Spatio-temporal control of asymmetric septum positioning during sporulation in Bacillus subtilis.

During sporulation, Bacillus subtilis forms an asymmetric septum, dividing the cell into two compartments, a mother cell and a forespore. The site of asymmetric septation is linked to the membrane where FtsZ and SpoIIE initiate the formation of the Z-ring and the E-ring, respectively. These rings then serve as a scaffold for the other cell division and peptidoglycan synthesizing proteins needed to build the septum. However, despite decades of research, not enough is known about how the asymmetric septation site is determined. Here, we identified and characterized the interaction between SpoIIE and RefZ. We show that these two proteins transiently colocalize during the early stages of asymmetric septum formation when RefZ localizes primarily from the mother cell side of the septum. We propose that these proteins and their interplay with the spatial organization of the chromosome play a role in controlling asymmetric septum positioning.

Cardiolipin-Containing Lipid Membranes Attract the Bacterial Cell Division Protein DivIVA.

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.

Linking the Peptidoglycan Synthesis Protein Complex with Asymmetric Cell Division during Bacillus subtilis Sporulation.

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.

Chromate tolerance and removal of bacterial strains isolated from uncontaminated and chromium-polluted environments.

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.

Clostridial DivIVA and MinD interact in the absence of MinJ.

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.

Physical interaction and assembly of Bacillus subtilis spore coat proteins CotE and CotZ studied by atomic force microscopy.

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.

Diverse supramolecular structures formed by self-assembling proteins of the Bacillus subtilis spore coat.

Bacterial spores (endospores), such as those of the pathogens Clostridium difficile and Bacillus anthracis, are uniquely stable cell forms, highly resistant to harsh environmental insults. Bacillus subtilis is the best studied spore-former and we have used it to address the question of how the spore coat is assembled from multiple components to form a robust, protective superstructure. B. subtilis coat proteins (CotY, CotE, CotV and CotW) expressed in Escherichia coli can arrange intracellularly into highly stable macro-structures through processes of self-assembly. Using electron microscopy, we demonstrate the capacity of these proteins to generate ordered one-dimensional fibres, two-dimensional sheets and three-dimensional stacks. In one case (CotY), the high degree of order favours strong, cooperative intracellular disulfide cross-linking. Assemblies of this kind could form exquisitely adapted building blocks for higher-order assembly across all spore-formers. These physically robust arrayed units could also have novel applications in nano-biotechnology processes.

Investigating interactions of the Bacillus subtilis spore coat proteins CotY and CotZ using single molecule force spectroscopy.

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.

Study of the interactions between the key spore coat morphogenetic proteins CotE and SpoVID.

The capability of Bacillus subtilis spores to withstand extreme environmental conditions is thought to be conferred especially by their outermost proteinaceous protective layer, called the spore coat. 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. In this study we investigated the interaction between two spore coat morphogenetic proteins SpoVID and CotE. SpoVID is involved in the process of spore surface encirclement by individual coat proteins, these include CotE, which controls the assembly of the outer coat layer. Both proteins were proposed to be recruited to a common protein scaffold, but their direct association has not been previously shown. Here we studied the interactions between CotE and SpoVID in vitro for the first time by using molecule recognition force spectroscopy, which allows the detection of piconewton forces between conjugated biological pairs and also facilitates the investigation of dynamic processes. The most probable CotE-CotE unbinding force was 49.4±0.1pN at a loading rate of 3.16×10³ pN/s while that of SpoVID-CotE was 26.5±0.6pN at a loading rate of 7.8×10² pN/s. We further analyzed the interactions with the bacterial two hybrid system and pull-down experiments, which also indicate that SpoVID interacts directly with CotE. In combination with the previously identified direct contacts among SpoIVA, SpoVID and SafA, our data imply that the physical association of key morphogenetic proteins forms a basic skeleton where other coat proteins could be attached.

Control of Bacillus subtilis cell shape by RodZ.

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.

The role of lipid domains in bacterial cell processes.

Membranes are vital structures for cellular life forms. As thin, hydrophobic films, they provide a physical barrier separating the aqueous cytoplasm from the outside world or from the interiors of other cellular compartments. They maintain a selective permeability for the import and export of water-soluble compounds, enabling the living cell to maintain a stable chemical environment for biological processes. Cell membranes are primarily composed of two crucial substances, lipids and proteins. Bacterial membranes can sense environmental changes or communication signals from other cells and they support different cell processes, including cell division, differentiation, protein secretion and supplementary protein functions. The original fluid mosaic model of membrane structure has been recently revised because it has become apparent that domains of different lipid composition are present in both eukaryotic and prokaryotic cell membranes. In this review, we summarize different aspects of phospholipid domain formation in bacterial membranes, mainly in Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. We describe the role of these lipid domains in membrane dynamics and the localization of specific proteins and protein complexes in relation to the regulation of cellular function.

The structure and interactions of SpoIISA and SpoIISB, a toxin-antitoxin system in Bacillus subtilis.

Spore formation in Bacillus subtilis begins with an asymmetric cell division, following which differential gene expression is established by alternative compartment-specific RNA polymerase σ factors. The spoIISAB operon of B. subtilis was identified as a locus whose mutation leads to increased activity of the first sporulation-specific sigma factor, σ(F). Inappropriate spoIISA expression causes lysis of vegetatively growing B. subtilis cells and Escherichia coli cells when expressed heterologously, effects that are countered by co-expression of spoIISB, identifying SpoIISA-SpoIISB as a toxin-antitoxin system. SpoIISA has three putative membrane-spanning segments and a cytoplasmic domain. Here, the crystal structure of a cytoplasmic fragment of SpoIISA (CSpoIISA) in complex with SpoIISB has been determined by selenomethionine-multiwavelength anomalous dispersion phasing to 2.5 Å spacing, revealing a CSpoIISA(2)·SpoIISB(2) heterotetramer. CSpoIISA has a single domain α/ß structure resembling a GAF domain with an extended α-helix at its N terminus. The two CSpoIISA protomers form extensive interactions through an intermolecular four-helix bundle. Each SpoIISB chain is highly extended and lacking tertiary structure. The SpoIISB chains wrap around the CSpoIISA dimer, forming extensive interactions with both CSpoIISA protomers. CD spectroscopy experiments indicate that SpoIISB is a natively disordered protein that adopts structure only in the presence of CSpoIISA, whereas surface plasmon resonance experiments revealed that the CSpoIISA·SpoIISB complex is stable with a dissociation constant in the nanomolar range. The results are interpreted in relation to sequence conservation and mutational data, and possible mechanisms of cell killing by SpoIISA are discussed.

An oscillating Min system in Bacillus subtilis influences asymmetrical septation during sporulation.

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.

Implantation of Bacillus pseudomycoides Chromate Transporter Increases Chromate Tolerance in Bacillus subtilis.

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 pseudomycoides environmental strain into non-pathogenic but only transiently Cr tolerant Bacillus subtilis strain. In our study, we compared the two Bacillus spp. strains harboring evolutionarily diverged chromate efflux proteins. We have found that individual cells of the Cr-resistant B. pseudomycoides environmental strain accumulate less Cr than the cells of B. subtilis strain. Further, we found that survival of the B. subtilis strain during the Cr stress can be increased by the introduction of the chromate transporter from the Cr resistant environmental strain into its genome. Additionally, the expression of B. pseudomycoides chromate transporter ChrA in B. subtilis seems to be activated by the presence of chromate, hinting at versatility of Cr-efflux proteins. This study outlines the future direction for increasing the Cr-tolerance of non-pathogenic species and safe bioremediation using soil bacteria.

A novel phage-encoded endolysin EN534-C active against clinical strain Streptococcus agalactiae GBS.

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.

Megahertz pulse trains enable multi-hit serial femtosecond crystallography experiments at X-ray free electron lasers.

The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 µs, yield up to 2.1 Å resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX.

Special Issue "Bacillus subtilis as a Model Organism to Study Basic Cell Processes".

Bacillus subtilis has served as a model microorganism for many decades [...].

Interactions of Bacillus subtilis Basement Spore Coat Layer Proteins.

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.

Diffraction pattern of Bacillus subtilis CotY spore coat protein 2D crystals.

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 Å and 5.0 Å, respectively. Obtaining diffractions of 2D crystals pave the way to determination of 3D structure of coat proteins by using strong X-ray sources.

The XBI BioLab for life science experiments at the European XFEL.

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.