RESUMEN
Cell wall homeostasis in bacteria is tightly regulated by balanced synthesis and degradation of peptidoglycan (PG), allowing cells to expand their sacculus during growth while maintaining physical integrity. In rod-shaped bacteria, actin-like MreB proteins are key players of the PG elongation machinery known as the Rod complex. In the Gram-positive model bacterium Bacillus subtilis depletion of the essential MreB leads to loss of rod shape and cell lysis. However, millimolar concentrations of magnesium in the growth medium rescue the viability and morphological defects of mreB mutants by an unknown mechanism. Here, we used a combination of cytological, biochemical and biophysical approaches to investigate the cell surface properties of mreB null mutant cells and the interactions of Mg2+ with the cell wall of B. subtilis. We show that ∆mreB cells have rougher and softer surfaces, and changes in PG composition indicative of increased DL- and DD-endopeptidase activities as well as increased deacetylation of the sugar moieties. Increase in DL-endopeptidase activity is mitigated by excess Mg2+ while DD-endopeptidase activity remains high. Visualization of PG degradation in pulse-chase experiments showed anisotropic PG hydrolase activity along the sidewalls of ∆mreB cells, in particular at the sites of increased cell width and bulging, while PG synthesis remained isotropic. Overall, our data support a model in which divalent cations maintain rod shape in ∆mreB cells by inhibiting PG hydrolases, possibly through the formation of crosslinks with carboxyl groups of the PG meshwork that affect the capacity of PG hydrolases to act on their substrate.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Magnesio/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Mutación , N-Acetil Muramoil-L-Alanina Amidasa/genéticaRESUMEN
The Min system negatively regulates the position of the Z ring, which serves as a scaffold for the divisome that mediates bacterial cytokinesis. In Escherichia coli, this system consists of MinC, which antagonizes assembly of the tubulin homologue FtsZ. MinC is recruited to the membrane by MinD and induced by MinE to oscillate between the cell poles. MinC is a dimer with each monomer consisting of functionally distinct MinCN and MinCC domains, both of which contact FtsZ. According to one model, MinCC/MinD binding to the FtsZ tail positions MinCN at the junction of two GDP-containing subunits in the filament, leading to filament breakage. Others posit that MinC sequesters FtsZ-GDP monomers or that MinCN caps the minus end of FtsZ polymers and that MinCC interferes with lateral interactions between FtsZ filaments. Here, we isolated minC mutations that impair MinCN function and analyzed FtsZ mutants resistant to MinC/MinD. Surprisingly, we found mutations in both minC and ftsZ that differentiate inhibition by MinC from inhibition by MinC/MinD. Analysis of these mutations suggests that inhibition of the Z ring by MinC alone is due to sequestration, whereas inhibition by MinC/MinD is not. In conclusion, our genetic and biochemical data support the model that MinC/MinD fragments FtsZ filaments.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Escherichia coli K12/química , Escherichia coli K12/citología , Escherichia coli K12/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conformación Proteica , Multimerización de ProteínaRESUMEN
The ability of excess Mg2+ to compensate the absence of cell wall related genes in Bacillus subtilis has been known for a long time, but the mechanism has remained obscure. Here, we show that the rigidity of wild-type cells remains unaffected with excess Mg2+ , but the proportion of amidated meso-diaminopimelic (mDAP) acid in their peptidoglycan (PG) is significantly reduced. We identify the amidotransferase AsnB as responsible for mDAP amidation and show that the gene encoding it is essential without added Mg2+ . Growth without excess Mg2+ causes ΔasnB mutant cells to deform and ultimately lyse. In cell regions with deformations, PG insertion is orderly and indistinguishable from the wild-type. However, PG degradation is unevenly distributed along the sidewalls. Furthermore, ΔasnB mutant cells exhibit increased sensitivity to antibiotics targeting the cell wall. These results suggest that absence of amidated mDAP causes a lethal deregulation of PG hydrolysis that can be inhibited by increased levels of Mg2+ . Consistently, we find that Mg2+ inhibits autolysis of wild-type cells. We suggest that Mg2+ helps to maintain the balance between PG synthesis and hydrolysis in cell wall mutants where this balance is perturbed in favor of increased degradation.
Asunto(s)
Ácido Diaminopimélico/metabolismo , Peptidoglicano/metabolismo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Hidrólisis , Magnesio/metabolismo , Peptidoglicano/biosíntesisRESUMEN
UNLABELLED: A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZ(G193D) allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZ(G193D) filaments are more twisted and shorter than wild-type filaments. In vivo, M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. IMPORTANCE: The mechanisms by which bacteria generate and maintain even the simplest cell shape remain an elusive but fundamental question in microbiology. In the absence of examples of coccus-to-rod transitions, the spherical shape has been suggested to be an evolutionary dead end in morphogenesis. We describe the first observation of the generation of elongated cells from truly spherical cocci, occurring in a Staphylococcus aureus mutant containing a single point mutation in its genome, in the gene encoding the bacterial tubulin homologue FtsZ. We demonstrate that FtsZ-dependent cell elongation is possible, even in the absence of dedicated elongation machinery.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Staphylococcus aureus/citología , Staphylococcus aureus/genética , Microscopía , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
In prokaryotes, about one third of cellular proteins are translocated across the plasma membrane or inserted into it by concerted action of the cytoplasmic ATPase SecA and the universally conserved SecYEG heterotrimeric polypeptide-translocating pore. Secretion complexes have been reported to localize in specific subcellular sites in Bacillus subtilis. In this work, we used a combination of total internal reflection microscopy, scanning fluorescence correlation spectroscopy, and pair correlation function to study the localization and dynamics of SecA and SecY in growing Bacillus subtilis cells. Both SecA and SecY localized in transient and dynamic foci in the cytoplasmic membrane, which displayed no higher-level organization in helices. Foci of SecA and SecY were in constant flux with freely diffusing SecA and SecY molecules. Scanning FCS confirmed the existence of populations of cellular SecA and SecY molecules with a wide range of diffusion coefficients. Diffusion of SecY as an uncomplexed molecular species was short-lived and only local while SecY complexed with its protein partners traversed distances of over half a micrometer in the cell.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Canales de Translocación SEC/metabolismo , Citosol/metabolismo , Unión Proteica , Transporte de Proteínas , Proteína SecARESUMEN
A key event in bacterial cytokinesis is the formation of the Z ring, which serves as a mechanical scaffold that recruits other cytokinetic proteins to establish functional divisomes. This scaffolding function of Z rings is essential throughout cytokinesis, but the underlying molecular interactions are poorly understood. Here we report that a widely conserved FtsZ binding protein, ZapA, has cytological, biochemical and biophysical properties that argue for the importance of cross-linking interactions between FtsZ polymers in the coherence of Z rings. Escherichia coli zapA null mutant cells have Z rings that are structurally looser and many helical precursors of Z rings fail to coalesce into coherent rings. Biophysical behaviour of FtsZ in the presence of ZapA reveals that ZapA not only bundles, but also cross-links FtsZ polymers, which makes it the first cross-linking protein of the bacterial cytoskeleton. Cross-linking in vitro occurs at the stoichiometry of FtsZ-ZapA interaction at the Z rings in vivo, where nearly all intracellular ZapA is dynamically associated. ZapA also stabilizes longitudinal bonds between FtsZ monomers since it promotes the polymerization of FtsZ mutants with lesions at the polymerization interface and since it reverses the inhibitory effects of SulA, a known antagonist of FtsZ longitudinal interactions.
Asunto(s)
Proteínas Bacterianas/metabolismo , Citocinesis , Proteínas del Citoesqueleto/metabolismo , Escherichia coli/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Escherichia coli/química , Escherichia coli/citología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Polimerizacion , Unión ProteicaRESUMEN
FtsZ is a tubulin homolog essential for prokaryotic cell division. In living bacteria, FtsZ forms a ringlike structure (Z-ring) at the cell midpoint. Cell division coincides with a gradual contraction of the Z-ring, although the detailed molecular structure of the Z-ring is unknown. To reveal the structural properties of FtsZ, an understanding of FtsZ filament and bundle formation is needed. We develop a kinetic model that describes the polymerization and bundling mechanism of FtsZ filaments. The model reveals the energetics of the FtsZ filament formation and the bundling energy between filaments. A weak lateral interaction between filaments is predicted by the model. The model is able to fit the in vitro polymerization kinetics data of another researcher, and explains the cooperativity observed in FtsZ kinetics and the critical concentration in different buffer media. The developed model is also applicable for understanding the kinetics and energetics of other bundling biopolymer filaments.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopolímeros/metabolismo , Proteínas del Citoesqueleto/metabolismo , Tampones (Química) , Cinética , Modelos Biológicos , Proteínas Mutantes/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Cytokinesis in bacteria is mediated by a cytokinetic ring, termed the Z ring, which forms a scaffold for recruitment of other cell-division proteins. The Z ring is composed of FtsZ filaments, but their organization in the Z ring is poorly understood. In Escherichia coli, the Min system contributes to the spatial regulation of cytokinesis by preventing the assembly of the Z ring away from midcell. The effector of the Min system, MinC, inhibits Z ring assembly by a mechanism that is not clear. RESULTS: Here, we report that MinC controls the scaffolding function of FtsZ by antagonizing the mechanical integrity of FtsZ structures. Specifically, MinC antagonizes the ability of FtsZ filaments to be in a solid-like gel state. MinC is a modular protein whose two domains (MinC(C) and MinC(N)) synergize to inhibit FtsZ function. MinC(C) interacts directly with FtsZ polymers to target MinC to Z rings. MinC(C) also prevents lateral interactions between FtsZ filaments, an activity that seems to be unique among cytoskeletal proteins. Because MinC(C) is inhibitory in vivo, it suggests that lateral interactions between FtsZ filaments are important for the structural integrity of the Z ring. MinC(N) contributes to MinC activity by weakening the longitudinal bonds between FtsZ molecules in a filament leading to a loss of polymer rigidity and consequent polymer shortening. On the basis of our results, we develop the first computational model of the Z ring and study the effects of MinC. CONCLUSIONS: Control over the scaffolding activity of FtsZ probably represents a universal regulatory mechanism of bacterial cytokinesis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Citocinesis/fisiología , Proteínas del Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Unión Periplasmáticas/metabolismo , Proteínas Bacterianas/ultraestructura , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/ultraestructura , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , GTP Fosfohidrolasas/metabolismo , Geles , Modelos Biológicos , Polímeros/metabolismoRESUMEN
In Escherichia coli FtsZ organizes into a cytoskeletal ring structure, the Z ring, which effects cell division. FtsZ is a GTPase, but the free energy of GTP hydrolysis does not appear to be used for generation of the constriction force, leaving open the question of the function of the GTPase activity of FtsZ. Here we study the mechanism by which SulA, an inhibitor of FtsZ induced during the SOS response, inhibits FtsZ function. We studied the effects of SulA on the in vitro activities of FtsZ, on Z rings in vivo, and on a kinetic model for FtsZ polymerization in silico. We found that the binding of SulA to FtsZ is necessary but not sufficient for inhibition of polymerization, since the assembly of FtsZ polymers in the absence of the GTPase activity was not inhibited by SulA. We developed a new model for FtsZ polymerization that accounts for the cooperativity of FtsZ and could account for cooperativity observed in other linear polymers. When SulA was included in the kinetic scheme, simulations revealed that SulA with strong affinity for FtsZ delayed, but did not prevent, the assembly of polymers when they were not hydrolyzing GTP. Furthermore, the simulations indicated that SulA controls the assembly of FtsZ by binding to a polymerization-competent form of the FtsZ molecule and preventing it from participating in assembly. In vivo stoichiometry of the disruption of Z rings by SulA suggests that FtsZ may undergo two cooperative transitions in forming the Z ring.
Asunto(s)
Algoritmos , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Dimerización , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Prueba de Complementación Genética , Guanosina Trifosfato/metabolismo , Immunoblotting , Cinética , Modelos Moleculares , Mutación , Nucleótidos/metabolismo , Proteínas de Unión Periplasmáticas/química , Proteínas de Unión Periplasmáticas/genética , Proteínas de Unión Periplasmáticas/metabolismo , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
It has become apparent that bacteria possess ancestors of the major eukaryotic cytoskeletal proteins. FtsZ, the ancestral homologue of tubulin, assembles into a cytoskeletal structure associated with cell division, designated the Z ring. Formation of the Z ring represents a major point of both spatial and temporal regulation of cell division. Here we discuss findings concerning the structure and the formation of the ring as well as its spatial and temporal regulation.