dynamics between CotY and CotZ at the single-molecule level. The results show that homotypic interactions of CotY and CotZ and the heterotypic interaction between CotY and CotZ exist. Furthermore, the dissociation kinetics of the complexes were studied by monitoring the relationship between the unbinding forces and the loading rates at different pulling velocities. In this way, a series of kinetic parameters regarding the three different complexes were obtained. It revealed the strong interactions between CotY and CotZ, CotY and CotY, and a relatively weak interaction of CotZ and CotZ.

Vegetative cell division in Bacillus subtilis takes place precisely at the middle of the cell to ensure that two viable daughter cells are formed. The first event in cell division is the positioning of the FtsZ Z-ring at the correct site. This is controlled by the coordinated action of both negative and positive regulators. The existence of positive regulators has been inferred, but none have presently been identified in B. subtilis. Noc and the Min system belong to negative regulators; Noc prevents division from occurring over the chromosomes, and the Min system inhibits cell division at the poles. Here we report that the morphogenic protein, RodZ, an essential cell shape determinant, is also required for proper septum positioning during vegetative growth. In rodZ mutant cells, the vegetative septum is positioned off center, giving rise to small, round, DNA-containing cells. Searching for the molecular mechanism giving rise to this phenotype led us to discover that RodZ directly interacts with MinJ. We hypothesize that RodZ may aid the Min system in preventing non-medial vegetative division.

One of the key regulators ensuring proper Z-ring placement in rod-shaped bacteria is the Min system. It does so by creating a concentration gradient of the MinC septation inhibitor along the cell axis. In Escherichia coli, this gradient is established by a MinE-mediated pole-to-pole oscillation of the MinCDE complex. In Bacillus subtilis, the creation of an inhibitory gradient relies on the MinJ and DivIVA pair of topological determinants, which target MinCD to the newly formed cell poles. Introducing the E. coli oscillating Min system into B. subtilis leads to a sporulation defect, suggesting that oscillation is incompatible with sporulation. However, Clostridia, close endospore-forming relatives of Bacilli, do encode oscillating Min homologues in various combinations together with homologues from the less dynamic B. subtilis Min system. Here we address the questions of how these two systems could exist side-by-side and how they influence one another by studying the Clostridium beijerinckii and Clostridium difficile Min systems. The toolbox of genetic manipulations and fluorescent protein fusions in Clostridia is limited, therefore B. subtilis and E. coli were chosen as heterologous systems for studying these proteins. In B. subtilis, MinD and DivIVA interact through MinJ; here, however, we discovered that the MinD and DivIVA proteins of both C. difficile, and C. beijerinckii, interact directly, which is surprising in the latter case, since that organism also encodes a MinJ homologue. We confirm this interaction using both in vitro and in vivo methods. We also show that C. beijerinckii MinJ is probably not in direct contact with DivIVACb and, unlike B. subtilis MinJ, does not mediate the MinDCb and DivIVACb interaction. Our results suggest that the Clostridia Min system uses a new mechanism of function.

Bacillus subtilis spores, representatives of an exceptionally resistant dormant cell type, are encircled by a thick proteinaceous layer called the spore coat. More than 80 proteins assemble into four distinct coat layers: a basement layer, an inner coat, an outer coat and a crust. As the spore develops inside the mother cell, spore coat proteins synthesized in the cytoplasm are gradually deposited onto the prespore surface. A small set of morphogenetic proteins necessary for spore coat morphogenesis are thought to form a scaffold to which the rest of the coat proteins are attached. Extensive localization and proteomic studies using wild type and mutant spores have revealed the arrangement of individual proteins within the spore coat layers. In this study we examined the interactions between the proteins localized to the outer coat and crust using a bacterial two hybrid system. These two layers are composed of at least 25 components. Self-interactions were observed for most proteins and numerous novel interactions were identified. The most interesting contacts are those made with the morphogenetic proteins CotE, CotY and CotZ; these could serve as a basis for understanding the specific roles of particular proteins in spore coat morphogenesis.

Endospores (also referred to as bacterial spores) are bacterial structures formed by several bacterial species of the phylum Firmicutes. Spores form as a response to environmental stress. These structures exhibit remarkable resistance to harsh environmental conditions such as exposure to heat, desiccation, and chemical oxidants. The spores include several layers of protein and peptidoglycan that surround a core harboring DNA as well as high concentrations of calcium and dipicolinic acid (DPA). A combination of scanning transmission X-ray microscopy, scanning transmission electron microscopy, and energy dispersive spectroscopy was used for the direct quantitative characterization of bacterial spores. The concentration and localization of DPA, Ca(2+) , and other elements were determined and compared for the core and cortex of spores from two distinct genera: Bacillus subtilis and Desulfotomaculum reducens. This micro-spectroscopic approach is uniquely suited for the direct study of individual bacterial spores, while classical molecular and biochemical methods access only bulk characteristics.

The bacterial cell wall ensures the structural integrity of the cell and is the main determinant of cell shape. In Bacillus subtilis, three cytoskeletal proteins, MreB, MreBH and Mbl, are thought to play a crucial role in maintaining the rod cell shape. These proteins are thought to be linked with the transmembrane proteins MreC, MreD and RodA, the peptidoglycan hydrolases, and the penicillin-binding proteins that are essential for peptidoglycan elongation. Recently, a well-conserved membrane protein RodZ was discovered in most Gram-negative and Gram-positive bacteria. This protein seems to be an additional member of the elongation complex. Here, we examine the role of RodZ in B. subtilis cells. Our results indicate that RodZ is an essential protein and that downregulation of RodZ expression causes the formation of shorter and rounder cells. We also found a direct interaction between RodZ and the cytoskeletal and morphogenetic proteins MreB, MreBH, Mbl and MreD. Taken together, we demonstrated that RodZ is an important part of the cell shape determining network in B. subtilis.

Spores of a number of clostridial species, and their resistance to thermal treatment is a major concern for the food industry. Spore resistance to wet heat is related to the level of spore hydration, which is inversely correlated with the content of calcium and dipicolinic acid (DPA) in the spore core. It is widely believed that the accumulation of DPA and calcium in the spore core is a fundamental component of the sporulation process for all endospore forming species. We have noticed heterogeneity in the heat resistance capacity and overall DPA/calcium content among the spores of several species belonging to Clostridium sensu stricto group: two C. acetobutylicum strains (DSM 792 and 1731), two C. beijerinckii strains (DSM 791 and NCIMB 8052), and a C. collagenovorans strain (DSM 3089). A C. beijerinckii strain (DSM 791) and a C. acetobutylicum strain (DSM 792) display low Ca and DPA levels. In addition, these two species, with the lowest average Ca/DPA content amongst the strains considered, also exhibit minimal heat resistance. There appears to be no correlation between the Ca/DPA content and the phylogenetic distribution of the C. acetobutylicum and C. beijerinckii species based either on the 16S rRNA or the spoVA gene. This finding suggests that a subset of Clostridium sensu stricto species produce spores with low resistance to wet heat. Additionally, analysis of individual spores using STEM-EDS and STXM revealed that DPA and calcium levels can also vary amongst individual spores in a single spore population.

Bacillus subtilis can form a spore, which is a dormant type of cell, when its external environment becomes unsuitable for vegetative growth. The spore is surrounded by a multilayered proteinaceous shell called a spore coat, which plays a crucial role in dormancy and germination. Of the over 70 proteins that form the spore coat, only a small subset of them affect its morphogenesis; they are referred to as morphogenetic proteins. How these morphogenetic proteins interact, and furthermore, how they build the ordered, functional coat layers is not well understood. Elucidating the self-assembly mechanism of individual proteins into such a complex structure may contribute to its potential use in nano-biotechnology applications for preparing highly organized, robust, and resistant proteinaceous layers. Herein, direct, noncovalent, low-affinity interactions between the spore-coat morphogenetic proteins SpoIVA, SpoVID, and SafA were studied by using single-molecule recognition force spectroscopy in vitro for the first time. Based on the real-time examination of interactions between these three proteins, a series of dynamic kinetic data were obtained. It was also observed that the SafA-SpoVID interaction was stronger than that of SafA-SpoIVA.

The Min system plays an important role in ensuring that cell division occurs at mid-cell in rod-shaped bacteria. In Escherichia coli, pole-to-pole oscillation of the Min proteins specifically inhibits polar septation. This system also prevents polar division in Bacillus subtilis during vegetative growth; however, the Min proteins do not oscillate in this organism. The Min system of B. subtilis plays a distinct role during sporulation, a process of differentiation which begins with an asymmetrical cell division. Here, we show that oscillation of the E. coli Min proteins can be reproduced following their introduction into B. subtilis cells. Further, we present evidence that the oscillatory behaviour of the Min system inhibits sporulation. We propose that an alternative Min system mechanism avoiding oscillation is evolutionarily important because oscillation of the Min system is incompatible with efficient asymmetrical septum formation and sporulation.

Sporulation in Bacillus subtilis begins with an asymmetric cell division producing two genetically identical cells with different fates. SpoIIE is a membrane protein that localizes to the polar cell division sites where it causes FtsZ to relocate from mid-cell to form polar Z-rings. Following polar septation, SpoIIE establishes compartment-specific gene expression in the smaller forespore cell by dephosphorylating the anti-sigma factor antagonist SpoIIAA, leading to the release of the RNA polymerase sigma factor σF from an inhibitory complex with the anti-sigma factor SpoIIAB. SpoIIE therefore couples morphological development to differential gene expression. Here, we determined the crystal structure of the phosphatase domain of SpoIIE to 2.6 Å spacing, revealing a domain-swapped dimer. SEC-MALLS (size-exclusion chromatography with multi-angle laser light scattering) analysis however suggested a monomer as the principal form in solution. A model for the monomer was derived from the domain-swapped dimer in which 2 five-stranded β-sheets are packed against one another and flanked by α-helices in an αββα arrangement reminiscent of other PP2C-type phosphatases. A flap region that controls access of substrates to the active site in other PP2C phosphatases is diminished in SpoIIE, and this observation correlates with the presence of a single manganese ion in the active site of SpoIIE in contrast to the two or three metal ions present in other PP2C enzymes. Mapping of a catalogue of mutational data onto the structure shows a clustering of sites whose point mutation interferes with the proper coupling of asymmetric septum formation to sigma factor activation and identifies a surface involved in intramolecular signaling., Graphical Abstract Highlights ► We have determined the crystal structure of the PP2C phosphatase domain of SpoIIE. ► We observe a single bound manganese ion in contrast to the two or three seen in other PP2C enzymes. ► An intramolecular signaling surface is identified by mapping mutational data onto the structure.

The cell wall is responsible for cell integrity and the maintenance of cell shape in bacteria. The Gram-positive bacterial cell wall consists of a thick peptidoglycan layer located on the outside of the cytoplasmic membrane. Bacterial cell membranes, like eukaryotic cell membranes, are known to contain domains of specific lipid and protein composition. Recently, using the membrane-binding fluorescent dye FM4-64, helix-like lipid structures extending along the long axis of the cell and consisting of negatively charged phospholipids were detected in the rod-shaped bacterium Bacillus subtilis. It was also shown that the cardiolipin-specific dye, nonyl acridine orange (NAO), is preferentially distributed at the cell poles and in the septal regions in both Escherichia coli and B. subtilis. These results suggest that phosphatidylglycerol is the principal component of the observed spiral domains in B. subtilis. Here, using the fluorescent dyes FM4-64 and NAO, we examined whether these lipid domains are linked to the presence of cell wall peptidoglycan. We show that in protoplasted cells, devoid of the peptidoglycan layer, helix-like lipid structures are not preserved. Specific lipid domains are also missing in cells depleted of MurG, an enzyme involved in peptidoglycan synthesis, indicating a link between lipid domain formation and peptidoglycan synthesis.

Bacterial membranes are known to form domains with specific lipid compositions and functions. Recently, using membrane binding fluorescent dyes, lipid spiral structures extending along the long axis of the cell were detected. These spirals were absent when the synthesis of phosphatidylglycerol and cardiolipin was disrupted, suggesting that the spirals are enriched in anionic phospholipids. It was also shown that the cardiolipin-specific NAO dye is preferentially distributed at the cell poles and in the septal regions. These results suggest that phoshatidylglycerol may be the principal component of the observed spiral domains. Additionally, GFP fusions of the cell division protein MinD also form spiral structures which are coincident with the lipid spirals, indicating their involvement in cell division. Here, using fluorescent dyes FM4-64 and NAO, we demonstrate the existence of lipid domains in Bacillus subtilis cells with inhibited DNA replication. The lipid domains observed are similar to those in the wild type, indicating that either formation of these domains is not affected by inhibition of replication or that structures already established are relatively stable. The results further suggest that the GFP-MinD spirals exist in these strains as well.

Pathogenic bacteria employ many strategies to overcome the host immune system for extended survival and propagation in their hosts. Components of the bacterial outer-membrane play an important role in this process. When invading the host, Gram-negative bacteria often use a strategy, known as phase variation, that involves a reversible change in antigenic determinants, frequently polysaccharides. This means that the genes encoding the outer-membrane antigens undergo reversible changes within repeated simple DNA sequence motifs. The antigenic structure of the bacterial outer-membrane is influenced by the character of the host immune system, as well as by the targets for bacterial invasion. When the selection pressure of the immune system is absent or weak, bacteria can fail to synthesise the outer-membrane antigens, which are not needed at that time. Smooth-to-rough (S-R) mutation, an economical and often irreversible process in some Gram-negative bacteria, involves the gradual shortening of the lipopolysaccharide (LPS) O-chain. Under certain conditions, e.g., propagation in embryonated eggs or cell lines, some bacteria will cease synthesis of the complete LPS O-chain because it is an energy-demanding process. A type of gradual shortening of the LPS O-chain by Coxiella burnetii, traditionally called phase variation, is used in serological tests for the diagnosis of Q fever. This review discusses the role and function of polysaccharides, especially LPS produced by some Gram-negative bacteria, in bacterial survival.

Ticks are well-known vectors for a wide range of pathogenic microorganisms. We examined the presence of Rickettsia spp., Anaplasma spp., Borrelia spp., Coxiella burnetii and Francisella tularensis in Ixodes ricinus ticks collected in central Slovakia using oligo-chip based assay. Rickettsiae were detected in 5.6% of examined ticks. Borreliae and anaplasmae were identified in 2.1% and 2.8% ticks, respectively. All tested samples were negative for presence of Coxiella burnetii and Francisella tularensis. All these results were compared with those obtained by PCR analysis, and a close correlation between them was found. In addition, rickettsiae of spotted fever group (SFG), Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato were found in ticks using genera or species-specific PCR methods. They are circulating in 10 out of 18 studied localities.

SpoIISA and SpoIISB proteins from Bacillus subtilis belong to a recently described bacterial programmed-cell death system. The current work demonstrates that the toxin-antitoxin module is also functional in Escherichia coli cells, where the expression of SpoIISA toxin leads to transient growth arrest coupled with cell lysis, and SpoIISA-induced death can be prevented by coexpression of its cognate antitoxin, SpoIISB. Escherichia coli cells appear to be able to escape the SpoIISA killing by activation of a specific, as yet unidentified protease that cleaves out the cytosolic part of the protein. Analysis of the toxic effects of the transmembrane and cytosolic portions of SpoIISA showed that neither of them separately can function as a toxin; therefore, both parts of the protein have to act in concert to exert the killing. This work also identifies genes encoding putative homologues of SpoIISA and SpoIISB proteins on chromosomes of other Bacilli species. The SpoIISA-like proteins from Bacillus anthracis and Bacillus cereus were shown to manifest the same effect on the viability of E. coli as their homologue from B. subtilis. Moreover, expression of the proposed spoIISB-like gene rescues E. coli cells from death induced by the SpoIISA homologue.

Bacillus subtilis, a Gram-positive bacterium commonly found in soil, is an excellent model organism for the study of basic cell processes, such as cell division and cell differentiation, called sporulation. In B. subtilis the essential genetic information is carried on a single circular chromosome, the correct segregation of which is crucial for both vegetative growth and sporulation. The proper completion of life cycle requires each daughter cell to obtain identical genetic information. The consequences of inaccurate chromosome segregation can lead to formation of anucleate cells, cells with two chromosomes, or cells with incomplete chromosomes. Although bacteria miss the classical eukaryotic mitotic apparatus, the chromosome segregation is undeniably an active process tightly connected to other cell processes as DNA replication and compaction. To fully understand the chromosome segregation, it is necessary to study this process in a wider context and to examine the role of different proteins at various cell life cycle stages. The life cycle of B. subtilis is characteristic by its specific cell differentiation process where, two slightly different segregation mechanisms exist, specialized in vegetative growth and in sporulation.