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Kristallstruktur eines mycobacteriellen PorinsFaller, Michael. January 2004 (has links) (PDF)
Freiburg (Breisgau), Universiẗat, Diss., 2004.
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Expression der Poren bildenden Hämolysine Listeriolysin und TlyA in Mycobacterium smegmatisBerger, Sven. January 2002 (has links) (PDF)
Köln, Universiẗat, Diss., 2002.
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Characterization of LYTM domain containing proteins in mycobacterium smegmatisShaku, Tube Moagi January 2017 (has links)
A dissertation submitted to the Faculty of Health Science, University of the Witwatersrand,
Johannesburg, in fulfillment of the requirements for the degree of Master of Science in
Medicine.
2017 / Mycobacterium tuberculosis assembles a complex cell wall consisting of mycolic acids, arabinogalactan and peptidoglycan layers. The peptidoglycan is important for structural maintenance and osmotic protection of the cell. Beta-lactam antibiotics, such as penicillin, perturb biogenesis of cross-linked peptidoglycan by inhibition of penicillin-binding proteins and cause cell death. As a result, penicillin-binding proteins have been extensively used in antimicrobial development. However, penicillin insensitive enzymes involved in peptidoglycan biogenesis such as amidases, transglycosylases and endopeptidases remain to be exploited for anti-TB drug development, a field that urgently requires new drugs in light of the rapid emergence of drug resistant strains. In this study, we functionally characterize a novel class of LytM domain containing peptidoglycan endopeptidases (also known as M23 peptidases) in mycobacteria. Bio-informatics tools were used to identify LytM domain-containing homologues in Mycobacterium smegmatis, designated MepB1-MepB4. These were deleted using standard allelic exchange mutagenesis and recombination techniques and the resulting mutants were assessed for cell wall related defects. We found that mycobacterial LytM endopeptidases have important roles in bacterial growth as demonstrated by delayed cell growth kinetics in a ΔmepB1 deletion mutant. We noted no growth defects in ΔmepB2 and ΔmepB3 single deletion mutants but observed defective cell division in a ΔmepB2 ΔmepB3 double deletion mutant. In this double mutant, spatial localization of new cell wall biosynthesis revealed the inability to degrade the septal bridge joining two daughter cells, pointing to a critical role for these enzymes in cell separation. MepB1 is sequestered from the peptidoglycan by cytosolic localization and its absence causes a septal and polar buldging phenotype. To further investigate the biological roles of these putative peptidoglycan endopeptidases, protein interaction studies were conducted using the bacterial two-hybrid mycobacterial protein fragment complementation assay. This analysis identified FtsX, a key cell division protein, as an interacting partner for both MepB2 and MepB3, thus identifying these proteins as novel components of the mycobacterial divisome. Collectively, these observations provide the first insight into a new group of potential drug targets for tuberculosis disease and notably enhance the overall understanding of peptidoglycan turnover, which is of general revelevance in bacterial pathogens. / MT2017
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Characterization of resuscitation promoting factors in Mycobacterium smegmatisMapela, Lusanda Thato 12 September 2012 (has links)
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) has
infected one third of the world’s population and continues to claim more lives
annually than any other infectious disease agent. A significant proportion of
individuals carry latent TB infection (LBTI) which is characterized by the absence
of clinical symptoms and it has been postulated that the tubercle bacilli are in a
dormant-like state during this type of infection. Resuscitation promoting factors
(Rpfs) are cell wall hydrolases which cleave glycosidic bonds within the
peptidoglycan (PG), a mechanism thought to result in reactivation of bacteria
from the state of dormancy. M. tuberculosis encodes five rpf-like homologues
which are collectively dispensable for growth but are required for reactivation
from dormancy in vitro, and for virulence in the mouse model of infection. LTBI
thus poses a huge threat to the global burden of active disease. The purpose of
this study was to further investigate the biological roles of Rpfs by assessing the
effects of rpf gene deletion in M. smegmatis, a model organism used for TB
research. M. smegmatis encodes four rpf-like genes designated rpfA, rpfB, rpfC
and rpfE, and deletion mutants that lack one or more of these genes were
constructed by allelic exchange mutagenesis. M. smegmatis mutant strains that
lack either rpfA or rpfB display no significant differences in growth both on solid
and in liquid medium when compared to wild type. However, loss of rpfA resulted
in bacterial clumping during stationary-phase growth in broth culture and
changes in cell morphology. Moreover; the rpfA rpfB double mutant and its
derivative strain lacking rpfC, rpfA rpfB rpfC displayed a ca. 2-4 log increase
in susceptibility to erythromycin and vancomycin. Furthermore, unusual colonial
morphologies with reduced serpentine cording and smooth peripheries were
observed for these multiple mutants. Cell surface defects and cell distortions
were evidenced as wrinkle-textured, bent cells and polar tip bulges for the
abovementioned mutants. The multiple deletion strains also displayed a defect in
biofilm formation revealing an inability for the mutants to form complex cell-cell
interactions. Collectively, the data are suggestive of a loss of bacterial cell wall integrity due to rpf gene deletion and it is proposed that rpfA, in combination with
other genes, is largely responsible for cell wall integrity maintenance. Our data
indicate that these factors play an important role in cell growth and division and
therefore represent an untapped source of novel targets for anti-tubercular drug
discovery.
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Biosynthesis of phospholipid in Mycobacterium smegmatis.Lee, Young Soon 01 January 1971 (has links) (PDF)
No description available.
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A preliminary investigation of a sialidase activity associated with M. smegmatisTrower, Carolyn Joy, 1975- January 2003 (has links)
Abstract not available
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Construção e estudo de mutantes envolvidos na biossíntese de aminoácidos aromáticos e purinas em Mycobacterium tuberculosis e Mycobacterium smegmatis por troca alélicaSchneider, Cristopher Zandoná January 2007 (has links)
A tuberculose (TB) é uma séria doença infecciosa causada por Mycobacterium tuberculosis, e constitui um importante problema de saúde pública em todo o mundo. Novas drogas e vacinas de segunda geração são urgentemente necessárias para o controle da TB, mas a complexa biologia de M. tuberculosis tem dificultado o desenvolvimento de estratégias terapêuticas inovadoras. A manipulação genética de M. tuberculosis também é complicada, mas, atualmente, técnicas novas e mais eficientes de troca alélica permitem o estudo detalhado de diversos genes micobacterianos. No presente trabalho, os genes da corismato mutase (CM) e fosforilase de nucleosídeos purínicos (PNP) de M. tuberculosis e Mycobacterium smegmatis foram estudados. A CM catalisa a conversão de corismato em prefenato na rota de biossíntese dos aminoácidos aromáticos fenilalanina e tirosina em bactérias, fungos e plantas. Dois genes de CMs monofuncionais (aroQ e *aroQ) foram identificados, clonados, expressos e bioquimicamente caracterizados em ambas as espécies de micobactérias. Esses genes também foram investigados usando uma metodologia de recombinação homóloga e ensaios de atividade promotora. Os resultados indicam que os genes aroQ são provavelmente essenciais ao crescimento in vitro de M. tuberculosis e M. smegmatis, enquanto uma linhagem mutante para o gene *aroQ de M. smegmatis pôde ser obtida. A PNP catalisa a interconversão e reciclagem de bases, nucleosídeos e nucleotídeos purínicos na via de salvamento das purinas. A construção de mutantes para o gene deoD (que codifica a PNP) de M. tuberculosis e M. smegmatis, usando um método eficiente de troca alélica em duas etapas, não foi possível. Assim, sugere-se que, nas condições testadas, o gene deoD seja essencial ao crescimento in vitro dessas micobactérias. A identificação de genes essenciais é de fundamental importância, pois indica tanto a relevância biológica dos mesmos como, no caso de M. tuberculosis, que seus produtos constituem alvos moleculares interessantes para o desenvolvimento de novas drogas antimicobacterianas. / Tuberculosis (TB), a serious infectious disease caused by Mycobacterium tuberculosis, still remains a public health problem in the world. New drugs and second generation vaccines are urgently required to control TB, but the complex biology of M. tuberculosis has hindered the development of novel therapeutic tools. Genetic manipulation of M. tuberculosis is also difficult, but currently new, more efficient gene replacement techniques have allowed detailed studies of many mycobacterial genes. In the present work, the chorismate mutase (CM) and purine nucleoside phosphorylase (PNP) genes from M. tuberculosis and Mycobacterium smegmatis were studied. CM catalyzes the rearrangement of chorismate to prephenate in the biosynthetic pathway that forms phenylalanine and tyrosine in bacteria, fungi, and plants. Two monofunctional CM genes (aroQ and *aroQ) were identified, cloned, expressed, and biochemically characterized in both mycobacteria. Those genes were also investigated by homologous recombination methods and promoter activity assays. AroQ genes seem to be essential for the growth of M. tuberculosis and M. smegmatis, while an *aroQ mutant strain could be generated in M. smegmatis. PNP promotes the interconversion and recycling of purine bases, nucleosides, and nucleotides in the purine salvage pathway. Construction of deoD (which codes for PNP) deletion mutants of M. tuberculosis and M. smegmatis using an efficient two-step gene replacement technique was not possible. Thus, it is suggested that deoD is also essential for mycobacterial growth under the conditions tested. The identification of essential genes is of great importance, both because it indicates their biological significance, and because, with pathogens like M. tuberculosis, these may also indicate attractive molecular targets for the development of new antimycobacterial drugs.
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Construção e estudo de mutantes envolvidos na biossíntese de aminoácidos aromáticos e purinas em Mycobacterium tuberculosis e Mycobacterium smegmatis por troca alélicaSchneider, Cristopher Zandoná January 2007 (has links)
A tuberculose (TB) é uma séria doença infecciosa causada por Mycobacterium tuberculosis, e constitui um importante problema de saúde pública em todo o mundo. Novas drogas e vacinas de segunda geração são urgentemente necessárias para o controle da TB, mas a complexa biologia de M. tuberculosis tem dificultado o desenvolvimento de estratégias terapêuticas inovadoras. A manipulação genética de M. tuberculosis também é complicada, mas, atualmente, técnicas novas e mais eficientes de troca alélica permitem o estudo detalhado de diversos genes micobacterianos. No presente trabalho, os genes da corismato mutase (CM) e fosforilase de nucleosídeos purínicos (PNP) de M. tuberculosis e Mycobacterium smegmatis foram estudados. A CM catalisa a conversão de corismato em prefenato na rota de biossíntese dos aminoácidos aromáticos fenilalanina e tirosina em bactérias, fungos e plantas. Dois genes de CMs monofuncionais (aroQ e *aroQ) foram identificados, clonados, expressos e bioquimicamente caracterizados em ambas as espécies de micobactérias. Esses genes também foram investigados usando uma metodologia de recombinação homóloga e ensaios de atividade promotora. Os resultados indicam que os genes aroQ são provavelmente essenciais ao crescimento in vitro de M. tuberculosis e M. smegmatis, enquanto uma linhagem mutante para o gene *aroQ de M. smegmatis pôde ser obtida. A PNP catalisa a interconversão e reciclagem de bases, nucleosídeos e nucleotídeos purínicos na via de salvamento das purinas. A construção de mutantes para o gene deoD (que codifica a PNP) de M. tuberculosis e M. smegmatis, usando um método eficiente de troca alélica em duas etapas, não foi possível. Assim, sugere-se que, nas condições testadas, o gene deoD seja essencial ao crescimento in vitro dessas micobactérias. A identificação de genes essenciais é de fundamental importância, pois indica tanto a relevância biológica dos mesmos como, no caso de M. tuberculosis, que seus produtos constituem alvos moleculares interessantes para o desenvolvimento de novas drogas antimicobacterianas. / Tuberculosis (TB), a serious infectious disease caused by Mycobacterium tuberculosis, still remains a public health problem in the world. New drugs and second generation vaccines are urgently required to control TB, but the complex biology of M. tuberculosis has hindered the development of novel therapeutic tools. Genetic manipulation of M. tuberculosis is also difficult, but currently new, more efficient gene replacement techniques have allowed detailed studies of many mycobacterial genes. In the present work, the chorismate mutase (CM) and purine nucleoside phosphorylase (PNP) genes from M. tuberculosis and Mycobacterium smegmatis were studied. CM catalyzes the rearrangement of chorismate to prephenate in the biosynthetic pathway that forms phenylalanine and tyrosine in bacteria, fungi, and plants. Two monofunctional CM genes (aroQ and *aroQ) were identified, cloned, expressed, and biochemically characterized in both mycobacteria. Those genes were also investigated by homologous recombination methods and promoter activity assays. AroQ genes seem to be essential for the growth of M. tuberculosis and M. smegmatis, while an *aroQ mutant strain could be generated in M. smegmatis. PNP promotes the interconversion and recycling of purine bases, nucleosides, and nucleotides in the purine salvage pathway. Construction of deoD (which codes for PNP) deletion mutants of M. tuberculosis and M. smegmatis using an efficient two-step gene replacement technique was not possible. Thus, it is suggested that deoD is also essential for mycobacterial growth under the conditions tested. The identification of essential genes is of great importance, both because it indicates their biological significance, and because, with pathogens like M. tuberculosis, these may also indicate attractive molecular targets for the development of new antimycobacterial drugs.
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Construção e estudo de mutantes envolvidos na biossíntese de aminoácidos aromáticos e purinas em Mycobacterium tuberculosis e Mycobacterium smegmatis por troca alélicaSchneider, Cristopher Zandoná January 2007 (has links)
A tuberculose (TB) é uma séria doença infecciosa causada por Mycobacterium tuberculosis, e constitui um importante problema de saúde pública em todo o mundo. Novas drogas e vacinas de segunda geração são urgentemente necessárias para o controle da TB, mas a complexa biologia de M. tuberculosis tem dificultado o desenvolvimento de estratégias terapêuticas inovadoras. A manipulação genética de M. tuberculosis também é complicada, mas, atualmente, técnicas novas e mais eficientes de troca alélica permitem o estudo detalhado de diversos genes micobacterianos. No presente trabalho, os genes da corismato mutase (CM) e fosforilase de nucleosídeos purínicos (PNP) de M. tuberculosis e Mycobacterium smegmatis foram estudados. A CM catalisa a conversão de corismato em prefenato na rota de biossíntese dos aminoácidos aromáticos fenilalanina e tirosina em bactérias, fungos e plantas. Dois genes de CMs monofuncionais (aroQ e *aroQ) foram identificados, clonados, expressos e bioquimicamente caracterizados em ambas as espécies de micobactérias. Esses genes também foram investigados usando uma metodologia de recombinação homóloga e ensaios de atividade promotora. Os resultados indicam que os genes aroQ são provavelmente essenciais ao crescimento in vitro de M. tuberculosis e M. smegmatis, enquanto uma linhagem mutante para o gene *aroQ de M. smegmatis pôde ser obtida. A PNP catalisa a interconversão e reciclagem de bases, nucleosídeos e nucleotídeos purínicos na via de salvamento das purinas. A construção de mutantes para o gene deoD (que codifica a PNP) de M. tuberculosis e M. smegmatis, usando um método eficiente de troca alélica em duas etapas, não foi possível. Assim, sugere-se que, nas condições testadas, o gene deoD seja essencial ao crescimento in vitro dessas micobactérias. A identificação de genes essenciais é de fundamental importância, pois indica tanto a relevância biológica dos mesmos como, no caso de M. tuberculosis, que seus produtos constituem alvos moleculares interessantes para o desenvolvimento de novas drogas antimicobacterianas. / Tuberculosis (TB), a serious infectious disease caused by Mycobacterium tuberculosis, still remains a public health problem in the world. New drugs and second generation vaccines are urgently required to control TB, but the complex biology of M. tuberculosis has hindered the development of novel therapeutic tools. Genetic manipulation of M. tuberculosis is also difficult, but currently new, more efficient gene replacement techniques have allowed detailed studies of many mycobacterial genes. In the present work, the chorismate mutase (CM) and purine nucleoside phosphorylase (PNP) genes from M. tuberculosis and Mycobacterium smegmatis were studied. CM catalyzes the rearrangement of chorismate to prephenate in the biosynthetic pathway that forms phenylalanine and tyrosine in bacteria, fungi, and plants. Two monofunctional CM genes (aroQ and *aroQ) were identified, cloned, expressed, and biochemically characterized in both mycobacteria. Those genes were also investigated by homologous recombination methods and promoter activity assays. AroQ genes seem to be essential for the growth of M. tuberculosis and M. smegmatis, while an *aroQ mutant strain could be generated in M. smegmatis. PNP promotes the interconversion and recycling of purine bases, nucleosides, and nucleotides in the purine salvage pathway. Construction of deoD (which codes for PNP) deletion mutants of M. tuberculosis and M. smegmatis using an efficient two-step gene replacement technique was not possible. Thus, it is suggested that deoD is also essential for mycobacterial growth under the conditions tested. The identification of essential genes is of great importance, both because it indicates their biological significance, and because, with pathogens like M. tuberculosis, these may also indicate attractive molecular targets for the development of new antimycobacterial drugs.
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Identification and isolation of growth-phase specific proteins of mycobacteriaBettoni, Jane Clementina 12 July 2017 (has links)
The aim of this project was to identify growth phase-specific heat shock proteins of Mycobacterium smegmatis LR222. A growth curve was constructed using the ATP assay. This method was shown by B. A. Ntolosi to be the most accurate indicator of when the organism entered the various phases of growth. It was possible to determine that M. smegmatis LR222 entered the exponential phase of growth after a short lag phase of 4 to 8 hours and persisted in this phase for 20 to 22 hours. It then reached the stationary phase, which lasted for 40 to 46 hours. Protein heat shock assays were performed on growth phase-specific samples. This allowed the identification of a 43-46 kDa in molecular weight stationary phase protein on one-dimensional SOS-PAGE. The protein was induced in cells entering the stationary phase of growth and not by heat shock as it was induced under both the control and the heat shock temperatures. The protein was further characterised by two-dimensional gel electrophoresis, which demonstrated resolution into two, strongly age-associated, proteins. Subtractive RNA hybridisation was attempted in order to obtain a subtraction cDNA probe from a stationary phase RNA sample depleted of sequences common in both the exponential and the stationary phase samples. Ribosomal RNA was removed from the total RNA by the process of photobiotinilation. The mRNA was then used as a template to synthesise with reverse transcriptase single stranded cDNA. cDNA/mRNA denatured hybrids were hybridised to the exponential phase RNA sample. The new hybrids were "subtracted" by chemical cross-linking with DZQ and the unique cDNA used to produce by random primers a radioactively labelled probe. A M. smegmatis library was probed but unfortunately no signal was observed. Further adjustments and improvements to this technique are required before it can be used effectively.
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