Imrich Barák

Lipid spirals in Bacillus subtilis and their role in cell division

The fluid mosaic model of membrane structure has been revised in recent years as it has become evident that domains of different lipid composition are present in eukaryotic and prokaryotic cells. Using membrane binding fluorescent dyes, we demonstrate the presence of lipid spirals extending along the long axis of cells of the rod‐shaped bacterium Bacillus subtilis. These spiral structures are absent from cells in which the synthesis of phosphatidylglycerol is disrupted, suggesting an enrichment in anionic phospholipids. Green fluorescent protein fusions of the cell division protein MinD also form spiral structures and these were shown by fluorescence resonance energy transfer to be coincident with the lipid spirals. These data indicate a higher level of membrane lipid organization than previously observed and a primary role for lipid spirals in determining the site of cell division in bacterial cells.

Maturation of the Coxiella burnetii parasitophorous vacuole requires bacterial protein synthesis but not replication

This study examined whether protein synthesis and replication are required for maturation and fusogenicity of the lysosomal‐like, large and spacious parasitophorous vacuole (PV) of Coxiella burnetii, an obligate intracellular bacterium. Large and spacious PV with multiple non‐replicating C. burnetii were observed by phase microscopy in Vero cells infected at a multiplicity of infection of ten and treated with a bacteriostatic concentration of nalidixic acid or carbenicillin, antimicrobics that inhibit DNA and cell wall biosynthesis respectively. Conversely, large and spacious PV were not observed in cells treated with a bacteriostatic concentration of the protein synthesis inhibitor chloramphenicol. Rather, fluorescence microscopy of individual cells revealed multiple, acidic PV harbouring a single organism tightly bounded by a LAMP‐1 positive vacuolar membrane. These vacuoles homotypically fused to form a large and spacious PV upon removal of the drug. Chloramphenicol also inhibited trafficking of latex beads to large and spacious PV and caused mature PV to collapse. Collectively, these results demonstrate that C. burnetii protein synthesis, but not replication, is required for fusion between nascent C. burnetii PV and latex bead phagosomes, and also for formation and maintenance of large and spacious, replicative PV. However, transit of nascent PV through the endocytic pathway to ultimately acquire lysosomal markers appears to occur irrespective of Coxiella protein synthesis.

Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes

BackgroundThe Gram-positive bacterium Bacillus subtilis is an important producer of high quality industrial enzymes and a few eukaryotic proteins. Most of these proteins are secreted into the growth medium, but successful examples of cytoplasmic protein production are also known. Therefore, one may anticipate that the high protein production potential of B. subtilis can be exploited for protein complexes and membrane proteins to facilitate their functional and structural analysis. The high quality of proteins produced with B. subtilis results from the action of cellular quality control systems that efficiently remove misfolded or incompletely synthesized proteins. Paradoxically, cellular quality control systems also represent bottlenecks for the production of various heterologous proteins at significant concentrations.ConclusionWhile inactivation of quality control systems has the potential to improve protein production yields, this could be achieved at the expense of product quality. Mechanisms underlying degradation of secretory proteins are nowadays well understood and often controllable. It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis that limit the production of high quality protein complexes and membrane proteins, and to enhance those systems that facilitate assembly of these proteins.

Oligomeric structure of the Bacillus subtilis cell division protein DivIVA determined by transmission electron microscopy

DivIVA from Bacillus subtilis is a bifunctional protein with distinct roles in cell division and sporulation. During vegetative growth, DivIVA regulates the activity of the MinCD complex, thus helping to direct cell division to the correct mid‐cell position. DivIVA fulfils a quite different role during sporulation in B. subtilis when it directs the oriC region of the chromosome to the cell pole before asymmetric cell division. DivIVA is a 19.5 kDa protein with a large part of its structure predicted to form a tropomyosin‐like α‐helical coiled‐coil. Here, we present a model for the quaternary structure of DivIVA, based on cryonegative stain transmission electron microscopy images. The purified protein appears as an elongated particle with lateral expansions at both ends producing a form that resembles a ‘doggy‐bone’. The particle mass estimated from these images agrees with the value of 145 kDa measured by analytical ultracentrifugation suggesting 6‐ to 8‐mers. These DivIVA oligomers serve as building blocks in the formation of higher order assemblies giving rise to strings, wires and, finally, two‐dimensional lattices in a time‐dependent manner.

The SpoIIE phosphatase, the sporulation septum and the establishment of forespore-specific transcription in Bacillus subtilis: a reassessment.

Making a spore in Bacillus subtilis requires the formation of two cells, the forespore and the mother cell, which follow dissimilar patterns of gene expression. Cell specificity is first established in the forespore under the control of the σF factor, which is itself activated through the action of the SpoIIE serine phosphatase, an enzyme targeted to the septum between the two cells. Deletion of the 10 transmembrane segments of the SpoIIE protein leads to random distribution of SpoIIE in the cytoplasm. Activation of σF is slightly delayed and less efficient than in wild type, but it remains restricted to the forespore in a large proportion of cells and the bacteria sporulate with 30% efficiency. Overexpression of the complete SpoIIE protein in a divIC mutant leads to significant σF activity, indicating that the septum requirement for activating σF can be bypassed. In contradiction to current models, we propose that genetic asymmetry is not created by unequal distribution of SpoIIE within the sporangium, but by exclusion of an inhibitor of SpoIIE from the forespore. This putative inhibitor would be a cytoplasmic molecule that interacts with SpoIIE and shuts off its phosphatase activity until it disappears specifically from the forespore.

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 trans-activation domain of the sporulation response regulator Spo0A revealed by X-ray crystallography.

Sporulation in Bacillus involves the induction of scores of genes in a temporally and spatially co‐ordinated programme of cell development. Its initiation is under the control of an expanded two‐component signal transduction system termed a phosphorelay. The master control element in the decision to sporulate is the response regulator, Spo0A, which comprises a receiver or phosphoacceptor domain and an effector or transcription activation domain. The receiver domain of Spo0A shares sequence similarity with numerous response regulators, and its structure has been determined in phosphorylated and unphosphorylated forms. However, the effector domain (C‐Spo0A) has no detectable sequence similarity to any other protein, and this lack of structural information is an obstacle to understanding how DNA binding and transcription activation are controlled by phosphorylation in Spo0A. Here, we report the crystal structure of C‐Spo0A from Bacillus stearothermophilus revealing a single α‐helical domain comprising six α‐helices in an unprecedented fold. The structure contains a helix–turn–helix as part of a three α‐helical bundle reminiscent of the catabolite gene activator protein (CAP), suggesting a mechanism for DNA binding. The residues implicated in forming the σA‐activating region clearly cluster in a flexible segment of the polypeptide on the opposite side of the structure from that predicted to interact with DNA. The structural results are discussed in the context of the rich array of existing mutational data.

Fusogenicity of the Coxiella burnetii Parasitophorous Vacuole

Abstract: This study investigated whether C. burnetii protein synthesis and replication is required for maintenance of the fusogenic character of the Coxiella parasitophorous vacuole (PV). Vero cells were infected with C. burnetii, (Nine Mile strain in phase II) at a multiplicity of infection of approximately 10 and simultaneously treated with bacteriostatic concentrations of chloramphenicol or carbenicillin. At 96 h post‐infection, cells were viewed by phase contrast microscopy for PV maturation. Mature, spacious PV containing multiple nonreplicating C. burnetii were clearly visible in infected Vero cells treated with the cell wall inhibitor carbenicillin. Conversely, mature, spacious PV did not form in cells treated with the protein synthesis inhibitor chloramphenicol. Rather, immunofluorescence microscopy revealed individual C. burnetii in small, tight PV scattered throughout the cytoplasm. Like mature PV, these PV localized with the lysosomal glycoprotein LAMP‐1, but not the early endosome protein EEA.1. This result suggests that de novo C. burnetii protein synthesis, but not replication, is required for homotypic fusion and maturation of nascent C. burnetii PV. We next examined whether sustained C. burnetii protein synthesis is necessary for maintenance of PV fusogenicity. J774A.1 murine macrophage‐like cells with mature C. burnetii PV were incubated with latex beads and the trafficking of beads to PV was quantified. Fusion of PV with bead‐laden vacuoles was severely inhibited in cells treated with chloramphenicol. These results suggest that sustained C. burnetii protein synthesis is required for PV fusion with other vacuoles of the endocytic pathway.

Oligomerization of the Bacillus subtilis division protein DivIVA

DivIVA appears to be a mediator of inhibition by MinCD of division at the cell poles in Bacillus subtilis. Gel permeation and ultracentrifugation techniques were used to show self-association of DivIVA into a form consisting of 10-12 monomers in vitro. Western blot analysis of non-denaturating polyacrylamide gels confirms the presence of similar oligomers in B. subtilis cell lysates. These oligomers persist in a B. subtilis strain containing the divIVA1 mutation, in which proper vegetative septum positioning is abolished. In contrast, the divIVA2 mutation, which has a similar biological impact, appears to produce a protein with different oligomerization properties. The results of the present study suggest that oligomerization of DivIVA is important, but not sufficient for its function in the cell division process.

Comparison of different Bacillus subtilis expression systems

Bacillus subtilis is considered to have great potential as a host for the production and secretion of recombinant proteins. Many different expression systems have been developed for B. subtilis. Here we compare two widely used expression systems, the IPTG-inducible derivative of spac system (hyper-spank) and the xylose-inducible (xyl) to the SURE (subtilin-regulated gene expression) system. Western blot analysis of the membrane protein SpoIISA together with its protein partner SpoIISB showed that the highest expression level of this complex is obtained using the SURE system. Measurement of β-galactosidase activities of the promoter-lacZ fusions in individual expression systems confirmed that the P(spaS) promoter of the SURE system is the strongest of those compared, although the induction/repression ratio reached only 1.84. Based on these results, we conclude that the SURE system is the most efficient of these three B. subtilis expression systems in terms of the amount of expressed product. Remarkably, the yield of the SpoIISA-SpoIISB complex obtained from B. subtilis was comparable to that normally obtained from the Escherichia coli arabinose-inducible expression system.