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Identificação e caracterização de novos moduladores da divisão em Bacillus subtilis / Identification and characterization of new modulators of division in B. subtilisTavares, José Roberto 31 July 2009 (has links)
Em procariotos, a principal forma de reprodução é a divisão binária, que permite à célula-mãe dar origem a duas outras células-filhas, com conteúdo genético idêntico ao da progenitora. Em Bacillus subtilis este processo acontece graças ao divisomo, um complexo formado por aproximadamente dezesseis proteínas, que leva à constrição da membrana e da parede, formando o septo de divisão. A montagem do divisomo é coordenada por FtsZ, um homólogo de tubulina, que polimeriza na região central da bactéria e serve de arcabouço para a montagem do divisomo. Partindo de um levantamento detalhado da distribuição dos genes envolvidos em divisão em genomas completos de procariotos detectamos que divIVA, um gene de divisão já bem caracterizado, apresentava um gene parálogo em B. subtilis, conhecido como ypsB. Para determinarmos se YpsB seria um novo componente do divisomo foi realizada uma caracterização citológica e funcional desta proteína. Utilizamos microscopia de fluorescência e fusões de YpsB a GFP para determinar a localização subcelular de YpsB. Estes experimentos revelaram que YpsB está presente no divisomo, apresentando um padrão de localização semelhante mas não idêntico ao de DivIVA. Medindo-se a taxa de co-localização entre o anel Z e YpsB ficou demonstrado que estas proteínas co-localizam em aproximadamente 50%, sugerindo que YpsB é recrutada depois que o anel Z é montado. Para determinar quando YpsB chega ao divisomo, usamos mutantes termo-sensíveis das proteínas de divisão que revelaram a dependência de YpsB pelo sub complexo DivIB-DivIC-FtsL-FtsW-PBP2B. Já na ausência de DivIVA, YpsB continua associado ao divisomo, indicando que não depende do seu parálogo para localizar. Além disso, análises de deleções de YpsB mostraram que a porção N-terminal da proteína é a mais importante para o seu recrutamento ao divisomo. Para determinarmos o papel de YpsB durante a divisão foi construído um mutante com deleção completa do gene. DivIVA é uma proteína responsável por localizar o sistema Min nos pólos da bactéria e assim contribui para a precisão espacial da divisão. Apesar de serem parálogos, a função de YpsB, no entanto, parece ser diferente da de DivIVA. Análise do mutante ypsB- mostrou que na sua ausência, o divisomo é montado e o seu posicionamento tanto em fase vegetativa como em esporulação não são afetados. Como a ausência de YpsB não afeta perceptivelmente a divisão, combinamos a mutação em ypsB com mutações em outros genes envolvidos em divisão. A análise destes duplos mutantes revelou que a ausência simultânea de YpsB e FtsA produz exacerbada lise celular e letalidade. Com base neste fenótipo e em evidências evolutivas, sugerimos que YpsB esteja envolvida na regulação da síntese de peptideoglicano do septo. Mais especificamente, YpsB seria responsável por modular a atividade de PBP1, uma enzima necessária para a síntese de peptideoglicano septal. / In prokaryotes, the main form of reproduction is binary fission, which allows the mother-cell to give origin the two daughter-cells, with identical genetic material. In Bacillus subtilis, this process is performed by the divisome, a complex formed for approximately sixteen proteins that leads to the constriction of the membrane and the wall, creating the division septum. The assembly of the divisome is coordinated by FtsZ, a homolog of tubulin, that polymerizes in the central region of the bacteria and serves as the base for the assembly of the divisome. From a detailed survey of the distribution of the genes involved in division in complete genomes of prokaryotes, we detected that divIVA, a well characterized division gene, showed a paralog in B. subtilis, known as YpsB. To determine if YpsB would be a new component of the divisome, a cytological and functional characterization of this protein was carried out. We used fluorescence microscopy and fusion of YpsB to GFP to determine the subcellular localization of YpsB. These experiments displayed that YpsB is present in the divisome, with similar but not identical localization as DivIVA. Measuring co-localization between the Z ring and YpsB demonstrated that this happened in approximately 50% of the cells, suggesting that YpsB go to the divisome after the Z ring is formed. To determine when YpsB goes to the divisome, we used temperature-sensitive mutants of the division proteins. This showed that YpsB depends on the DivIB-DivIC-FtsL-FtsW-PBP2B sub-complex to associate with the divisome. In the absence of DivIVA, YpsB is still present in the divisome, indicating that it does not depend on its paralog to localize. Moreover, deletion analyses of YpsB showed that the N-terminal portion of the protein is the most important for its recruitment to the divisome. To determine the role of YpsB during division, we constructed a ypsB- mutant. DivIVA is the protein responsible for localization of the Min system in polar regions of B. subtilis and, thus, contributes for the spatial precision of division. Our results showed that the function of YpsB must be different from that of DivIVA, since analysis of the ypsB- mutant showed that in the absence this protein the divisome is assembled and septum position in vegetatively growing or sporulating cells is not affected. Since the absence of YpsB does not affect division, we combined the ypsB- mutant with mutants involved in division. Analysis of these double mutants showed that the simultaneous absence of YpsB and FtsA caused cellular lysis and lethality. Based on this phenotype and evolutionary evidences, we suggest that YpsB is involved in the regulation of peptidoglycan synthesis in the septum. More specifically, YpsB would be responsible for modulating the activity of PBP1, a necessary enzyme for septum peptidoglycan synthesis.
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Identificação e caracterização de novos moduladores da divisão em Bacillus subtilis / Identification and characterization of new modulators of division in B. subtilisJosé Roberto Tavares 31 July 2009 (has links)
Em procariotos, a principal forma de reprodução é a divisão binária, que permite à célula-mãe dar origem a duas outras células-filhas, com conteúdo genético idêntico ao da progenitora. Em Bacillus subtilis este processo acontece graças ao divisomo, um complexo formado por aproximadamente dezesseis proteínas, que leva à constrição da membrana e da parede, formando o septo de divisão. A montagem do divisomo é coordenada por FtsZ, um homólogo de tubulina, que polimeriza na região central da bactéria e serve de arcabouço para a montagem do divisomo. Partindo de um levantamento detalhado da distribuição dos genes envolvidos em divisão em genomas completos de procariotos detectamos que divIVA, um gene de divisão já bem caracterizado, apresentava um gene parálogo em B. subtilis, conhecido como ypsB. Para determinarmos se YpsB seria um novo componente do divisomo foi realizada uma caracterização citológica e funcional desta proteína. Utilizamos microscopia de fluorescência e fusões de YpsB a GFP para determinar a localização subcelular de YpsB. Estes experimentos revelaram que YpsB está presente no divisomo, apresentando um padrão de localização semelhante mas não idêntico ao de DivIVA. Medindo-se a taxa de co-localização entre o anel Z e YpsB ficou demonstrado que estas proteínas co-localizam em aproximadamente 50%, sugerindo que YpsB é recrutada depois que o anel Z é montado. Para determinar quando YpsB chega ao divisomo, usamos mutantes termo-sensíveis das proteínas de divisão que revelaram a dependência de YpsB pelo sub complexo DivIB-DivIC-FtsL-FtsW-PBP2B. Já na ausência de DivIVA, YpsB continua associado ao divisomo, indicando que não depende do seu parálogo para localizar. Além disso, análises de deleções de YpsB mostraram que a porção N-terminal da proteína é a mais importante para o seu recrutamento ao divisomo. Para determinarmos o papel de YpsB durante a divisão foi construído um mutante com deleção completa do gene. DivIVA é uma proteína responsável por localizar o sistema Min nos pólos da bactéria e assim contribui para a precisão espacial da divisão. Apesar de serem parálogos, a função de YpsB, no entanto, parece ser diferente da de DivIVA. Análise do mutante ypsB- mostrou que na sua ausência, o divisomo é montado e o seu posicionamento tanto em fase vegetativa como em esporulação não são afetados. Como a ausência de YpsB não afeta perceptivelmente a divisão, combinamos a mutação em ypsB com mutações em outros genes envolvidos em divisão. A análise destes duplos mutantes revelou que a ausência simultânea de YpsB e FtsA produz exacerbada lise celular e letalidade. Com base neste fenótipo e em evidências evolutivas, sugerimos que YpsB esteja envolvida na regulação da síntese de peptideoglicano do septo. Mais especificamente, YpsB seria responsável por modular a atividade de PBP1, uma enzima necessária para a síntese de peptideoglicano septal. / In prokaryotes, the main form of reproduction is binary fission, which allows the mother-cell to give origin the two daughter-cells, with identical genetic material. In Bacillus subtilis, this process is performed by the divisome, a complex formed for approximately sixteen proteins that leads to the constriction of the membrane and the wall, creating the division septum. The assembly of the divisome is coordinated by FtsZ, a homolog of tubulin, that polymerizes in the central region of the bacteria and serves as the base for the assembly of the divisome. From a detailed survey of the distribution of the genes involved in division in complete genomes of prokaryotes, we detected that divIVA, a well characterized division gene, showed a paralog in B. subtilis, known as YpsB. To determine if YpsB would be a new component of the divisome, a cytological and functional characterization of this protein was carried out. We used fluorescence microscopy and fusion of YpsB to GFP to determine the subcellular localization of YpsB. These experiments displayed that YpsB is present in the divisome, with similar but not identical localization as DivIVA. Measuring co-localization between the Z ring and YpsB demonstrated that this happened in approximately 50% of the cells, suggesting that YpsB go to the divisome after the Z ring is formed. To determine when YpsB goes to the divisome, we used temperature-sensitive mutants of the division proteins. This showed that YpsB depends on the DivIB-DivIC-FtsL-FtsW-PBP2B sub-complex to associate with the divisome. In the absence of DivIVA, YpsB is still present in the divisome, indicating that it does not depend on its paralog to localize. Moreover, deletion analyses of YpsB showed that the N-terminal portion of the protein is the most important for its recruitment to the divisome. To determine the role of YpsB during division, we constructed a ypsB- mutant. DivIVA is the protein responsible for localization of the Min system in polar regions of B. subtilis and, thus, contributes for the spatial precision of division. Our results showed that the function of YpsB must be different from that of DivIVA, since analysis of the ypsB- mutant showed that in the absence this protein the divisome is assembled and septum position in vegetatively growing or sporulating cells is not affected. Since the absence of YpsB does not affect division, we combined the ypsB- mutant with mutants involved in division. Analysis of these double mutants showed that the simultaneous absence of YpsB and FtsA caused cellular lysis and lethality. Based on this phenotype and evolutionary evidences, we suggest that YpsB is involved in the regulation of peptidoglycan synthesis in the septum. More specifically, YpsB would be responsible for modulating the activity of PBP1, a necessary enzyme for septum peptidoglycan synthesis.
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Characterization of two Bacillus subtilis penicillin-binding protein-coding genes, ykuA (pbpH) and yrrR (pbpI)Wei, Yuping 06 September 2002 (has links)
Penicillin-binding proteins (PBPs) are required in the synthesis of the cell wall of bacteria. In Bacillus subtilis, PBPs play important roles in the life cycle, including both vegetative growth and sporulation, and contribute to the formation of the different structures of vegetative cell wall and spore cortex. The B. subtilis genome sequencing project revealed there were two uncharacterized genes, ykuA and yrrR, with extensive sequence similarity to class B PBPs. These two genes are renamed and referred to henceforth as pbpH and pbpI, respectively.
A sequence alignment of the predicted product of pbpH against the microbial protein database demonstrated that the most similar protein in B. subtilis is PBP2A and in E. coli is PBP2. This suggested that PbpH belongs to a group of the genes required for maintaining the rod shape of the cell. Study of a pbpH-lacZ fusion showed that pbpH was expressed weakly during vegetative growth and the expression reached the highest level at the transition from exponential phase to stationary phase. The combination of a pbpA deletion and the pbpH deletion was lethal and double mutant strains lacking pbpH and pbpC or pbpI (also named yrrR) were viable. The viable mutants were indistinguishable from the wild-type except that the vegetative PG of the pbpC pbpH strain had a slightly slightly lower amount of disaccharide tetrapeptide with 1 amidation and higher amount of disaccharide tripeptide tetrapeptide with 2 amidations when compared to others strains. This suggests that PbpC (PBP3) is involved in vegetative PG synthesis but only affects the PG structure with a very low efficiency.
A pbpA pbpH double mutant containing a xylose-regulated pbpH gene inserted into the chromosome at the amyE locus was constructed. Depletion of PbpH resulted in an arrest in cell growth and a dramatic morphological change in both vegetative cells and outgrowing spores. Vegetative cells lacking pbpA and pbpH expression swelled and cell elongation was arrested, leading to the formation of pleiomorphic spherical cells and eventual lysis. In these cells, cell septations were randomly localized, cell walls and septa were thicker than those seen in wild type cells, and the average cell width and volume were larger than those of cells expressing pbpA or pbpH. The vegetative PG had an increased abundance of one unidentified muropeptide. Spores produced by the pbpA pbpH double mutant were able to initiate germination but the transition of the oval-shaped spores to rod-shape cells was blocked. The outgrowing cells were spherical, gradually enlarged, and eventually lysed. Outgrowth of these spores in the presence of xylose led to the formation of helical cells. Thus, PbpH is apparently required for maintenance of cell shape, specifically for cell elongation. PbpH and PBP2a play a redundant role homologous to that of PBP2 in E. coli.
A sequence alignment of the predicted product of pbpI against the microbial protein database demonstrated that the most similar protein in B. subtilis is SpoVD and in E. coli is PBP3. This suggested that PbpI belongs to the group of the genes required for synthesis of the spore or septum PG. PbpI was identified using radio-labeled penicillin and found to run underneath PBP4 on SDS-PAGE. PbpI is therefore renamed PBP4b. Study of a pbpI-lacZ fusion showed that pbpI was expressed predominantly during early sporulation. A putative sigma F recognition site is present in the region upstream of pbpI and studies using mutant strains lacking sporulation-specific sigma factors demonstrated that the expression of pbpI is mainly dependent on sigma factor F. A pbpI single mutant, a pbpI pbpG double mutant, and a pbpI pbpF double mutant were indistinguishable from the wild-type. The sporulation defect of a pbpI pbpF pbpG triple mutant was indistinguishable from that of a pbpF pbpG double mutant. Structure parameters of the forespore PG in a pbpI spoVD strain are similar to that of a spoVD strain. These results indicate that PBP4b plays a unknown redundant role. / Master of Science
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Requirements for Compartmentalization of Penicillin-Binding Proteins during Sporulation in Bacillus subtilisDean, Amanda Marie 06 January 2003 (has links)
Penicillin-binding proteins (PBP's) are membrane-associated enzymes involved in the polymerization of peptidoglycan. PBP's are divided into three classes based upon their molecular weights and functional domains. Gene expression is regulated in the two differentiated cells in Bacillus subtilis, the mother cell and the forespore, by coordinated expression of different sigma factors that recognize specific promoters in each compartment. The functional and compartmental specificity of individual penicillin-binding proteins from the different classes of PBP's were examined during sporulation in B. subtilis. Analyses of three class A high molecular weight PBP's indicated that pbpF and pbpG must be expressed in the forespore to carry out their specific role during spore peptidoglycan synthesis. Expressing pbpD in either the forespore or the mother cell could not complement for the loss of pbpF and pbpG, suggesting that there must be additional sequence information in PBP2c and PBP2d that allows them to carry out their specific role during germ cell wall synthesis. Analyses of a low molecular weight PBP, PBP5*, suggested that expressing dacB in either the mother cell or in the forespore could regulate the level of spore peptidoglycan cross-linking to what is typical of wild type spore peptidoglycan. / Master of Science
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Using Live Cell Imaging to Probe Biogenesis of the Gram-Negative Cell EnvelopeYao, Zhizhong January 2012 (has links)
In Gram-negative bacteria, the three-layered cell envelope, including the cell wall, outer and inner membranes, is essential for cell survival in the changing, and often hostile environments. Conserved in all prokaryotes, the cell wall is incredibly thin, yet it functions to prevent osmotic lysis in diluted conditions. Based on observations obtained by genetic and chemical perturbations, time-lapse live cell imaging, quantitative imaging and statistical analysis, Part I of this dissertation explores the molecular and physical events leading to cell lysis induced by division-specific beta-lactams. We found that such lysis requires the complete assembly of all essential components of the cell division apparatus and the subsequent recruitment of hydrolytic amidases. We propose that division-specific beta-lactams lyze cells by inhibiting FtsI (PBP3) without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. On the other hand, we demonstrated that cell lysis by beta-lactams proceeds through four physical phases: elongation, bulge formation, bulge stagnation and lysis. Bulge formation dynamics is determined by the specific perturbation of the cell wall and outer membrane plays an independent role in stabilizing the bulge once it is formed. The stabilized bulge delays lysis, and allows escape and recovery upon drug removal. Asymmetrical in structure and unique to Gram-negative bacteria, outer membrane prevents the passage of many hydrophobic, toxic compounds. Together with inner membrane and the cell wall, three layers of the Gram-negative cell envelope must be well coordinated throughout the cell cycle to allow elongation and division. Part II of this dissertation explores the essentiality of the LPS layer, the outer leaflet of the outer membrane. Using a conditional mutant severely defective in LPS transport, we found that mutations in the initiation phase of fatty acid synthesis suppress cells defective in LPS transport. The suppressor cells are remarkably small with a 70% reduction in cell volume and a 50 % reduction in growth rate. They are also blind to nutrient excess with respect to cell size control. We propose a model where fatty acid synthesis regulates cell size in response to nutrient availability, thereby influencing growth rate. / Chemistry and Chemical Biology
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Antibiotic-induced Bacterial Toxin Release – Inhibition by Protein Synthesis InhibitorsHjerdt-Goscinski, Gunilla January 2004 (has links)
<p>Toxic products, such as endotoxin from the gram-negative and exotoxin from the gram-positive bacteria, are the most important initiators of the inflammatory host response in sepsis. In addition to antibacterial treatment, numerous attempts have been made to interfere with the exaggerated proinflammatory cascade initiated by the toxins. As most antitoxic and anti-inflammatory agents have shown no clear efficacy, an attractive alternative has been to prevent or minimise their release. Therefore, it was of interest to further study the antibiotic-induced release of toxins after exposure to antibiotics used for the treatment of the most severe infections, especially if protein synthesis inhibitors could reduce the release induced by PBP 3-specific β-lactam antibiotics.</p><p>There were significant reductions in endotoxin release from gram-negative bacteria when the combination of the PBP 3-specific β-lactam antibiotic, cefuroxime, and the protein synthesis inhibitor, tobramycin, was compared with cefuroxime alone. Increasing doses of tobramycin reduced endotoxin release and increased the killing rate. In a kinetic <i>in vitro</i> model the endotoxin release from <i>E.coli</i> was higher after the second dose of cefuroxime. Nevertheless, it was reduced after addition of tobramycin.</p><p>No binding of tobramycin to endotoxin was observed, either <i>in vivo</i> or <i>in vitro</i>. In a porcine sepsis model, a possible anti-inflammatory effect of ceftazidime and tobramycin, expressed as late cytokine inhibition, was seen.</p><p>The protein synthesis inhibitor, clindamycin, released less streptococcal pyrogenic exotoxin A (SpeA) from a group A streptococcus strain than penicillin, and addition of clindamycin to penicillin resulted in less toxin production than penicillin alone. The SpeA production was dependent on the bacterial number at the start of treatment. Higher doses of penicillin also led to less SpeA. </p><p>The choice of antibiotic class and dose may be important in the severely ill septic patient in whom an additional toxin release could be deleterious. A combination of a β-lactam antibiotic and a protein synthesis inhibitor seems beneficial but further investigations are needed.</p>
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Antibiotic-induced Bacterial Toxin Release – Inhibition by Protein Synthesis InhibitorsHjerdt-Goscinski, Gunilla January 2004 (has links)
Toxic products, such as endotoxin from the gram-negative and exotoxin from the gram-positive bacteria, are the most important initiators of the inflammatory host response in sepsis. In addition to antibacterial treatment, numerous attempts have been made to interfere with the exaggerated proinflammatory cascade initiated by the toxins. As most antitoxic and anti-inflammatory agents have shown no clear efficacy, an attractive alternative has been to prevent or minimise their release. Therefore, it was of interest to further study the antibiotic-induced release of toxins after exposure to antibiotics used for the treatment of the most severe infections, especially if protein synthesis inhibitors could reduce the release induced by PBP 3-specific β-lactam antibiotics. There were significant reductions in endotoxin release from gram-negative bacteria when the combination of the PBP 3-specific β-lactam antibiotic, cefuroxime, and the protein synthesis inhibitor, tobramycin, was compared with cefuroxime alone. Increasing doses of tobramycin reduced endotoxin release and increased the killing rate. In a kinetic in vitro model the endotoxin release from E.coli was higher after the second dose of cefuroxime. Nevertheless, it was reduced after addition of tobramycin. No binding of tobramycin to endotoxin was observed, either in vivo or in vitro. In a porcine sepsis model, a possible anti-inflammatory effect of ceftazidime and tobramycin, expressed as late cytokine inhibition, was seen. The protein synthesis inhibitor, clindamycin, released less streptococcal pyrogenic exotoxin A (SpeA) from a group A streptococcus strain than penicillin, and addition of clindamycin to penicillin resulted in less toxin production than penicillin alone. The SpeA production was dependent on the bacterial number at the start of treatment. Higher doses of penicillin also led to less SpeA. The choice of antibiotic class and dose may be important in the severely ill septic patient in whom an additional toxin release could be deleterious. A combination of a β-lactam antibiotic and a protein synthesis inhibitor seems beneficial but further investigations are needed.
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Pneumococcus morphogenesis and resistance to beta-lactams / Morphogenèse du pneumocoque et résistance aux bêta-lactaminesPhilippe, Jules 29 September 2014 (has links)
Streptococcus pneumoniae, le pneumocoque, est une bactérie pathogène qui entraîne le décès de plus d'un million et demi de personnes dans le monde chaque année. Les β-lactamines sont très utilisées pour traiter les infections à pneumocoques. Ces antibiotiques inhibent la synthèse du peptidoglycane, une molécule géante constituant un réseau de chaînes glycopeptidiques qui englobe la cellule, lui confère sa forme et lui permet de maintenir son intégrité face à la pression osmotique. Le mécanisme d'action des β-lactamines est bien connud'un point de vue biochimique. En revanche, la réponse physiologique empêchant la multiplication des bactéries traitées est mal connue. Au cours de ma thèse, j'ai étudié les mécanismes moléculaires de la morphogenèse du pneumocoque par des approches de biochimie et de microbiologie. Un modèle de morphogenèse est proposé intégrant mes résultats à la littérature et permettant de formuler des hypothèses sur la réponse physiologique de S. pneumoniae aux β-lactamines. / Streptococcus pneumoniae, the pneumococcus, is a bacterial pathogen that causes more than 1.5 million deaths each year in the world. β-Lactams are widely used to treat patients with pneumococcal infections. These antibiotics inhibit the synthesis of the peptidoglycan, a giant molecule constituting a mesh of aminosugar strands encasing the cell. This main constituent of the cell wall allows cells to maintain their integrity under the turgor pressure, and endows bacteria with their shape. The action of β-lactams is well understood from a biochemical point of view. However, a complete understanding of the physiological response of treated bacteria remains elusive. In this thesis, I investigated the molecular mechanisms of the morphogenesis of S. pneumoniae using methods of biochemistry and microbiology. A morphogenesis model is built based on my results and the literature, which permits to emit hypotheses concerning the response of the pneumococcus to β-lactams.
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