Establishing how bacterial cells position the division site

University of Technology, Sydney. Faculty of Science.In virtually all bacteria cell division is essential and tightly regulated both temporally and spatially to ensure that cells divide precisely at the centre between segregated chromosomes. Failure to do so can lead to cell death. The earliest event in bacterial cell division is the polymerization of the highly conserved tubulin-like protein, FtsZ, to form a contractile structure called the Z ring, on the inner side of the cytoplasmic membrane at midcell and between chromosomes. The Z ring subsequently contracts causing the cell envelope to invaginate, generating two newborn cells. Thus the Z ring defines the position of the division site in bacterial cells. How the Z ring is positioned precisely at midcell is a controversial topic that remains unresolved. Division site positioning has long been believed to occur via the combined action of two factors: the Min system and nucleoid occlusion. Both factors have been proposed to prevent Z ring assembly along the length of the cell, allowing it to assemble only once chromosomes segregate and nucleoid occlusion is relieved specifically at midcell. The research described in this thesis challenges this paradigm, providing compelling evidence that other mechanisms in addition to nucleoid occlusion and the Min system act to position the Z ring at midcell in B. subtilis. Moreover, this work also shows that nucleoid occlusion and the Min system do not define the Z ring position at midcell but rather ensure that the midcell division site is utilized efficiently. A clue to an additional mechanism for positioning the Z ring has emerged from studies investigating the relationship between chromosome replication and Z ring position. The nature of this relationship has remained obscure for years. Part of this thesis involves a closer examination of this relationship. It was found that the ability to position the Z ring at midcell is linked specifically to the progress of the initiation stage of DNA replication, such that the frequency of Z rings at midcell increases as this stage of DNA replication is progressively completed. Moreover, this link was found to be nucleoid occlusion independent. Spatial and temporal control of Z ring assembly has been widely attributed to the Min system and nucleoid occlusion. While inactivating both systems substantially affects cell division, it is currently unknown whether their absence affects precise midcell Z ring positioning. This thesis deals with this question, and it was found that the combined effect of MinCD and Noc proteins actually affects the timing and efficiency of Z ring assembly, but not its spatial precision between nucleoids at midcell. If Noc and MinCD proteins do not position the Z ring at midcell, what other factores may play this role? Two hypotheses were proposed to help explain the precise Z ring positioning observed in absence of nae and minCD: 1) Noc-independent nucleoid occlusion or 2) factors completely independent of nucleoid occlusion position the Z ring at midcell. Experiments designed to discriminate between these hypotheses showed that they are actually both valid: while the data obtained suggests that factors completely independent of nucleoid occlusion (Noc inclusive) and the Min system position the Z ring at midcell, it also suggested that other Noc-independent nucleoid occlusion factors prevent the Z ring from assembling at midcell over unreplicated DNA

The ParB homologs, Spo0J and Noc, together prevent premature midcell Z ring assembly when the early stages of replication are blocked in Bacillus subtilis

© 2019 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd Precise cell division in coordination with DNA replication and segregation is of utmost importance for all organisms. The earliest stage of cell division is the assembly of a division protein FtsZ into a ring, known as the Z ring, at midcell. What still eludes us, however, is how bacteria precisely position the Z ring at midcell. Work in B. subtilis over the last two decades has identified a link between the early stages of DNA replication and cell division. A recent model proposed that the progression of the early stages of DNA replication leads to an increased ability for the Z ring to form at midcell. This model arose through studies examining Z ring position in mutants blocked at different steps of the early stages of DNA replication. Here, we show that this model is unlikely to be correct and the mutants previously studied generate nucleoids with different capacity for blocking midcell Z ring assembly. Importantly, our data suggest that two proteins of the widespread ParB family, Noc and Spo0J are required to prevent Z ring assembly over the bacterial nucleoid and help fine tune the assembly of the Z ring at midcell during the cell cycle

A two-step transport pathway allows the mother cell to nurture the developing spore in Bacillus subtilis

© 2017 Ramírez-Guadiana et al. One of the hallmarks of bacterial endospore formation is the accumulation of high concentrations of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA) in the developing spore. This small molecule comprises 5–15% of the dry weight of dormant spores and plays a central role in resistance to both wet heat and desiccation. DPA is synthesized in the mother cell at a late stage in sporulation and must be translocated across two membranes (the inner and outer forespore membranes) that separate the mother cell and forespore. The enzymes that synthesize DPA and the proteins required to translocate it across the inner forespore membrane were identified over two decades ago but the factors that transport DPA across the outer forespore membrane have remained mysterious. Here, we report that SpoVV (formerly YlbJ) is the missing DPA transporter. SpoVV is produced in the mother cell during the morphological process of engulfment and specifically localizes in the outer forespore membrane. Sporulating cells lacking SpoVV produce spores with low levels of DPA and cells engineered to express SpoVV and the DPA synthase during vegetative growth accumulate high levels of DPA in the culture medium. SpoVV resembles concentrative nucleoside transporters and mutagenesis of residues predicted to form the substrate-binding pocket supports the idea that SpoVV has a similar structure and could therefore function similarly. These findings provide a simple two-step transport mechanism by which the mother cell nurtures the developing spore. DPA produced in the mother cell is first translocated into the intermembrane space by SpoVV and is then imported into the forespore by the SpoVA complex. This pathway is likely to be broadly conserved as DPA synthase, SpoVV, and SpoVA proteins can be found in virtually all endospore forming bacteria

Evidence that regulation of intramembrane proteolysis is mediated by substrate gating during sporulation in Bacillus subtilis

© 2018 Ramírez-Guadiana et al. http://creativecommons.org/licenses/by/4.0/. During the morphological process of sporulation in Bacillus subtilis two adjacent daughter cells (called the mother cell and forespore) follow different programs of gene expression that are linked to each other by signal transduction pathways. At a late stage in development, a signaling pathway emanating from the forespore triggers the proteolytic activation of the mother cell transcription factor σK. Cleavage of pro-σK to its mature and active form is catalyzed by the intramembrane cleaving metalloprotease SpoIVFB (B), a Site-2 Protease (S2P) family member. B is held inactive by two mother-cell membrane proteins SpoIVFA (A) and BofA. Activation of pro-σK processing requires a site-1 signaling protease SpoIVB (IVB) that is secreted from the forespore into the space between the two cells. IVB cleaves the extracellular domain of A but how this cleavage activates intramembrane proteolysis has remained unclear. Structural studies of the Methanocaldococcus jannaschii S2P homolog identified closed (substrate-occluded) and open (substrate-accessible) conformations of the protease, but the biological relevance of these conformations has not been established. Here, using co-immunoprecipitation and fluorescence microscopy, we show that stable association between the membrane-embedded protease and its substrate requires IVB signaling. We further show that the cytoplasmic cystathionine-β-synthase (CBS) domain of the B protease is not critical for this interaction or for pro-σK processing, suggesting the IVB-dependent interaction site is in the membrane protease domain. Finally, we provide evidence that the B protease domain adopts both open and closed conformations in vivo. Collectively, our data support a substrate-gating model in which IVB-dependent cleavage of A on one side of the membrane triggers a conformational change in the membrane-embedded protease from a closed to an open state allowing pro-σK access to the caged interior of the protease

A ring-shaped conduit connects the mother cell and forespore during sporulation in Bacillus subtilis

During spore formation in Bacillus subtilis a transenvelope complex is assembled across the double membrane that separates the mother cell and forespore. This complex (called the "A-Q complex") is required to maintain forespore development and is composed of proteins with remote homology to components of type II, III, and IV secretion systems found in Gram-negative bacteria. Here, we show that one of these proteins, SpoIIIAG, which has remote homology to ring-forming proteins found in type III secretion systems, assembles into an oligomeric ring in the periplasmic-like space between the two membranes. Three-dimensional reconstruction of images generated by cryo-electron microscopy indicates that the SpoIIIAG ring has a cup-and-saucer architecture with a 6-nm central pore. Structural modeling of SpoIIIAG generated a 24-member ring with dimensions similar to those of the EM-derived saucer. Point mutations in the predicted oligomeric interface disrupted ring formation in vitro and impaired forespore gene expression and efficient spore formation in vivo. Taken together, our data provide strong support for the model in which the A-Q transenvelope complex contains a conduit that connects the mother cell and forespore. We propose that a set of stacked rings spans the intermembrane space, as has been found for type III secretion systems

A dynamic, ring-forming MucB / RseB-like protein influences spore shape in Bacillus subtilis.

How organisms develop into specific shapes is a central question in biology. The maintenance of bacterial shape is connected to the assembly and remodelling of the cell envelope. In endospore-forming bacteria, the pre-spore compartment (the forespore) undergoes morphological changes that result in a spore of defined shape, with a complex, multi-layered cell envelope. However, the mechanisms that govern spore shape remain poorly understood. Here, using a combination of fluorescence microscopy, quantitative image analysis, molecular genetics and transmission electron microscopy, we show that SsdC (formerly YdcC), a poorly-characterized new member of the MucB / RseB family of proteins that bind lipopolysaccharide in diderm bacteria, influences spore shape in the monoderm Bacillus subtilis. Sporulating cells lacking SsdC fail to adopt the typical oblong shape of wild-type forespores and are instead rounder. 2D and 3D-fluorescence microscopy suggest that SsdC forms a discontinuous, dynamic ring-like structure in the peripheral membrane of the mother cell, near the mother cell proximal pole of the forespore. A synthetic sporulation screen identified genetic relationships between ssdC and genes involved in the assembly of the spore coat. Phenotypic characterization of these mutants revealed that spore shape, and SsdC localization, depend on the coat basement layer proteins SpoVM and SpoIVA, the encasement protein SpoVID and the inner coat protein SafA. Importantly, we found that the ΔssdC mutant produces spores with an abnormal-looking cortex, and abolishing cortex synthesis in the mutant largely supresses its shape defects. Thus, SsdC appears to play a role in the proper assembly of the spore cortex, through connections to the spore coat. Collectively, our data suggest functional diversification of the MucB / RseB protein domain between diderm and monoderm bacteria and identify SsdC as an important factor in spore shape development

Structural insights into ring-building motif domains involved in bacterial sporulation.

Components of specialized secretion systems, which span the inner and outer membranes in Gram-negative bacteria, include ring-forming proteins whose oligomerization was proposed to be promoted by domains called RBM for "Ring-Building Motifs". During spore formation in Gram-positive bacteria, a transport system called the SpoIIIA-SpoIIQ complex also assembles in the double membrane that surrounds the forespore following its endocytosis by the mother cell. The presence of RBM domains in some of the SpoIIIA proteins led to the hypothesis that they would assemble into rings connecting the two membranes and form a conduit between the mother cell and forespore. Among them, SpoIIIAG forms homo-oligomeric rings in vitro but the oligomerization of other RBM-containing SpoIIIA proteins, including SpoIIIAH, remains to be demonstrated. In this work, we identified RBM domains in the YhcN/YlaJ family of proteins that are not related to the SpoIIIA-SpoIIQ complex. We solved the crystal structure of YhcN from Bacillus subtilis, which confirmed the presence of a RBM fold, flanked by additional secondary structures. As the protein did not show any oligomerization ability in vitro, we investigated the structural determinants of ring formation in SpoIIIAG, SpoIIIAH and YhcN. We showed that in vitro, the conserved core of RBM domains alone is not sufficient for oligomerization while the β-barrel forming region in SpoIIIAG forms rings on its own. This work suggests that some RBMs might indeed participate in the assembly of homomeric rings but others might have evolved toward other functions

Regulation of immunity during visceral Leishmania infection

Unicellular eukaryotes of the genus Leishmania are collectively responsible for a heterogeneous group of diseases known as leishmaniasis. The visceral form of leishmaniasis, caused by L. donovani or L. infantum, is a devastating condition, claiming 20,000 to 40,000 lives annually, with particular incidence in some of the poorest regions of the world. Immunity to Leishmania depends on the development of protective type I immune responses capable of activating infected phagocytes to kill intracellular amastigotes. However, despite the induction of protective responses, disease progresses due to a multitude of factors that impede an optimal response. These include the action of suppressive cytokines, exhaustion of specific T cells, loss of lymphoid tissue architecture and a defective humoral response. We will review how these responses are orchestrated during the course of infection, including both early and chronic stages, focusing on the spleen and the liver, which are the main target organs of visceral Leishmania in the host. A comprehensive understanding of the immune events that occur during visceral Leishmania infection is crucial for the implementation of immunotherapeutic approaches that complement the current anti-Leishmania chemotherapy and the development of effective vaccines to prevent disease.The research leading to these results has received funding from the European Community’s Seventh Framework Programme under grant agreement No.602773 (Project KINDRED). VR is supported by a post-doctoral fellowship granted by the KINDReD consortium. RS thanks the Foundation for Science and Technology (FCT) for an Investigator Grant (IF/00021/2014). This work was supported by grants to JE from ANR (LEISH-APO, France), Partenariat Hubert Curien (PHC) (program Volubilis, MA/11/262). JE acknowledges the support of the Canada Research Chair Program

The New Kid on the Block: A Specialized Secretion System during Bacterial Sporulation

© 2018 Elsevier Ltd The transport of proteins across the bacterial cell envelope is mediated by protein complexes called specialized secretion systems. These nanomachines exist in both Gram-positive and Gram-negative bacteria and have been categorized into different types based on their structural components and function. Interestingly, multiple studies suggest the existence of a protein complex in endospore-forming bacteria that appears to be a new type of specialized secretion system. This protein complex is called the SpoIIIA-SpoIIQ complex and is an exception to the categorical norm since it appears to be a hybrid composed of different parts from well-defined specialized secretion systems. Here we summarize and discuss the current understanding of this complex and its potential role as a specialized secretion system