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University of Rochester
1.
Martinelli, Daniel J.
Examination of the Roles of the Endopeptidase RipA and
the Penicillin Binding Protein PonA2 in Mycobacterial Cell Wall
Maintenance.
Degree: PhD, 2015, University of Rochester
URL: http://hdl.handle.net/1802/29783 
► Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has infected nearly one-third of the world’s populace with millions of new cases arising each year. Recently,…
(more)
▼ Mycobacterium tuberculosis (Mtb), the causative
agent of tuberculosis, has infected nearly one-third of the world’s
populace with millions of new cases arising each year. Recently,
there has been an emergence of multi-drug resistant and
extensively-drug resistant strains, which hasten our need to find
new drug targets to fight Mtb.
Mtb has a complex and lipid rich
cell envelope, comprised of the
mycolyl-arabinogalactan-peptidoglycan complex, the biosynthesis of
which is the target of several antibiotics. However, most
antitubercular drugs are only effective against growing cells, and
thus do not affect non-replicating Mtb bacilli present in latent
tuberculosis infections. We propose that enzymes involved with the
maintenance of the cell envelope could be exploited as potential
drug targets.
Drug targets that fit this criterion are
peptidoglycan (PG) endopeptidases. These enzymes cleave peptide
bonds between glycan strands to remodel PG. The two most
well-studied mycobacterial endopeptidases are RipA and RipB, which
are encoded in a bicistronic operon. There is some controversy in
the field about the essentiality of the ripA gene in mycobacteria.
It has been shown that the endopeptidase RipA interacts with a
class A penicillin binding protein (PBP) PonA1, which synthesizes
PG. This interaction between opposing role proteins is antagonistic
whereby the PonA1 inhibits RipA activity through interactions
between both proteins’ C-terminal domains. PonA2, another class A
PBP, also maintains a similar C-terminal domain to that of PonA1, a
proline rich region (PRR). An in-frame ponA2 deletion strain
created in our lab displays phenotypes that could be a result of
unregulated cell wall degradation due to appearance of a spherical
morphology and inability to recover from stationary phase. We think
that RipA may be responsible for this phenotype.
Here we show that
neither ripA nor ripB is individually essential to M. smegmatis,
but that the organism requires at least one of the genes to
survive. We characterized the ripA and ripB deletion mutants, and
our data suggests that RipA interacts with both PonA1 and PonA2 via
PRRs found in the C-termini of both enzymes. Additionally, we found
that RipA and RipB were responsible for the ΔponA2 phenotype in
conditions of non-replication.
Subjects/Keywords: Peptidoglycan; Mycobacterium Smegmatis
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APA (6th Edition):
Martinelli, D. J. (2015). Examination of the Roles of the Endopeptidase RipA and
the Penicillin Binding Protein PonA2 in Mycobacterial Cell Wall
Maintenance. (Doctoral Dissertation). University of Rochester. Retrieved from http://hdl.handle.net/1802/29783
Chicago Manual of Style (16th Edition):
Martinelli, Daniel J. “Examination of the Roles of the Endopeptidase RipA and
the Penicillin Binding Protein PonA2 in Mycobacterial Cell Wall
Maintenance.” 2015. Doctoral Dissertation, University of Rochester. Accessed December 10, 2019.
http://hdl.handle.net/1802/29783.
MLA Handbook (7th Edition):
Martinelli, Daniel J. “Examination of the Roles of the Endopeptidase RipA and
the Penicillin Binding Protein PonA2 in Mycobacterial Cell Wall
Maintenance.” 2015. Web. 10 Dec 2019.
Vancouver:
Martinelli DJ. Examination of the Roles of the Endopeptidase RipA and
the Penicillin Binding Protein PonA2 in Mycobacterial Cell Wall
Maintenance. [Internet] [Doctoral dissertation]. University of Rochester; 2015. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/1802/29783.
Council of Science Editors:
Martinelli DJ. Examination of the Roles of the Endopeptidase RipA and
the Penicillin Binding Protein PonA2 in Mycobacterial Cell Wall
Maintenance. [Doctoral Dissertation]. University of Rochester; 2015. Available from: http://hdl.handle.net/1802/29783
Cornell University
2.
Hawkins, Benjamin.
Characterization And Design Of Insulator-Based Dielectrophoresis Devices For Continuous-Flow Particle Enrichment And Automated Electrode-Based Dielectrophoretic Characterization Of Mycobacterium Smegmatis
.
Degree: 2011, Cornell University
URL: http://hdl.handle.net/1813/29232
► In this work we detail the development of insulator-based dielectrophoresis devices to be used for enrichment of cellular subpopulations with phenotypic differences in membrane composition.…
(more)
▼ In this work we detail the development of insulator-based dielectrophoresis devices to be used for enrichment of cellular subpopulations with phenotypic differences in membrane composition. We present a novel insulator-based dielectrophoresis device design that incorporates coherently patterned three-dimensional channel constrictions and demonstrate continuous flow particle separation based on dielectrophoretic mobility. Experimental and numerical techniques were used to characterize the effects of channel geometry on particle motion and distilled to a set of design criteria for similar devices. We also detail further characterization of fluid motion due to electrothermal fluid body forces via numeric multiphysics simulations. These simulations model heat transport via convection through the channel and conduction through the substrate material and consider the coupling between fluid, electrical, and thermal equation systems via temperature-dependent material properties. Finally, we discuss the development of an automated electrode-based dielectrophoresis device for cell characterization and its application to Escherichia coli as well as wild-type and ethambutol-treated
Mycobacterium smegmatis. We discuss these results in the context of Mycobacterial physiology and the mechanism of action of ethambutol.
Advisors/Committee Members: Koch, Donald L (committeeMember), Russell, David G (committeeMember).
Subjects/Keywords: Dielectrophoresis;
Microfluidics;
Mycobacterium smegmatis
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APA (6th Edition):
Hawkins, B. (2011). Characterization And Design Of Insulator-Based Dielectrophoresis Devices For Continuous-Flow Particle Enrichment And Automated Electrode-Based Dielectrophoretic Characterization Of Mycobacterium Smegmatis
. (Thesis). Cornell University. Retrieved from http://hdl.handle.net/1813/29232
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Hawkins, Benjamin. “Characterization And Design Of Insulator-Based Dielectrophoresis Devices For Continuous-Flow Particle Enrichment And Automated Electrode-Based Dielectrophoretic Characterization Of Mycobacterium Smegmatis
.” 2011. Thesis, Cornell University. Accessed December 10, 2019.
http://hdl.handle.net/1813/29232.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Hawkins, Benjamin. “Characterization And Design Of Insulator-Based Dielectrophoresis Devices For Continuous-Flow Particle Enrichment And Automated Electrode-Based Dielectrophoretic Characterization Of Mycobacterium Smegmatis
.” 2011. Web. 10 Dec 2019.
Vancouver:
Hawkins B. Characterization And Design Of Insulator-Based Dielectrophoresis Devices For Continuous-Flow Particle Enrichment And Automated Electrode-Based Dielectrophoretic Characterization Of Mycobacterium Smegmatis
. [Internet] [Thesis]. Cornell University; 2011. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/1813/29232.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Hawkins B. Characterization And Design Of Insulator-Based Dielectrophoresis Devices For Continuous-Flow Particle Enrichment And Automated Electrode-Based Dielectrophoretic Characterization Of Mycobacterium Smegmatis
. [Thesis]. Cornell University; 2011. Available from: http://hdl.handle.net/1813/29232
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Otago
3.
Weimar, Marion Ricarda.
Regulation of Cytochrome bd Expression in Mycobacterium smegmatis
.
Degree: 2010, University of Otago
URL: http://hdl.handle.net/10523/359
► The metabolism and generation of energy by the majority of pathogenic bacteria in the host remains enigmatic and emerging evidence suggests that the identification of…
(more)
▼ The metabolism and generation of energy by the majority of pathogenic bacteria in the host remains enigmatic and emerging evidence suggests that the identification of essential bacterial metabolic pathways that have no human counterparts may represent a virtually untapped source of novel drug targets for antibiotic development. The aim of this study was to determine how mycobacteria metabolize and generate energy microaerobically. To identify potential regulatory circuits that control the response to low oxygen tension, we targeted the enzyme cytochrome bd oxidase, purportedly involved in the generation of energy under these conditions, and isolated mutants that fail to activate expression of this enzyme. Instead of finding regulators of cytochrome bd oxidase, our screen revealed enzymes and regulators involved in redox homeostasis and defence against electrophiles (oxidative stress). We discovered ProR, a novel regulator of proline dehydrogenase and 1-pyrroline-5-carboxylate dehydrogenase, two enzymes involved in proline degradation. Evidence is presented that ProR is essential for growth on proline as a sole carbon source and that a proR mutant experiences DNA damage due to methylglyoxal, an endogenously produced toxic intermediate of glycolysis and glycerol metabolism. It is shown that proline confers resistance to methylglyoxal. Furthermore, we characterized the sodium/proline symporter PutP as a high-affinity transporter for proline and show that it is the major uptake system for proline in
Mycobacterium smegmatis. A new model for methylglyoxal detoxification in Mycobacteria is presented.
Advisors/Committee Members: Cook, Gregory M (advisor).
Subjects/Keywords: Mycobacterium smegmatis;
cytochrome bd
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APA (6th Edition):
Weimar, M. R. (2010). Regulation of Cytochrome bd Expression in Mycobacterium smegmatis
. (Masters Thesis). University of Otago. Retrieved from http://hdl.handle.net/10523/359
Chicago Manual of Style (16th Edition):
Weimar, Marion Ricarda. “Regulation of Cytochrome bd Expression in Mycobacterium smegmatis
.” 2010. Masters Thesis, University of Otago. Accessed December 10, 2019.
http://hdl.handle.net/10523/359.
MLA Handbook (7th Edition):
Weimar, Marion Ricarda. “Regulation of Cytochrome bd Expression in Mycobacterium smegmatis
.” 2010. Web. 10 Dec 2019.
Vancouver:
Weimar MR. Regulation of Cytochrome bd Expression in Mycobacterium smegmatis
. [Internet] [Masters thesis]. University of Otago; 2010. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/10523/359.
Council of Science Editors:
Weimar MR. Regulation of Cytochrome bd Expression in Mycobacterium smegmatis
. [Masters Thesis]. University of Otago; 2010. Available from: http://hdl.handle.net/10523/359
Universidade do Rio Grande do Sul
4.
Schneider, Cristopher Zandoná.
Construção e estudo de mutantes envolvidos na biossíntese de aminoácidos aromáticos e purinas em Mycobacterium tuberculosis e Mycobacterium smegmatis por troca alélica.
Degree: 2007, Universidade do Rio Grande do Sul
URL: http://hdl.handle.net/10183/30206
► 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…
(more)
▼ 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…
Advisors/Committee Members: Santos, Diogenes Santiago, Basso, Luiz Augusto.
Subjects/Keywords: Mycobacterium tuberculosis; Mycobacterium smegmatis; Aminoácidos; Purinas
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APA (6th Edition):
Schneider, C. Z. (2007). Construção e estudo de mutantes envolvidos na biossíntese de aminoácidos aromáticos e purinas em Mycobacterium tuberculosis e Mycobacterium smegmatis por troca alélica. (Thesis). Universidade do Rio Grande do Sul. Retrieved from http://hdl.handle.net/10183/30206
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Schneider, Cristopher Zandoná. “Construção e estudo de mutantes envolvidos na biossíntese de aminoácidos aromáticos e purinas em Mycobacterium tuberculosis e Mycobacterium smegmatis por troca alélica.” 2007. Thesis, Universidade do Rio Grande do Sul. Accessed December 10, 2019.
http://hdl.handle.net/10183/30206.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Schneider, Cristopher Zandoná. “Construção e estudo de mutantes envolvidos na biossíntese de aminoácidos aromáticos e purinas em Mycobacterium tuberculosis e Mycobacterium smegmatis por troca alélica.” 2007. Web. 10 Dec 2019.
Vancouver:
Schneider CZ. Construção e estudo de mutantes envolvidos na biossíntese de aminoácidos aromáticos e purinas em Mycobacterium tuberculosis e Mycobacterium smegmatis por troca alélica. [Internet] [Thesis]. Universidade do Rio Grande do Sul; 2007. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/10183/30206.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Schneider CZ. Construção e estudo de mutantes envolvidos na biossíntese de aminoácidos aromáticos e purinas em Mycobacterium tuberculosis e Mycobacterium smegmatis por troca alélica. [Thesis]. Universidade do Rio Grande do Sul; 2007. Available from: http://hdl.handle.net/10183/30206
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
5.
Bakala n'goma, Jean-claude.
Etude In vitro de Phospholipases mycobactériennes impliquées dans la virulence : In vitro study of micobactérial Phospholipases involved in virulence.
Degree: Docteur es, Microbiologie, biologie végétale et biotechnologies, 2010, Aix-Marseille 2
URL: http://www.theses.fr/2010AIX22027
► Les phospholipases et en particulier les phospholipases C sont d'importants facteurs de virulence chez de nombreuses bactéries pathogènes (C. perfringens, B. Cereus et P. aeruginosa).…
(more)
▼ Les phospholipases et en particulier les phospholipases C sont d'importants facteurs de virulence chez de nombreuses bactéries pathogènes (C. perfringens, B. Cereus et P. aeruginosa). Cependant, peu de choses sont connues sur l'implication de ces enzymes dans le processus de virulence des mycobactéries. Bien que l'étude des mutants des phospholipases C de M. tuberculosis dans un modèle d'infection chez la souris ait permis de proposer une implication de ces protéines dans la virulence de ce bacille, leurs propriétés biochimiques, leur mode d'action et leur rôle physiologique exact restent à élucider. Ce manque de données biochimiques sur les phospholipases mycobactériennes peuvent être attribuée à la difficulté à produire et à purifier des quantités importantes de ces enzymes. Dans le but de mieux caractériser le rôle physiologique des phospholipase mycobactériennes, l'objectif de ma thèse a été de mettre au point des conditions d'expression hétérologue permettant la production des phospholipases C mycobactériennes recombinantes (rPLC) dans différents systèmes d'expression (E. coli, Pichia pastoris et baculovirus/cellules d'insectes). Ces systèmes d'expression n'ayant pas donné des résultats satisfaisants, nous avons développé une méthode efficace d'expression de ces protéines en utilisant M. smegmatis.Ce système d'expression nous a permis de produire et de purifier les quate PLC (PLC-A, PLC-B, PLC-C et PLC-D) de M. tuberculosis et la PLC de M. Abscessus sous forme soluble et active. Nous avons pour la première fois montré que ces protéines purifiées avaient un effet cytotoxique sur les macrophages de souris en culture mais ne présentaient aucune activité hémolytique. en utilisant des marquages radioactifs, nous avons confirmé que l'effet cytotoxique observé était lité à l'hydrolyse des phospholipides des membranaires des cellules hôtes. Pour la première fois, nous avons pu confirmer que ces PLC sont directement impliquées dans le processus d'infection et de virulence.Un autre aspect de mon travail de thèse a concerné l'étude de deux autres protéines sécrétées par M. tuberculosis appartenant à la famille des cutinases : la Rv1984c et la Rv3452. Après les avoir produites et purifiées chez E. Coli, nouq avons montré que malgré ces deux protéines présentent 50% d'identité de séquence en acides aminés, elles ont des spécificités de substrat différentes et probablement un rôle physiologique différent. La Rv1984c est une lipase capacle d'hydrolyser des lipides à chaines moyennes, alors que la Rv3452 est une phospholipase de type A2 et est capable d'induire la lyse de macrophage de souris en culture.
Phospholipases, particularly phospholipases C, are important virulence factors in several pathogenic bacteria (C. perfringens, B. cereus, L. monocytogenese and P. aeruginosa). However, little is know on the involvement of thses enzymes in mycobacteria pathogenesis. Although study on M. tuberculosis phospholipases C mutants in a mouse aerosol model of infection gave rise to the contribution of these proteins in virulence…
Advisors/Committee Members: Canaan, Stéphane (thesis director).
Subjects/Keywords: Mycobacterium tuberculosis; Mycobacterium abscessus; Mycobacterium smegmatis; Phospholipase; Lipase; Cytotoxicité; Macrophages; Virulence
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APA (6th Edition):
Bakala n'goma, J. (2010). Etude In vitro de Phospholipases mycobactériennes impliquées dans la virulence : In vitro study of micobactérial Phospholipases involved in virulence. (Doctoral Dissertation). Aix-Marseille 2. Retrieved from http://www.theses.fr/2010AIX22027
Chicago Manual of Style (16th Edition):
Bakala n'goma, Jean-claude. “Etude In vitro de Phospholipases mycobactériennes impliquées dans la virulence : In vitro study of micobactérial Phospholipases involved in virulence.” 2010. Doctoral Dissertation, Aix-Marseille 2. Accessed December 10, 2019.
http://www.theses.fr/2010AIX22027.
MLA Handbook (7th Edition):
Bakala n'goma, Jean-claude. “Etude In vitro de Phospholipases mycobactériennes impliquées dans la virulence : In vitro study of micobactérial Phospholipases involved in virulence.” 2010. Web. 10 Dec 2019.
Vancouver:
Bakala n'goma J. Etude In vitro de Phospholipases mycobactériennes impliquées dans la virulence : In vitro study of micobactérial Phospholipases involved in virulence. [Internet] [Doctoral dissertation]. Aix-Marseille 2; 2010. [cited 2019 Dec 10].
Available from: http://www.theses.fr/2010AIX22027.
Council of Science Editors:
Bakala n'goma J. Etude In vitro de Phospholipases mycobactériennes impliquées dans la virulence : In vitro study of micobactérial Phospholipases involved in virulence. [Doctoral Dissertation]. Aix-Marseille 2; 2010. Available from: http://www.theses.fr/2010AIX22027
Jawaharlal Nehru University
6.
Singh, Anirudh Kumar.
Identification and characterization of sigma factor F of
Mycobacterium smegmatis;.
Degree: Microbiology, 2010, Jawaharlal Nehru University
URL: http://shodhganga.inflibnet.ac.in/handle/10603/13339
None
Bibliography p. 98-107 and Appendix
included
Advisors/Committee Members: Singh, Bhupendra N.Subjects/Keywords: Microbiology; Mycobacterium smegmatis
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APA (6th Edition):
Singh, A. K. (2010). Identification and characterization of sigma factor F of
Mycobacterium smegmatis;. (Thesis). Jawaharlal Nehru University. Retrieved from http://shodhganga.inflibnet.ac.in/handle/10603/13339
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Singh, Anirudh Kumar. “Identification and characterization of sigma factor F of
Mycobacterium smegmatis;.” 2010. Thesis, Jawaharlal Nehru University. Accessed December 10, 2019.
http://shodhganga.inflibnet.ac.in/handle/10603/13339.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Singh, Anirudh Kumar. “Identification and characterization of sigma factor F of
Mycobacterium smegmatis;.” 2010. Web. 10 Dec 2019.
Vancouver:
Singh AK. Identification and characterization of sigma factor F of
Mycobacterium smegmatis;. [Internet] [Thesis]. Jawaharlal Nehru University; 2010. [cited 2019 Dec 10].
Available from: http://shodhganga.inflibnet.ac.in/handle/10603/13339.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Singh AK. Identification and characterization of sigma factor F of
Mycobacterium smegmatis;. [Thesis]. Jawaharlal Nehru University; 2010. Available from: http://shodhganga.inflibnet.ac.in/handle/10603/13339
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Otago
7.
Aung, Htin Lin.
CRP is a global regulator of carbon catabolism and energy metabolism in Mycobacterium smegmatis
.
Degree: 2013, University of Otago
URL: http://hdl.handle.net/10523/4381
► Members of the genus Mycobacterium respire using two terminal respiratory oxidases: a cytochrome bd-type menaquinol oxidase (encoded by cydABDC) or an aa3-type menaquinol-cytochrome c oxidase.…
(more)
▼ Members of the genus
Mycobacterium respire using two terminal respiratory oxidases: a cytochrome bd-type menaquinol oxidase (encoded by cydABDC) or an aa3-type menaquinol-cytochrome c oxidase. During the transition to hypoxic (< 0.1% air saturation) conditions, cytochrome bd is induced in
Mycobacterium smegmatis but the molecular mechanism governing this induction remains unknown. Analysis of the proposed cydABDC operon in M.
smegmatis under hypoxic conditions revealed that cydAB and cydDC were not polycistronic. A transposon-mediated random mutagenesis was performed and revealed genes involved in redox homeostasis and defense against oxidase stress including KatG and PruC were required for cydAB operon expression. DNA-binding experiments were performed using a cydA promoter as target DNA and a DNA-binding histone-like protein (Hlp) was identified as protein bound to the cydAB promoter region. A cAMP receptor protein (CRP)-binding site (GTGAN6CCACA) was identified in the regulatory region of the cydAB operon and mutations in this sequence caused a significant reduction in cydA-lacZ expression.
The M.
smegmatis mc2155 genome has two copies of CRP [Msmeg_0539 (Crp1) and Msmeg_6189 (Crp2)]. Phylogenetic analysis suggested that Crp1 is uniquely present in fast-growing mycobacteria, whilst Crp2 is closely related to CRP from other slow- growing mycobacteria. In addition, only Crp1 but not Crp2 could complement a Δcrp mutant of the Gram-negative bacterium, Pectobacterium atrosepticum. To dissect the physiological role of each Crp protein, transcriptional profiling studies using deletion mutants were conducted. Microarray analysis of the crp1 deletion mutant revealed 239 genes were differentially regulated including those involved in carbon catabolism and energy metabolism. However, a crp2-deletion mutant could not be obtained suggesting that this gene is essential for growth in M.
smegmatis. Therefore, crp2 was conditionally expressed and transcriptional profiling studies were performed. Microarray analysis of the crp2 overexpression strain showed 58 genes were differentially regulated including genes from many biological processes. Identification of the CRP promoter consensus in M.
smegmatis revealed that both Crp1 and Crp2 recognise the same consensus sequence yet the regulon of Crp1 is distinct from that of Crp2. Taken together, these data demonstrate that each Crp could be activated in response to unique signals. The mechanism of how each Crp is activated remains to be elucidated.
Advisors/Committee Members: Cook, Gregory (advisor).
Subjects/Keywords: CRP;
Mycobacterium;
tuberculosis;
smegmatis;
cAMP;
cytochromebd;
cydAB
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APA (6th Edition):
Aung, H. L. (2013). CRP is a global regulator of carbon catabolism and energy metabolism in Mycobacterium smegmatis
. (Doctoral Dissertation). University of Otago. Retrieved from http://hdl.handle.net/10523/4381
Chicago Manual of Style (16th Edition):
Aung, Htin Lin. “CRP is a global regulator of carbon catabolism and energy metabolism in Mycobacterium smegmatis
.” 2013. Doctoral Dissertation, University of Otago. Accessed December 10, 2019.
http://hdl.handle.net/10523/4381.
MLA Handbook (7th Edition):
Aung, Htin Lin. “CRP is a global regulator of carbon catabolism and energy metabolism in Mycobacterium smegmatis
.” 2013. Web. 10 Dec 2019.
Vancouver:
Aung HL. CRP is a global regulator of carbon catabolism and energy metabolism in Mycobacterium smegmatis
. [Internet] [Doctoral dissertation]. University of Otago; 2013. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/10523/4381.
Council of Science Editors:
Aung HL. CRP is a global regulator of carbon catabolism and energy metabolism in Mycobacterium smegmatis
. [Doctoral Dissertation]. University of Otago; 2013. Available from: http://hdl.handle.net/10523/4381
University of Sydney
8.
SHANAHAN, Erin Rose.
Molecular investigation of mycobacterium tuberculosis and the critical cell wall lipase culp6
.
Degree: 2014, University of Sydney
URL: http://hdl.handle.net/2123/10066
► Tuberculosis remains a leading challenge in global public health. An in depth understanding of physiological processes in Mycobacterium tuberculosis is required for the development of…
(more)
▼ Tuberculosis remains a leading challenge in global public health. An in depth understanding of physiological processes in Mycobacterium tuberculosis is required for the development of improved treatments. This study aimed to develop tools for disruption of mycobacterial genes utilising the group II intron based Targetron system. The putatively essential cell wall lipase Cutinase-like protein 6 (Culp6, Rv3802c) was also investigated to determine its biological function and role as a target for the antibiotic tetrahydrolipstatin (THL).
The Targetron was adapted for use in mycobacteria through incorporation into an inducible mycobacterial vector, and functional Targetron insertion sites identified in a number of mycobacterial genes. However, induction of Targetron expression under a range of growth conditions failed to result in successful gene disruption. Despite modifications including codon optimisation of the intron reverse transcriptase, the mycobacterial species tested have proved impervious to Targetron insertion. Further investigation of the Targetron in mycobacteria is required to develop this molecular tool.
This study has revealed the Mycobacterium smegmatis Culp6 ortholog MSMEG_6394 is required for optimal growth under conditions of stress, including increased temperature and presence of Tween. Loss of MSMEG_6394 leads to altered colony morphology and increased sensitivity to Rifampicin and Isoniazid. Complementation with M. tuberculosis Culp6 restored the phenotype of the MSMEG_6394 deletion mutant. THL disrupts mycolic acid synthesis and is known to inhibit purified Culp6, however does not reduce the growth of M. smegmatis. In this study, it was revealed that either over-expression of Culp6, or increased temperature, resulted in sensitisation of M. smegmatis to THL. Taken together, these results suggest an interaction between Culp6 and THL in mycobacteria. The data presented in this study is highly suggestive of a function for Culp6 in cell wall synthesis.
Subjects/Keywords: Mycobacterium tuberculosis;
Mycobacterium smegmatis;
Targetron.;
Culp6;
Rv3802c;
Tetrahydrolipstatin
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APA (6th Edition):
SHANAHAN, E. R. (2014). Molecular investigation of mycobacterium tuberculosis and the critical cell wall lipase culp6
. (Thesis). University of Sydney. Retrieved from http://hdl.handle.net/2123/10066
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
SHANAHAN, Erin Rose. “Molecular investigation of mycobacterium tuberculosis and the critical cell wall lipase culp6
.” 2014. Thesis, University of Sydney. Accessed December 10, 2019.
http://hdl.handle.net/2123/10066.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
SHANAHAN, Erin Rose. “Molecular investigation of mycobacterium tuberculosis and the critical cell wall lipase culp6
.” 2014. Web. 10 Dec 2019.
Vancouver:
SHANAHAN ER. Molecular investigation of mycobacterium tuberculosis and the critical cell wall lipase culp6
. [Internet] [Thesis]. University of Sydney; 2014. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/2123/10066.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
SHANAHAN ER. Molecular investigation of mycobacterium tuberculosis and the critical cell wall lipase culp6
. [Thesis]. University of Sydney; 2014. Available from: http://hdl.handle.net/2123/10066
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Virginia Tech
9.
Kocabas, Evren.
Identification of native co-factors of MshB and MCA from Mycobacterium species.
Degree: MS, Biochemistry, 2010, Virginia Tech
URL: http://hdl.handle.net/10919/44457
► Mycothiol (MSH), a low-molecular- weight thiol, is a primary reducing agent and essential for the survival of mycobacteria. The full pathway of MSH biosynthesis and…
(more)
▼ Mycothiol (MSH), a low-molecular- weight thiol, is a primary reducing agent and essential for the survival of mycobacteria. The full pathway of MSH biosynthesis and detoxification includes various promising drug targets. Several metalloenzymes are involved in this pathway, such as a deacetylase (MshB) and mycothiol S-conjugate amidase (MCA). MshB catalyzes the deacetylation of GlcNAc-Ins to form GlcN-Ins and acetate. Mycothiol S-conjugate amidase (MCA) cleaves the amide bond of mycothiol S-conjugates of various drugs and toxins. The identification of the native co-factor is critical for the design of potent and effective inhibitors. Therefore, in this study, we identified the possible native co-factors of MshB and MCA from M.
smegmatis and M. tuberculosis.
To reach our aim, we used a pull-down method to rapidly purify halo-MshB and halo-MCA under anaerobic conditions. Our data indicates that the metal bound to MshB and MCA anaerobically purified from E. coli grown in minimal medium is mainly Fe(II), while proteins purified under aerobic conditions contain bound Zn (II) and Fe(II) that varies with the metal content of the medium. For a further clarification of the metal ion preferences of MshB and MCA, we determined the MshB and MCA affinity for Zn(II) to be in the picomolar range and Ms MshB affinity for Fe(II) in nanomolar range. These results indicate that MshB and MCA can be found bound with either iron or zinc and this is independent to their affinities for these metal ions.
Advisors/Committee Members: Hernick, Marcy (committeechair), Helm, Richard Frederick (committee member), Klemba, Michael (committee member).
Subjects/Keywords: Mycobacterium tuberculosis; metal-dependent deacetylase; MCA; MshB; iron; zinc; Mycobacterium smegmatis
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APA (6th Edition):
Kocabas, E. (2010). Identification of native co-factors of MshB and MCA from Mycobacterium species. (Masters Thesis). Virginia Tech. Retrieved from http://hdl.handle.net/10919/44457
Chicago Manual of Style (16th Edition):
Kocabas, Evren. “Identification of native co-factors of MshB and MCA from Mycobacterium species.” 2010. Masters Thesis, Virginia Tech. Accessed December 10, 2019.
http://hdl.handle.net/10919/44457.
MLA Handbook (7th Edition):
Kocabas, Evren. “Identification of native co-factors of MshB and MCA from Mycobacterium species.” 2010. Web. 10 Dec 2019.
Vancouver:
Kocabas E. Identification of native co-factors of MshB and MCA from Mycobacterium species. [Internet] [Masters thesis]. Virginia Tech; 2010. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/10919/44457.
Council of Science Editors:
Kocabas E. Identification of native co-factors of MshB and MCA from Mycobacterium species. [Masters Thesis]. Virginia Tech; 2010. Available from: http://hdl.handle.net/10919/44457
Arizona State University
10.
Maarsingh, Jason.
Genetic and Biochemical Insights into the Mycobacterial
PrrAB System as a Regulator of Respiration and Central
Metabolism.
Degree: Microbiology, 2019, Arizona State University
URL: http://repository.asu.edu/items/53852
► Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the 10th leading cause of death, worldwide. The prevalence of drug-resistant clinical isolates and the paucity…
(more)
▼ Mycobacterium tuberculosis (Mtb), the causative agent
of tuberculosis, is the 10th leading cause of death, worldwide. The
prevalence of drug-resistant clinical isolates and the paucity of
newly-approved antituberculosis drugs impedes the successful
eradication of Mtb. Bacteria commonly use two-component systems
(TCS) to sense their environment and genetically modulate adaptive
responses. The prrAB TCS is essential in Mtb, thus representing an
auspicious drug target; however, the inability to generate an Mtb
ΔprrAB mutant complicates investigating how this TCS contributes to
pathogenesis. Mycobacterium smegmatis, a commonly used M.
tuberculosis genetic surrogate was used here. This work shows that
prrAB is not essential in M. smegmatis. During ammonium stress, the
ΔprrAB mutant excessively accumulates triacylglycerol lipids, a
phenotype associated with M. tuberculosis dormancy and chronic
infection. Additionally, triacylglycerol biosynthetic genes were
induced in the ΔprrAB mutant relative to the wild-type and
complementation strains during ammonium stress. Next, RNA-seq was
used to define the M. smegmatis PrrAB regulon. PrrAB regulates
genes participating in respiration, metabolism, redox balance, and
oxidative phosphorylation. The M. smegmatis ΔprrAB mutant is
compromised for growth under hypoxia, is hypersensitive to cyanide,
and fails to induce high-affinity respiratory genes during hypoxia.
Furthermore, PrrAB positively regulates the hypoxia-responsive dosR
TCS response regulator, potentially explaining the hypoxia-mediated
growth defects in the ΔprrAB mutant. Despite inducing genes
encoding the F1F0 ATP synthase, the ΔprrAB mutant accumulates
significantly less ATP during aerobic, exponential growth compared
to the wild-type and complementation strains. Finally, the M.
smegmatis ΔprrAB mutant exhibited growth impairment in media
containing gluconeogenic carbon sources. M. tuberculosis mutants
unable to utilize these substrates fail to establish chronic
infection, suggesting that PrrAB may regulate Mtb central carbon
metabolism in response to chronic infection. In conclusion, 1)
prrAB is not universally essential in mycobacteria; 2) M. smegmatis
PrrAB regulates genetic responsiveness to nutrient and oxygen
stress; and 3) PrrAB may provide feed-forward control of the DosRS
TCS and dormancy phenotypes. The data generated in these studies
provide insight into the mycobacterial PrrAB TCS transcriptional
regulon, PrrAB essentiality in Mtb, and how PrrAB may mediate
stresses encountered by Mtb during the transition to chronic
infection.
Subjects/Keywords: Microbiology; metabolism; Mycobacterium smegmatis; Mycobacterium tuberculosis; PrrAB; respiration; two-component systems
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APA ·
Chicago ·
MLA ·
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CSE |
Export
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Manager
APA (6th Edition):
Maarsingh, J. (2019). Genetic and Biochemical Insights into the Mycobacterial
PrrAB System as a Regulator of Respiration and Central
Metabolism. (Doctoral Dissertation). Arizona State University. Retrieved from http://repository.asu.edu/items/53852
Chicago Manual of Style (16th Edition):
Maarsingh, Jason. “Genetic and Biochemical Insights into the Mycobacterial
PrrAB System as a Regulator of Respiration and Central
Metabolism.” 2019. Doctoral Dissertation, Arizona State University. Accessed December 10, 2019.
http://repository.asu.edu/items/53852.
MLA Handbook (7th Edition):
Maarsingh, Jason. “Genetic and Biochemical Insights into the Mycobacterial
PrrAB System as a Regulator of Respiration and Central
Metabolism.” 2019. Web. 10 Dec 2019.
Vancouver:
Maarsingh J. Genetic and Biochemical Insights into the Mycobacterial
PrrAB System as a Regulator of Respiration and Central
Metabolism. [Internet] [Doctoral dissertation]. Arizona State University; 2019. [cited 2019 Dec 10].
Available from: http://repository.asu.edu/items/53852.
Council of Science Editors:
Maarsingh J. Genetic and Biochemical Insights into the Mycobacterial
PrrAB System as a Regulator of Respiration and Central
Metabolism. [Doctoral Dissertation]. Arizona State University; 2019. Available from: http://repository.asu.edu/items/53852
Harvard University
11.
Wu, Katherine J.
Functional characterization of an essential mycobacterial protease.
Degree: PhD, 2019, Harvard University
URL: http://nrs.harvard.edu/urn-3:HUL.InstRepos:41121323
► Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, remains a global health threat due to its engimatic ability to withstand diverse environmental stresses in the…
(more)
▼ Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, remains a global health threat due to its engimatic ability to withstand diverse environmental stresses in the host. The widespread phenomenon of antibiotic resistance can be at least partially attributed to Mtb’s incredible adaptibility. As such, an increased understanding of how this bacterium grows and survives under different conditions is ncessary to continue developing tools to prevent the spread of disease.
In Chapter 1 of my dissertation, I present a brief overview of mycobacterial proteases. In Chapter 2, we explore the previously uncharacterized function of the predicted essential protease HtrA. We find that HtrA is essential and interacts with another essential protein of unknown function, LppZ. Loss of HtrA/LppZ leads to accumulation of the amidase Ami3, which is toxic when mannosylated. In the presence of HtrA/LppZ, Ami3 has a shorter half-life and accumulates to lower levels. These data suggest HtrA-LppZ blocks the toxicity of a cell wall enzyme. In Chapter 3, we explore another set of essential genes, FtsQLB, which we show to be critical for mediating proper mycobacterial cell division. We identify and characterize homologs of the conserved cell division regulators FtsL and FtsB, adding to a previous body of work characterizing their partner FtsQ, and show that, as a set, these enzymes appear to function similarly to their homologs in E. coli. We then identify a number of previously undescribed septally-localized factors which could be involved in cell wall regulation, including SepIVA. Finally, in Chapter 4, we present preliminary findings on another conserved mycobacterial protease, FtsH. We show that FtsH is not essential for viability in Mycobacterium smegmatis or Mycobacterium tuberculosis. However, FtsH may play a small role in the viability of mycobacteria under conditions of oxidative stress. Additionally, we present candidate substrates for this protease.
Characterizing the critical proteins that allow mycobacteria to survive extreme environments is a crucial pursuit in the push for new tubericidal agents. This work forms the foundation for future work on the stress-responsive growth patterns of mycobacteria, particularly through the lens of regulated proteolysis.
Medical Sciences
Advisors/Committee Members: Dove, Simon (advisor), Bernhardt, Thomas (committee member), Higgins, Darren (committee member), Sassetti, Christopher (committee member).
Subjects/Keywords: HtrA; Mycobacterium tuberculosis; LppZ; Pmt; Ami3; Protease; Periplasm; Mycobacterium smegmatis
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APA ·
Chicago ·
MLA ·
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CSE |
Export
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Manager
APA (6th Edition):
Wu, K. J. (2019). Functional characterization of an essential mycobacterial protease. (Doctoral Dissertation). Harvard University. Retrieved from http://nrs.harvard.edu/urn-3:HUL.InstRepos:41121323
Chicago Manual of Style (16th Edition):
Wu, Katherine J. “Functional characterization of an essential mycobacterial protease.” 2019. Doctoral Dissertation, Harvard University. Accessed December 10, 2019.
http://nrs.harvard.edu/urn-3:HUL.InstRepos:41121323.
MLA Handbook (7th Edition):
Wu, Katherine J. “Functional characterization of an essential mycobacterial protease.” 2019. Web. 10 Dec 2019.
Vancouver:
Wu KJ. Functional characterization of an essential mycobacterial protease. [Internet] [Doctoral dissertation]. Harvard University; 2019. [cited 2019 Dec 10].
Available from: http://nrs.harvard.edu/urn-3:HUL.InstRepos:41121323.
Council of Science Editors:
Wu KJ. Functional characterization of an essential mycobacterial protease. [Doctoral Dissertation]. Harvard University; 2019. Available from: http://nrs.harvard.edu/urn-3:HUL.InstRepos:41121323
Universidade Federal de Santa Maria
12.
Ritiel Corrêa da Cruz.
COMPOSTOS FENÓLICOS E ATIVIDADE ANTIMICOBACTERIANA DAS FOLHAS DE Ficus benjamina L. e Ficus luschnathiana (MIQ.) MIQ.
Degree: 2011, Universidade Federal de Santa Maria
URL: http://coralx.ufsm.br/tede/tde_busca/arquivo.php?codArquivo=4815
► The following work presents an evaluation of the antimycobacterial activity (against Mycobacterium smegmatis) of the extracts, fractions and some phenolic compounds present in the leaves…
(more)
▼ The following work presents an evaluation of the antimycobacterial activity (against Mycobacterium smegmatis) of the extracts, fractions and some phenolic compounds present in the leaves of Ficus benjamina L. and Ficus luschnathiana (Miq.) Miq., along with an estimation of the total phenolic content and the quantification of some of these compounds by High Performance Liquid Chromatography (HPLC). The phenolic estimation (Folin-Ciocalteu method) revealed that the crude extracts and the high polarity fractions from these extracts (ethyl acetate and n-butanol) are rich in polyphenols, although, its amount are not directly related to the antimycobacterial activity. The evaluation of this biological activity was performed by broth microdilution method, furnishing the minimum inhibitory concentration (MIC) of the extracts, fractions and tested compounds. The better biological activity was verified with butanolic fraction from F. luschnathiana (MIC = 156,25 μg/mL) and ethyl acetate fraction from F. benjamina (MIC = 312,50 μg/mL). However, the correlation of these good results with specific compounds was not possible, since technical difficulties prevented the isolation of substances from these fractions; and concerning the screened polyphenols only quercetin was encountered in butanolic fraction. In addition, quercetin exerted weak antimycobacterial activity against M. smegmatis (MIC = 625,00 μg/mL), much weaker than that observed for the fraction. The other investigated standards that were encountered in the extracts and fractions (caffeic and chlorogenic acids, rutin and kaempferol), also exhibited weak inhibitory effect. Thus, the good antimycobacterial activity observed for the fractions above mentioned are probably not related to these phenolics, still it may be related to compounds of the same nature. Many polyphenols, such as flavonoids, have been reported as mycobacterial growth inhibitors, thus it is also possible to conclude that the structural characteristics of quercetin, rutin and kaempferol do not support the biological activity studied.
A seguinte dissertação apresenta uma avaliação da atividade antimicobacteriana (frente à Mycobacterium smegmatis) de extratos, frações e substâncias fenólicas presentes nas folhas de Ficus benjamina L. e Ficus luschnathiana (Miq.) Miq., juntamente com uma estimativa do teor de polifenóis totais e a quantificação de alguns destes compostos por Cromatografia Líquida de Alta Eficiência (CLAE). O doseamento de polifenóis (método de Folin-Ciocalteu) revelou que os extratos brutos e as frações mais polares destes extratos (acetato de etila e n-butanol) são bastante providos de substâncias fenólicas, ainda que este teor não esteja diretamente relacionado à atividade antimicobacteriana. A avaliação desta atividade biológica foi realizada por método de microdiluição em caldo, que fornece a concentração inibitória mínima (CIM) dos extratos, frações e substâncias testadas. Uma boa atividade inibitória foi constatada para a fração butanólica de F. luschnathiana (MIC =…
Advisors/Committee Members: Margareth Linde Athayde, Sydney Hartz Alves, Cibele Rosa Gracioli.
Subjects/Keywords: CLAE; flavonóides; polifenóis; Mycobacterium smegmatis; Ficus luschnathiana; Ficus benjamina; FARMACIA; Keywords: Ficus benjamina; Ficus luschnathiana; Mycobacterium smegmatis; polyphenols; flavonoids; HPLC
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APA (6th Edition):
Cruz, R. C. d. (2011). COMPOSTOS FENÓLICOS E ATIVIDADE ANTIMICOBACTERIANA DAS FOLHAS DE Ficus benjamina L. e Ficus luschnathiana (MIQ.) MIQ. (Thesis). Universidade Federal de Santa Maria. Retrieved from http://coralx.ufsm.br/tede/tde_busca/arquivo.php?codArquivo=4815
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Cruz, Ritiel Corrêa da. “COMPOSTOS FENÓLICOS E ATIVIDADE ANTIMICOBACTERIANA DAS FOLHAS DE Ficus benjamina L. e Ficus luschnathiana (MIQ.) MIQ.” 2011. Thesis, Universidade Federal de Santa Maria. Accessed December 10, 2019.
http://coralx.ufsm.br/tede/tde_busca/arquivo.php?codArquivo=4815.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Cruz, Ritiel Corrêa da. “COMPOSTOS FENÓLICOS E ATIVIDADE ANTIMICOBACTERIANA DAS FOLHAS DE Ficus benjamina L. e Ficus luschnathiana (MIQ.) MIQ.” 2011. Web. 10 Dec 2019.
Vancouver:
Cruz RCd. COMPOSTOS FENÓLICOS E ATIVIDADE ANTIMICOBACTERIANA DAS FOLHAS DE Ficus benjamina L. e Ficus luschnathiana (MIQ.) MIQ. [Internet] [Thesis]. Universidade Federal de Santa Maria; 2011. [cited 2019 Dec 10].
Available from: http://coralx.ufsm.br/tede/tde_busca/arquivo.php?codArquivo=4815.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Cruz RCd. COMPOSTOS FENÓLICOS E ATIVIDADE ANTIMICOBACTERIANA DAS FOLHAS DE Ficus benjamina L. e Ficus luschnathiana (MIQ.) MIQ. [Thesis]. Universidade Federal de Santa Maria; 2011. Available from: http://coralx.ufsm.br/tede/tde_busca/arquivo.php?codArquivo=4815
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Indian Institute of Science
13.
Padiadpu, Jyothi.
An Integrated Systems Biology Approach to Study Drug Resistance in Mycobacteria.
Degree: 2015, Indian Institute of Science
URL: http://etd.iisc.ernet.in/2005/3750
;
http://etd.iisc.ernet.in/abstracts/4621/G26950-Abs.pdf
► Emergence of drug resistance is a major problem in the treatment of many diseases including tuberculosis. To tackle the problem, it is essential to obtain…
(more)
▼ Emergence of drug resistance is a major problem in the treatment of many diseases including tuberculosis. To tackle the problem, it is essential to obtain a global perspective of the molecular mechanisms by which bacteria acquire drug resistance. Systems biology approaches therefore become necessary. This work aims to understand pathways to drug resistance and strategies for inhibition of the resistant strains by using a combination of experimental genomics and computational molecular systems approaches.
Laboratory evolution of
Mycobacterium smegmatis MC2 155 by treatment with isoniazid (INH), a front-line anti-tubercular drug, resulted in a drug-resistant strain (4XR), capable of growth even at about 10-times the minimum inhibitory concentration of the drug. Whole genome sequence of the 4XR was determined, which indicated only 31 variations in the whole genome, including 3 point mutations, 17 indels and 11 frame-shifts. Two mutations were in proteins required for the pharmacological action of the drug, albeit in regions distant from the drug binding site. The variations however were insufficient to explain the observed resistance to isoniazid.
For a better understanding of the global changes associated with drug resistance, whole genome-wide gene expression data was obtained for the resistant strain and compared with that of the WT strain. 716 genes were found to be differentially regulated in 4XR, spanning different biochemical, signaling and regulatory pathways. From this, some explanations for the emergence of drug resistance were obtained, such as the up-regulation of the enzymes in the mycolic acid biosynthesis pathway and also of the drug efflux pumps. In addition, enrichment analysis indicated that up-regulated genes belong to functional categories of response to stress, carbohydrate metabolism, oxidation-reduction process, ion transport, signaling as well as lipid metabolism. The differential gene regulations seemed to be partially responsible for conferring the phenotype to the organism.
Alterations in the metabolic pathways in 4XR were characterized using the phenotypic
microarray technology, which experimentally scanned the respiratory ability of the resistant bacteria under 280 different nutrient conditions and 96 different inhibitors. Phenotypic gain, where the resistant strain grows significantly better than the wild type and phenotypic loss, where the growth of the resistant strain is compromised as compared to the sensitive strains were derived from the comparison of the phenotypic responses. Differences in survival ability and growth rates in different nutrient sources in the resistant phenotype as compared to the wild type were observed, suggesting rewiring in the metabolic network of the drug-resistant strain. In particular, the pathways of central carbon metabolism and amino acid biosynthesis exhibit significant differences. The strain-specific metabolic pathway differences may guide in devising strategies to tackle the drug-resistant strains selectively and in a rational manner.
…
Advisors/Committee Members: Chandra, Nagasuma.
Subjects/Keywords: Drug Resistance Tuberculosis; Drug Resistance Mycobacteria; M. smegmatis; MC2 155; Mycobacterium smegmatis; M. tuberculosis; Anti-tubercular Drugs; Mycobacterium tuberculosis; Biochemistry
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APA (6th Edition):
Padiadpu, J. (2015). An Integrated Systems Biology Approach to Study Drug Resistance in Mycobacteria. (Thesis). Indian Institute of Science. Retrieved from http://etd.iisc.ernet.in/2005/3750 ; http://etd.iisc.ernet.in/abstracts/4621/G26950-Abs.pdf
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Padiadpu, Jyothi. “An Integrated Systems Biology Approach to Study Drug Resistance in Mycobacteria.” 2015. Thesis, Indian Institute of Science. Accessed December 10, 2019.
http://etd.iisc.ernet.in/2005/3750 ; http://etd.iisc.ernet.in/abstracts/4621/G26950-Abs.pdf.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Padiadpu, Jyothi. “An Integrated Systems Biology Approach to Study Drug Resistance in Mycobacteria.” 2015. Web. 10 Dec 2019.
Vancouver:
Padiadpu J. An Integrated Systems Biology Approach to Study Drug Resistance in Mycobacteria. [Internet] [Thesis]. Indian Institute of Science; 2015. [cited 2019 Dec 10].
Available from: http://etd.iisc.ernet.in/2005/3750 ; http://etd.iisc.ernet.in/abstracts/4621/G26950-Abs.pdf.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Padiadpu J. An Integrated Systems Biology Approach to Study Drug Resistance in Mycobacteria. [Thesis]. Indian Institute of Science; 2015. Available from: http://etd.iisc.ernet.in/2005/3750 ; http://etd.iisc.ernet.in/abstracts/4621/G26950-Abs.pdf
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Indian Institute of Science
14.
Rajan Prabhu, J.
Structural Studies On Mycobacterial RecA And RuvA.
Degree: 2009, Indian Institute of Science
URL: http://hdl.handle.net/2005/924
► Homologous recombination is a fundamental cellular process evolved to maintain genomic integrity and to generate genetic diversity. It plays a crucial role in DNA repair,…
(more)
▼ Homologous recombination is a fundamental cellular process evolved to maintain genomic integrity and to generate genetic diversity. It plays a crucial role in DNA repair, correct segregation of meiotic chromosomes and resumption of the stalled replication forks. In vitro, the homologous recombination pathway is kinetically separable into a four step process involving initiation, homologous pairing, branch migration and junction resolution. The process of pairing and strand exchange between two homologous double-stranded DNA molecules leads to the formation of an intermediate structure called the Holliday junction (HJ). The crucial enzyme involved in this step in bacteria is RecA. In eubacteria, the junction is processed by three proteins, collectively referred to as the RuvABC protein complex. RuvA binds to the HJ, while RuvB, a helicase, binds to the RuvA-HJ complex and pumps the duplex DNA thus facilitating branch migration. The work reported here is concerned with structural studies on mycobacterial RecA and RuvA.
X-ray crystallography was used to solve the protein crystal structures. The hanging drop vapour diffusion method was used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator except for two data sets collected using synchrotron radiation. The data were processed mostly using Mosflm and Scala and few data sets were processed using the HKL program suite. The molecular replacement method using programs Phaser and AMoRe was used for structure solution. Structure refinements were carried out using programs CNS and PHENIX. Model building was performed using COOT and O. PROCHECK, MOLPROBITY, ALIGN and NACCESS were used for structure validation and analysis of the refined structures.
Mycobacterium smegmatis RecA (MsRecA) and its nucleotide complexes crystallize in three different, but closely related, forms characterized by specific ranges of unit cell dimensions. The six crystals discussed in the earlier part of the thesis and the five reported earlier, all grown under the same or very similar conditions, belong to these three forms, all in space group P61. They include one obtained by reducing the relative humidity around the crystal. In all crystals, RecA monomers form filaments around a 61 screw axis. Thus, the c-dimension of the crystal corresponds to the pitch of the RecA filament. As reported in the case of E.coli RecA, the variation in the pitch among the three forms correlate well with the motion of the C-terminal domain of the RecA monomers with respect to the main domain. The domain motion is compatible with formation of inactive as well as active RecA filaments involving monomers with a fully ordered C-domain. It does not appear to influence the movement upon nucleotide-binding of the switch residue Gln 196, which is believed to provide the trigger for transmitting the effect of nucleotide-binding to the DNA-binding region. Interestingly, partial dehydration of the crystal results in the movement of…
Advisors/Committee Members: Vijayan, M.
Subjects/Keywords: Mycobacterium - Recombination; Mycobacterium Smegmatis RecA; Protien; Mycobacterium Tuberculosis RecA; RuvA Protein; Mycobacterial Proteins; Mycobacterial RecA; Mycobacterium Tuberculosis RuvA; Microbiology
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APA (6th Edition):
Rajan Prabhu, J. (2009). Structural Studies On Mycobacterial RecA And RuvA. (Thesis). Indian Institute of Science. Retrieved from http://hdl.handle.net/2005/924
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Rajan Prabhu, J. “Structural Studies On Mycobacterial RecA And RuvA.” 2009. Thesis, Indian Institute of Science. Accessed December 10, 2019.
http://hdl.handle.net/2005/924.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Rajan Prabhu, J. “Structural Studies On Mycobacterial RecA And RuvA.” 2009. Web. 10 Dec 2019.
Vancouver:
Rajan Prabhu J. Structural Studies On Mycobacterial RecA And RuvA. [Internet] [Thesis]. Indian Institute of Science; 2009. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/2005/924.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Rajan Prabhu J. Structural Studies On Mycobacterial RecA And RuvA. [Thesis]. Indian Institute of Science; 2009. Available from: http://hdl.handle.net/2005/924
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Indian Institute of Science
15.
Roy, Sougata.
Transcriptional Analysis Of The Principal Cell Division Gene ftsZ Of Mycobacterium Tuberculosis And Mycobacterium Smegmatis.
Degree: 2006, Indian Institute of Science
URL: http://hdl.handle.net/2005/400
► The success of Mycobacterium tuberculosis as a pathogen is due to its remarkable ability to: (i). adapt to and survive inside activated macrophages under nonproliferating…
(more)
▼ The success of
Mycobacterium tuberculosis as a pathogen is due to its remarkable
ability to: (i). adapt to and survive inside activated macrophages under nonproliferating condition, (ii). put up drug resistance and (iii). enter into hypoxia-induced dormancy and remain in nonproliferating condition, be resistant to drugs, and get reactivated into
proliferation when favourable conditions arise. Thus, regulation of cell division (arrest and resumption) is an obligatory event that is critical to the pathogen for the establishment of successful infection, latency and reactivation process in human host. Therefore, in order to understand and combat the successful survival strategy of the bacterium inside the host macrophages or in granuloma, a basic knowledge of the
regulation of cell division in tubercle bacillus is essential. Bacterial cytokinetic protein FtsZ (a tubulin homologue) is the key regulatory molecule for cell division and its
intracellular level is critical for initiation of cell division in bacteria. Therefore, in order to understand the regulation cell division by expression and maintenance of ftsZ mRNA and protein, we initiated studies on the transcriptional regulation of ftsZ gene in the slow growing pathogen, M. tuberculosis, and in the fast-growing saprophyte M.
smegmatis.
Identification of regions containing ftsZMt promoter activity
In order to identify promoter activity-containing regions of ftsZ gene of M. tuberculosis H37Rv (ftsZMt) in vivo, different regions upstream of ftsZMt namely, the ftsQ-ftsZ intergenic region, the ftsQ open reading frame (ORF), and different regions of ftsQ ORF, were cloned in a gfp reporter plasmid and analyzed for gfp expression in M.
smegmatis mc2155 cells. Flow cytometric analysis of exponentially grown M.
smegmatis mc2155 cells containing these transcription fusion constructs revealed GFP expression in the cells harbouring ftsQ-ftsZ intergenic region (172 bp), the entire ftsQ ORF (945 bp), and 5’ 467 bp and 3’ 217 bp regions of ftsQ ORF. RT-PCR analyses on RNA from M.
smegmatis mc2155 cell transformants carrying the entire ftsQ ORF-ftsQ-ftsZ intergenic region containing construct, as well as on total RNA from M. tuberculosis confirmed that the
regions identified indeed elicit promoter activity. RT-PCR analysis on M. tuberculosis RNA as well as semi-quantitative RT-PCR analyses of gfp transcripts driven by cloned MtftsZ promoter regions in M.
smegmatis cells showed that about 70% of the total
promoter activity comes from ftsQ ORF and there is co-transcription of ftsQ-ftsZ genes.
Multiple transcripts code for ftsZMt
Primer extension analysis, using primers annealing at different positions in the
ftsQ-ftsZ chromosomal region, on RNA from M. tuberculosis as well as from M.
smegmatis transformants containing 1.117 kb ftsZMtpromoter region in a promoter probe
vector, identified origin of six different transcripts (T1-T6)…
Advisors/Committee Members: Ajithkumar, Parthasarathi.
Subjects/Keywords: Gene Transcription; Gene Regulation; Mycobacterium Tuberculosis; Mycobacterium Smegmatis; ftsZ; Cell Division Gene; Molecular Biology
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APA ·
Chicago ·
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CSE |
Export
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APA (6th Edition):
Roy, S. (2006). Transcriptional Analysis Of The Principal Cell Division Gene ftsZ Of Mycobacterium Tuberculosis And Mycobacterium Smegmatis. (Thesis). Indian Institute of Science. Retrieved from http://hdl.handle.net/2005/400
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Roy, Sougata. “Transcriptional Analysis Of The Principal Cell Division Gene ftsZ Of Mycobacterium Tuberculosis And Mycobacterium Smegmatis.” 2006. Thesis, Indian Institute of Science. Accessed December 10, 2019.
http://hdl.handle.net/2005/400.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Roy, Sougata. “Transcriptional Analysis Of The Principal Cell Division Gene ftsZ Of Mycobacterium Tuberculosis And Mycobacterium Smegmatis.” 2006. Web. 10 Dec 2019.
Vancouver:
Roy S. Transcriptional Analysis Of The Principal Cell Division Gene ftsZ Of Mycobacterium Tuberculosis And Mycobacterium Smegmatis. [Internet] [Thesis]. Indian Institute of Science; 2006. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/2005/400.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Roy S. Transcriptional Analysis Of The Principal Cell Division Gene ftsZ Of Mycobacterium Tuberculosis And Mycobacterium Smegmatis. [Thesis]. Indian Institute of Science; 2006. Available from: http://hdl.handle.net/2005/400
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Pretoria
16.
Mothiba, Maborwa Tebogo.
The effects of
clofazimine on mycobacterium smegmatis biofilm
formation.
Degree: Immunology, 2013, University of Pretoria
URL: http://hdl.handle.net/2263/31569
► Chemotherapy of tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (M. tuberculosis), is successful against actively-growing bacilli but ineffective against dormant/persistent organisms, found mainly in…
(more)
▼ Chemotherapy of tuberculosis (TB), a disease caused by
Mycobacterium tuberculosis (M. tuberculosis), is successful against
actively-growing bacilli but ineffective against dormant/persistent
organisms, found mainly in a protective lipid-laden granuloma,
possibly necessitating the use of lipophilic antibiotics. In vitro,
these bacilli are encased in lipid-rich biofilms. In this study,
the antimycobacterial activity of one such agent, clofazimine, and
its nanoparticle formulation, have been investigated against
Mycobacterium smegmatis (M.
smegmatis), as a surrogate for M.
tuberculosis, by determining the bacteriostatic and bactericidal
activities of the native (NC) and spray-dried (SDC) preparations of
this agent on planktonic and biofilm populations, as well as their
effects on biofilm formation and its lipid compositions,
specifically free mycolic acid (FM) content. Both preparations were
comparable, being bacteriostatic for rapidly-proliferating bacilli,
bactericidal for slow-growing, biofilm-producing sessile bacteria,
but ineffective against non-replicating, biofilm-encased M.
smegmatis organisms. However, similar studies in M. tuberculosis
are required.
Advisors/Committee Members: Cholo, Moloko C. (advisor).
Subjects/Keywords: Free mycolic
acid;
Biofilm;
Planktonic;
Antimycobacterial activity;
Nanoparticle;
Clofazimine;
Chemotherapy;
Granuloma; Mycobacterium
smegmatis; Mycobacterium
tuberculosis
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Mothiba, M. T. (2013). The effects of
clofazimine on mycobacterium smegmatis biofilm
formation. (Masters Thesis). University of Pretoria. Retrieved from http://hdl.handle.net/2263/31569
Chicago Manual of Style (16th Edition):
Mothiba, Maborwa Tebogo. “The effects of
clofazimine on mycobacterium smegmatis biofilm
formation.” 2013. Masters Thesis, University of Pretoria. Accessed December 10, 2019.
http://hdl.handle.net/2263/31569.
MLA Handbook (7th Edition):
Mothiba, Maborwa Tebogo. “The effects of
clofazimine on mycobacterium smegmatis biofilm
formation.” 2013. Web. 10 Dec 2019.
Vancouver:
Mothiba MT. The effects of
clofazimine on mycobacterium smegmatis biofilm
formation. [Internet] [Masters thesis]. University of Pretoria; 2013. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/2263/31569.
Council of Science Editors:
Mothiba MT. The effects of
clofazimine on mycobacterium smegmatis biofilm
formation. [Masters Thesis]. University of Pretoria; 2013. Available from: http://hdl.handle.net/2263/31569
Virginia Tech
17.
Riggs, Sarah Danielle.
Construction and Use of a Transposon for Identification of Essential Genes in Mycobacteria.
Degree: MS, Biological Sciences, 2011, Virginia Tech
URL: http://hdl.handle.net/10919/76973
► The continuing emergence of multi-drug resistant Mycobacterium tuberculosis is threatening the ability to treat tuberculosis (TB) worldwide. The development of new anti-TB drugs requires new…
(more)
▼ The continuing emergence of multi-drug resistant
Mycobacterium tuberculosis is threatening the ability to treat tuberculosis (TB) worldwide. The development of new anti-TB drugs requires new approaches and new drug targets. In this study, a mariner-based transposon, TnQuoVadis, was constructed to identify essential genes as potential drug targets. This transposon has an outward-facing anhydrotetracycline (ATc)-inducible promoter at each end. A mutant with TnQuoVadis inserted upstream of an essential gene may display normal growth in the presence of ATc, but exhibit no growth or severely diminished growth in the absence of ATc. TnQuoVadis was placed onto a vector with a temperature sensitive replication origin for more efficient mutagenesis of mycobacteria. In a preliminary genetic screen using the model organism
Mycobacterium smegmatis, 13 mutants with ATc-dependent growth were identified. Identification of the insertion sites by cloning and sequencing indicated that there were nine genetic loci containing transposon insertions upstream of essential gene candidates in M.
smegmatis. Further analysis of these genes indicated that many were previously known essential in both M.
smegmatis and M. tuberculosis. These results demonstrate that TnQuoVadis and its delivery system can be utilized for the identification of essential genes in mycobacteria
Advisors/Committee Members: Yang, Zhaomin (committeechair), Popham, David L. (committee member), Falkinham, Joseph O. III (committeecochair).
Subjects/Keywords: transposon; Mycobacterium smegmatis; Tuberculosis; TetR; essential genes; genetics
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Riggs, S. D. (2011). Construction and Use of a Transposon for Identification of Essential Genes in Mycobacteria. (Masters Thesis). Virginia Tech. Retrieved from http://hdl.handle.net/10919/76973
Chicago Manual of Style (16th Edition):
Riggs, Sarah Danielle. “Construction and Use of a Transposon for Identification of Essential Genes in Mycobacteria.” 2011. Masters Thesis, Virginia Tech. Accessed December 10, 2019.
http://hdl.handle.net/10919/76973.
MLA Handbook (7th Edition):
Riggs, Sarah Danielle. “Construction and Use of a Transposon for Identification of Essential Genes in Mycobacteria.” 2011. Web. 10 Dec 2019.
Vancouver:
Riggs SD. Construction and Use of a Transposon for Identification of Essential Genes in Mycobacteria. [Internet] [Masters thesis]. Virginia Tech; 2011. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/10919/76973.
Council of Science Editors:
Riggs SD. Construction and Use of a Transposon for Identification of Essential Genes in Mycobacteria. [Masters Thesis]. Virginia Tech; 2011. Available from: http://hdl.handle.net/10919/76973
Indian Institute of Science
18.
Roy, Siddhartha.
Structural Studies On Mycobacterium Smegmatis Dps Molecules.
Degree: 2007, Indian Institute of Science
URL: http://hdl.handle.net/2005/642
► Oxidative stress is a universal phenomenon experienced by both aerobic and anaerobic organisms. Reactive oxygen species (ROS) are generated during the stress, which can damage…
(more)
▼ Oxidative stress is a universal phenomenon experienced by both aerobic and anaerobic organisms. Reactive oxygen species (ROS) are generated during the stress, which can damage most cellular components including proteins, lipids and DNA. Naturally, organisms have evolved defence mechanisms to prevent oxidative damage. In prokaryotic systems, Dps (DNA binding protein from stationary phase cells) forms an important component of the mechanisms. Dps is known to be produced maximally during the stationary phase of bacterial growth. They exhibit ferroxidase activity as well. Dps homologs have been identified in a variety of distantly related bacteria, thus implying that this protein has a crucial function. The crystal structures of these proteins from a few bacteria are available. The work reported here is concerned with structural studies on Dps molecules from
Mycobacterium smegmatis.
Well-established X-ray crystallographic techniques were used to study the structures reported here. Hanging drop vapour diffusion and microbatch methods were used for crystallization. X-ray intensity data were collected on MAR Research imaging plates mounted on Rigaku X-ray generators. The data were processed using the HKL program suite. All the structures were solved by the molecular replacement method using the programs AMoRe and PHASER. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using FRODO and COOT. PROCHECK, ALIGN, INSIGHT, NACCESS, HBPLUS, CONTACT and ESCET were used for validation and analysis of the refined structures. Figures were prepared using MOLSCRIPT, BOBSCRIPT, RASTER3D and PYMOL.
The structure of the first Dps identified in M.
smegmatis has been determined in three crystal forms and has been compared with those of similar proteins from other sources. The dodecameric molecule can be described as a distorted icosahedron. The interfaces among subunits are such that the dodecameric molecule appears to have been made up of stable trimers. The situation is similar in the proteins from Escherichia coli and Agrobacterium tumefaciens, which are closer to the M.
smegmatis protein in sequence and structure than those from other sources, which appear to form a dimer first. Trimerisation is aided in the three proteins by the additional N-terminal stretches they possess. The M.
smegmatis protein has an additional C-terminal stretch compared to other related proteins. The stretch, known to be involved in DNA binding, is situated on the surface of the molecule. A comparison of the available structures permits a delineation of the rigid and flexible regions in the molecule. The subunit interfaces around the molecular dyads, where the ferroxidation centres are located, are relatively rigid. Regions in the vicinity of the acidic holes centred around molecular threefold axes, are relatively flexible. So are the DNA binding regions. The crystal structures of the protein from M.
smegmatis confirm that DNA molecules can occupy spaces within the crystal without disturbing the…
Advisors/Committee Members: Vijayan, M.
Subjects/Keywords: Mycobacterium Smegmatis; Molecular Biology; DNA Binding Proteins; DNA Binding; Oxidative Stress; Mycobacterium Smegmatis Dps - Crystallization; Mycobacterium Proteins - Structural Biology; Dps; MsDps1; MsDps2; Structural Biology
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Roy, S. (2007). Structural Studies On Mycobacterium Smegmatis Dps Molecules. (Thesis). Indian Institute of Science. Retrieved from http://hdl.handle.net/2005/642
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Roy, Siddhartha. “Structural Studies On Mycobacterium Smegmatis Dps Molecules.” 2007. Thesis, Indian Institute of Science. Accessed December 10, 2019.
http://hdl.handle.net/2005/642.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Roy, Siddhartha. “Structural Studies On Mycobacterium Smegmatis Dps Molecules.” 2007. Web. 10 Dec 2019.
Vancouver:
Roy S. Structural Studies On Mycobacterium Smegmatis Dps Molecules. [Internet] [Thesis]. Indian Institute of Science; 2007. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/2005/642.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Roy S. Structural Studies On Mycobacterium Smegmatis Dps Molecules. [Thesis]. Indian Institute of Science; 2007. Available from: http://hdl.handle.net/2005/642
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Indian Institute of Science
19.
Arumugam, Muthu.
Structural, Functional And Transcriptional Analysis Of Nucleoside Diphosphate Kinase From Mycobacterium Smegmatis mc2 155.
Degree: 2009, Indian Institute of Science
URL: http://hdl.handle.net/2005/1338
► Maintenance of the levels of nucleoside triphosphates (NTPs) as well as their corresponding deoxy derivatives (dNTPs) is crucial to all growth and developmental processes. The…
(more)
▼ Maintenance of the levels of nucleoside triphosphates (NTPs) as well as their corresponding deoxy derivatives (dNTPs) is crucial to all growth and developmental processes. The enzyme nucleoside diphosphate kinase (NDK) utilises an autophosporylated enzyme intermediate to catalyse the transfer of 5’ terminal phosphate from NTPs (mostly ATP) to nucleoside diphosphates (NDPs) via a reversible mechanism as given below.
N1TP + NDK ↔N1DP+ −NDK-His* (1)
N2DP + NDK-His* P ↔N2TP + NDK−His. (2) In the γ-phosphoryl group transfer, the highly conserved His 117 active site residue becomes autocatalytically phosphorylated, in the enzyme intermediate (NDK-H*). This phosphoryl group is transferred to ribo-or deoxyribonucleotides (N2DP) in a substrate non-specific manner. In addition to its fundamental role in nucleotide metabolism, NDP kinase is also involved in a number of cellular regulatory functions such as growth and developmental control, tumor metastasis suppression, signal transduction and so on. From mycobacterial genera, NDK of
Mycobacterium tuberculosis (MtNDK) has been crystallised, structure was solved and biochemical functions were elucidated. However, there has not been any such study on the NDK of
Mycobacterium smegmatis, except on the possible interaction with other proteins which modulates the NTP synthesising activity of MsNDK, towards specific NTPs. M.
smegmatis, being a saprophytic, fast growing and non-pathogenic
mycobacterium that is widely used as an experimental model mycobacterial system to study various biological processes in mycobacteria, it was thought appropriate to study NDK from this organism.
The outcome of current study is presented in five chapters. The First Chapter gives a detailed introduction on the structural and functional aspects of NDK from diverse organisms, from bacteria to humans.
Chapter 2. Molecular Cloning, Expression and Characterisation of Biochemical Activities of Nucleoside Diphosphate Kinase from
Mycobacterium smegmatis mc 155
The research work starts with the molecular cloning, overexpression, purification, and characterisation of biochemical activities of recombinant MsNDK protein. In brief, ndk gene from M.
smegmatis (Msndk) has been cloned, efficiently overexpressed as a soluble 6xHis-tagged recombinant protein, purified through affinity chromatography, and its biochemical characterisation for ATPase, GTPase and NTP synthesising activities have been demonstrated. Catalytic mutant of MsNDK, MsNDK-H117Q, was generated using site-directed mutagenesis approach and H117 was shown to be essential for the catalytic activity. Further experiments revealed that it is the same H117 that is required for mediating autophosphorylation as well, which is an intermediate in the transphosphorylation reaction of NDK.
Chapter 3. Characterisation of Oligomerisation Property of M.
smegmatis Nucleoside Diphosphate Kinase: the Possible Role of Hydrogen Bond and Hydrophobic Interactions
The present study revealed that presence of homodimer of MsNDK could be observed in the presence of heat…
Advisors/Committee Members: Ajitkumar, P.
Subjects/Keywords: Nucleoside Sequence; Micobacterium Smegmatis; Structural Analysis; Transcriptional Analysis; Mycobacterial Nucleoside Diphosphate Kinase; Mycobacterium Smegmatis Nucleoside Diphosphate Kinase; Nucleoside Diphosphate Kinase; mc2 155; FtsZ; M. smegmatis; Biochemical Genetics
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Arumugam, M. (2009). Structural, Functional And Transcriptional Analysis Of Nucleoside Diphosphate Kinase From Mycobacterium Smegmatis mc2 155. (Thesis). Indian Institute of Science. Retrieved from http://hdl.handle.net/2005/1338
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Arumugam, Muthu. “Structural, Functional And Transcriptional Analysis Of Nucleoside Diphosphate Kinase From Mycobacterium Smegmatis mc2 155.” 2009. Thesis, Indian Institute of Science. Accessed December 10, 2019.
http://hdl.handle.net/2005/1338.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Arumugam, Muthu. “Structural, Functional And Transcriptional Analysis Of Nucleoside Diphosphate Kinase From Mycobacterium Smegmatis mc2 155.” 2009. Web. 10 Dec 2019.
Vancouver:
Arumugam M. Structural, Functional And Transcriptional Analysis Of Nucleoside Diphosphate Kinase From Mycobacterium Smegmatis mc2 155. [Internet] [Thesis]. Indian Institute of Science; 2009. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/2005/1338.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Arumugam M. Structural, Functional And Transcriptional Analysis Of Nucleoside Diphosphate Kinase From Mycobacterium Smegmatis mc2 155. [Thesis]. Indian Institute of Science; 2009. Available from: http://hdl.handle.net/2005/1338
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Indian Institute of Science
20.
Arumugam, Muthu.
Structural, Functional And Transcriptional Analysis Of Nucleoside Diphosphate Kinase From Mycobacterium Smegmatis mc2 155.
Degree: 2009, Indian Institute of Science
URL: http://etd.iisc.ernet.in/handle/2005/1338
;
http://etd.ncsi.iisc.ernet.in/abstracts/1731/G23694-Abs.pdf
► Maintenance of the levels of nucleoside triphosphates (NTPs) as well as their corresponding deoxy derivatives (dNTPs) is crucial to all growth and developmental processes. The…
(more)
▼ Maintenance of the levels of nucleoside triphosphates (NTPs) as well as their corresponding deoxy derivatives (dNTPs) is crucial to all growth and developmental processes. The enzyme nucleoside diphosphate kinase (NDK) utilises an autophosporylated enzyme intermediate to catalyse the transfer of 5’ terminal phosphate from NTPs (mostly ATP) to nucleoside diphosphates (NDPs) via a reversible mechanism as given below.
N1TP + NDK ↔N1DP+ −NDK-His* (1)
N2DP + NDK-His* P ↔N2TP + NDK−His. (2) In the γ-phosphoryl group transfer, the highly conserved His 117 active site residue becomes autocatalytically phosphorylated, in the enzyme intermediate (NDK-H*). This phosphoryl group is transferred to ribo-or deoxyribonucleotides (N2DP) in a substrate non-specific manner. In addition to its fundamental role in nucleotide metabolism, NDP kinase is also involved in a number of cellular regulatory functions such as growth and developmental control, tumor metastasis suppression, signal transduction and so on. From mycobacterial genera, NDK of
Mycobacterium tuberculosis (MtNDK) has been crystallised, structure was solved and biochemical functions were elucidated. However, there has not been any such study on the NDK of
Mycobacterium smegmatis, except on the possible interaction with other proteins which modulates the NTP synthesising activity of MsNDK, towards specific NTPs. M.
smegmatis, being a saprophytic, fast growing and non-pathogenic
mycobacterium that is widely used as an experimental model mycobacterial system to study various biological processes in mycobacteria, it was thought appropriate to study NDK from this organism.
The outcome of current study is presented in five chapters. The First Chapter gives a detailed introduction on the structural and functional aspects of NDK from diverse organisms, from bacteria to humans.
Chapter 2. Molecular Cloning, Expression and Characterisation of Biochemical Activities of Nucleoside Diphosphate Kinase from
Mycobacterium smegmatis mc 155
The research work starts with the molecular cloning, overexpression, purification, and characterisation of biochemical activities of recombinant MsNDK protein. In brief, ndk gene from M.
smegmatis (Msndk) has been cloned, efficiently overexpressed as a soluble 6xHis-tagged recombinant protein, purified through affinity chromatography, and its biochemical characterisation for ATPase, GTPase and NTP synthesising activities have been demonstrated. Catalytic mutant of MsNDK, MsNDK-H117Q, was generated using site-directed mutagenesis approach and H117 was shown to be essential for the catalytic activity. Further experiments revealed that it is the same H117 that is required for mediating autophosphorylation as well, which is an intermediate in the transphosphorylation reaction of NDK.
Chapter 3. Characterisation of Oligomerisation Property of M.
smegmatis Nucleoside Diphosphate Kinase: the Possible Role of Hydrogen Bond and Hydrophobic Interactions
The present study revealed that presence of homodimer of MsNDK could be observed in the presence of heat…
Advisors/Committee Members: Ajitkumar, P.
Subjects/Keywords: Nucleoside Sequence; Micobacterium Smegmatis; Structural Analysis; Transcriptional Analysis; Mycobacterial Nucleoside Diphosphate Kinase; Mycobacterium Smegmatis Nucleoside Diphosphate Kinase; Nucleoside Diphosphate Kinase; mc2 155; FtsZ; M. smegmatis; Biochemical Genetics
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APA (6th Edition):
Arumugam, M. (2009). Structural, Functional And Transcriptional Analysis Of Nucleoside Diphosphate Kinase From Mycobacterium Smegmatis mc2 155. (Thesis). Indian Institute of Science. Retrieved from http://etd.iisc.ernet.in/handle/2005/1338 ; http://etd.ncsi.iisc.ernet.in/abstracts/1731/G23694-Abs.pdf
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Arumugam, Muthu. “Structural, Functional And Transcriptional Analysis Of Nucleoside Diphosphate Kinase From Mycobacterium Smegmatis mc2 155.” 2009. Thesis, Indian Institute of Science. Accessed December 10, 2019.
http://etd.iisc.ernet.in/handle/2005/1338 ; http://etd.ncsi.iisc.ernet.in/abstracts/1731/G23694-Abs.pdf.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Arumugam, Muthu. “Structural, Functional And Transcriptional Analysis Of Nucleoside Diphosphate Kinase From Mycobacterium Smegmatis mc2 155.” 2009. Web. 10 Dec 2019.
Vancouver:
Arumugam M. Structural, Functional And Transcriptional Analysis Of Nucleoside Diphosphate Kinase From Mycobacterium Smegmatis mc2 155. [Internet] [Thesis]. Indian Institute of Science; 2009. [cited 2019 Dec 10].
Available from: http://etd.iisc.ernet.in/handle/2005/1338 ; http://etd.ncsi.iisc.ernet.in/abstracts/1731/G23694-Abs.pdf.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Arumugam M. Structural, Functional And Transcriptional Analysis Of Nucleoside Diphosphate Kinase From Mycobacterium Smegmatis mc2 155. [Thesis]. Indian Institute of Science; 2009. Available from: http://etd.iisc.ernet.in/handle/2005/1338 ; http://etd.ncsi.iisc.ernet.in/abstracts/1731/G23694-Abs.pdf
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
21.
Lamrabet, Otmane.
Modifications génétiques de Mycobacterium tuberculosis : interactions avec les organismes hôtes : Synthesis and characterization of new electron transfer {Fe(µ-CN)M} (M = Fe, Mn, Co) binuclear complexes.
Degree: Docteur es, Sciences de la vie et de la sante, 2012, Aix Marseille Université
URL: http://www.theses.fr/2012AIXM5027
► Les mycobactéries sont classées parmi les bactéries contenant des acides mycoliques dans leur paroi et un haut GC% dans leur génome. Elles peuvent être isolées…
(more)
▼ Les mycobactéries sont classées parmi les bactéries contenant des acides mycoliques dans leur paroi et un haut GC% dans leur génome. Elles peuvent être isolées à partir du sol ou d'environnement d'eau douce où vivent aussi les protozoaires libres. Plusieurs études ont montré une possibilité de co-isolement des mycobactéries et des amibes à partir de ces sources environnementales. Il a été montré également que la plupart des mycobactéries de l'environnement ont la capacité à survivre dans les trophozoites et les kystes d'amibes et dans certaines cellules eucaryotes, y compris les macrophages. Les manipulations génétiques des mycobactéries en général et des mycobactéries du complexe Mycobacterium tuberculosis en particulier sont compliquées et aucune étude de modification génétique des mycobactéries (pathogènes ou non pathogènes) n'avait été réalisée dans notre laboratoire avant notre travail de thèse. Dans notre travail de thèse, nous avons montré que les amibes ou d'autres organismes phagocytaires peuvent servir comme sources et lieu de transfert des gènes chez les mycobactéries. Ce transfert des gènes peut avoir contribué à l'adaptation des mycobactéries à un mode de vie intracellulaire. Nous avons développé ensuite deux systèmes de coculture: Mycobacterium smegmatis-Acanthamoeba polyphaga et Mycobacterium gilvum-A. polyphaga et nous avons clarifié le spectre des interactions des mycobactéries à croissance rapide avec les amibes. Ce modèle d'interaction mycobactéries-amibes a été utilisé pour tester l'hypothèse contraire au paradigme dominant que l'addition des gènes réduit la virulence des bactéries.
Mycobacteria are mycolic-acid containing, high GC% bacterial organisms which can be recovered from soil and fresh water environments where free-living protozoa also live. Co-isolation of mycobacteria and amoeba collected from such environmental sources has been reported. Several experiments further demonstrated the ability of most environmental mycobacteria to survive in the amoebal trophozoites and cysts and in some eukaryotic cells including macrophages. Genetic modification of mycobacteria in general and mycobacteria belonging to Mycobacterium tuberculosis complex in particular are complicated and no studies using genetic modification of mycobacteria (pathogenic or non-pathogenic) had been performed in our laboratory prior to our work. In our thesis work, we showed that amoebae or other phagocytic organisms can serve as sources and places for gene transfers in mycobacteria. Gene transfers may have contributed to the adaptation of mycobacteria to an intracellular lifestyle. In addition, we developed two co-culture systems: Mycobacterium smegmatis-Acanthamoeba polyphaga and Mycobacterium gilvum-A. polyphaga and we clarified the spectrum of rapid-growing mycobacteria and amoeba interactions. This model of mycobacteria-amoeba interactions was then used to test another hypothesis according to which unlike the prevailing paradigm, the addition of genes does not reduce the virulence of bacteria. For the first time in…
Advisors/Committee Members: Drancourt, Michel (thesis director).
Subjects/Keywords: Mycobactéries; Mycobacterium tuberculosis; Mycobacterium smegmatis; Mycobacterium gilvum; Amibes; Cellules eucaryotes; Transfert des gènes; Modifications génétiques; Porine MspA; Mycobacteria; Mycobacterium tuberculosis; Mycobacterium smegmatis; Mycobacterium gilvum; Amoeba; Eukaryotic cells; Gene transfer; Genetic modification; MspA porin
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APA ·
Chicago ·
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CSE |
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APA (6th Edition):
Lamrabet, O. (2012). Modifications génétiques de Mycobacterium tuberculosis : interactions avec les organismes hôtes : Synthesis and characterization of new electron transfer {Fe(µ-CN)M} (M = Fe, Mn, Co) binuclear complexes. (Doctoral Dissertation). Aix Marseille Université. Retrieved from http://www.theses.fr/2012AIXM5027
Chicago Manual of Style (16th Edition):
Lamrabet, Otmane. “Modifications génétiques de Mycobacterium tuberculosis : interactions avec les organismes hôtes : Synthesis and characterization of new electron transfer {Fe(µ-CN)M} (M = Fe, Mn, Co) binuclear complexes.” 2012. Doctoral Dissertation, Aix Marseille Université. Accessed December 10, 2019.
http://www.theses.fr/2012AIXM5027.
MLA Handbook (7th Edition):
Lamrabet, Otmane. “Modifications génétiques de Mycobacterium tuberculosis : interactions avec les organismes hôtes : Synthesis and characterization of new electron transfer {Fe(µ-CN)M} (M = Fe, Mn, Co) binuclear complexes.” 2012. Web. 10 Dec 2019.
Vancouver:
Lamrabet O. Modifications génétiques de Mycobacterium tuberculosis : interactions avec les organismes hôtes : Synthesis and characterization of new electron transfer {Fe(µ-CN)M} (M = Fe, Mn, Co) binuclear complexes. [Internet] [Doctoral dissertation]. Aix Marseille Université 2012. [cited 2019 Dec 10].
Available from: http://www.theses.fr/2012AIXM5027.
Council of Science Editors:
Lamrabet O. Modifications génétiques de Mycobacterium tuberculosis : interactions avec les organismes hôtes : Synthesis and characterization of new electron transfer {Fe(µ-CN)M} (M = Fe, Mn, Co) binuclear complexes. [Doctoral Dissertation]. Aix Marseille Université 2012. Available from: http://www.theses.fr/2012AIXM5027
Indian Institute of Science
22.
Verma, Amit Kumar.
Elucidating the Role of MsRbpA in Rifampicin Tolerance and Transcription Regulation of Mycobacterium Smegmatis.
Degree: 2013, Indian Institute of Science
URL: http://etd.iisc.ernet.in/2005/3397
;
http://etd.iisc.ernet.in/abstracts/4263/G25860-Abs.pdf
► RNA polymerase binding protein A (RbpA) was first discovered as a RNA polymerase binding protein from Streptomyces. coelicolor. It was shown to cause rifampicin tolerance…
(more)
▼ RNA polymerase binding protein A (RbpA) was first discovered as a RNA polymerase binding protein from Streptomyces. coelicolor. It was shown to cause rifampicin tolerance to RNA polymerase in vitro and leads to basal level of rifampicin resistance in vivo. This protein is exclusively present in the actinobacteria family with the nearest neighbour in mycobacteria. When null mutant of RbpA in S. coelicolor were transformed with the rbpA gene from
Mycobacterium tuberculosis the resistance level of rifampicin increased from 0.75 µgml-1 to 2 µg ml-1 suggesting analogous role of MtbRbpA (RbpA from M. tuberculosis). MsRbpA, RbpA from
Mycobacterium smegmatis was found to interact with the β-subunit of RNAP and its binding location on M.
smegmatis RNAP was shown to be 18 Å from the (i+1) site. MsRbpA was also shown to rescue the inhibitory effect of rifampicin in vitro. Furthermore, overexpression of MsRbpA in wild type M.
smegmatis resulted in the increase in the MIC of rifampicin to 85 µg ml-1 from 20 µg ml-1, which is the MIC of rifampicin for the wild type M.
smegmatis. On the other hand, MsRbpA was unable to augment transcription in the presence of rifampicin when the reaction was catalysed by rifampicin resistant RNAP. Recent reports have shown that MtbRbpA enhances the affinity σA to core RNAP thereby activates transcription. The N and C-termini of MtbRbpA interact with σA while the C-terminal region of MtbRbpA is required for the oligomerisation of MtbRbpA. However M. tuberculosis and S. coleicolor are part of same family actinobacteria, RbpA is essential for the former while it is dispensable in the later case.This work focuses on characterisation of rifampicin resistant RNAP from M.
smegmatis and elaborates on the roles played by MsRbpA. These include its effect on transcription activation, transcription rescue, its role in RNAP promoter closed and open complex formation, characterisation of its site of interaction with RNAP and σA, finding critical functional residues and establishing the essentiality of MsRbpA in M.
smegmatis.
Chapter 1 deals with the literature survey on structure of bacterial RNAP, promoters, sigma factors, RNAP inhibitors, transcriptional activators with the emphasis on the Mycobacteria.
Chapter 2 summarises the identification of the mutations in rpoB gene from the rifampicin resistant (RifR) mutant strains of M.
smegmatis, purification of RNAP from these strain, determining IC50 values of these RifR RNAP for rifampicin, finding kinetic parameters for the interaction of RifR RNAP with 3-formyl rifampicin and evaluating their interaction with MsRbpA.
Chapter 3 describes the function of MsRbpA in transcription initiation, particularly its role in RNAP-promoter closed and open complex formation. Furthermore, this chapter throws light on the role of MsRbpA in transcription activation vis a vis its effects on transcription rescue from the inhibitory effect of rifampicin.
Chapter 4 elucidates the function of a segment of MsRbpA from Arg58 to Lys 73 in activation of transcription activity,…
Advisors/Committee Members: Chatterji, Dipankar.
Subjects/Keywords: Mycrobacterium Smegmatis; Bacterial Transcription; Mycobacterium Smegmatis RNA Polymer Binding Protein; Bacterial RNA Polymerase; Mycobacterial Transcription Regulation; Mycobacterial Transcription; Mycobacterium Smegmatis - Rifampicin; MsRbpA; Mycobacterial RNA Polymerase; M. smegmatis; RNA Polymerase Binding Protein A (RbpA); Bacteriology
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Verma, A. K. (2013). Elucidating the Role of MsRbpA in Rifampicin Tolerance and Transcription Regulation of Mycobacterium Smegmatis. (Thesis). Indian Institute of Science. Retrieved from http://etd.iisc.ernet.in/2005/3397 ; http://etd.iisc.ernet.in/abstracts/4263/G25860-Abs.pdf
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Verma, Amit Kumar. “Elucidating the Role of MsRbpA in Rifampicin Tolerance and Transcription Regulation of Mycobacterium Smegmatis.” 2013. Thesis, Indian Institute of Science. Accessed December 10, 2019.
http://etd.iisc.ernet.in/2005/3397 ; http://etd.iisc.ernet.in/abstracts/4263/G25860-Abs.pdf.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Verma, Amit Kumar. “Elucidating the Role of MsRbpA in Rifampicin Tolerance and Transcription Regulation of Mycobacterium Smegmatis.” 2013. Web. 10 Dec 2019.
Vancouver:
Verma AK. Elucidating the Role of MsRbpA in Rifampicin Tolerance and Transcription Regulation of Mycobacterium Smegmatis. [Internet] [Thesis]. Indian Institute of Science; 2013. [cited 2019 Dec 10].
Available from: http://etd.iisc.ernet.in/2005/3397 ; http://etd.iisc.ernet.in/abstracts/4263/G25860-Abs.pdf.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Verma AK. Elucidating the Role of MsRbpA in Rifampicin Tolerance and Transcription Regulation of Mycobacterium Smegmatis. [Thesis]. Indian Institute of Science; 2013. Available from: http://etd.iisc.ernet.in/2005/3397 ; http://etd.iisc.ernet.in/abstracts/4263/G25860-Abs.pdf
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Indian Institute of Science
23.
Mattoo, Rohini.
Metallophosphoesterases In Mycobacteria Enigmatic Roles In Regulating Mycobacterial Physiology.
Degree: 2012, Indian Institute of Science
URL: http://hdl.handle.net/2005/2305
► Pathogenic bacteria such as M.tuberculosis have evolved several mechanisms to aid their intracellular survival and subvert host defenses. One of the contributing factors is thought…
(more)
▼ Pathogenic bacteria such as M.tuberculosis have evolved several mechanisms to aid their intracellular survival and subvert host defenses. One of the contributing factors is thought to be the production and secretion of large amount of cAMP, Mycobacterial genomes encode a large number of adenylyl cyclases distinct in their structure and regulatory mechanisms. The roles of these enzymes in the physiology and pathogenesis of virulent mycobacteria are only now being elucidated. The roles of phosphodiesterases (PDEs), which serve to lower cAMP levels through degradation are, however, relatively unexplored.
The Rv0805 gene was previously shown to code for an active phosphodiesterase from
Mycobacterium tuberculosis. Bioinformatics analysis revealed that orthologs of Rv0805 were found even in eukaryotes. Biochemical and structural characterization of Rv0805 revealed that it was a class III cAMP phosphodiesterase. Comparative genomics identified a close ortholog of Rv0805 in M. leprae (ML2210). The genome of M. leprae
Encodes only 1,604 predicted proteins and possesses the highest number of pseudogenes, 1,116. The retention of a functional PDE, the ortholog of Rv0805, in the minimal genome of M. leprae is indicative of its importance in cellular physiology. Biochemical characterization of proteins from M. leprae and use of heterologous hosts will help understand this human pathogen better, since there are no tools currently available to genetically manipulate this bacterium.
Sequence analysis of ML2210 revealed the presence of conserved motifs and residues known to be critical for catalysis and unique to class III phosphodiesterases. ML2210 shares 83% sequence identity with Rv0805 and 24% sequence identity with the phosphodiesterase from E. coli (cpdA). In vitro biochemical characterization of ML2210 using non-nucleotide colorigenic and cyclic nucleotide substrates revealed that it was an enzymatically active phosphodiesterase. Kinetic parameters of ML2210 with respect ot colorigenic substrates revealed that its catalytic properties were similar to that of Rv0805. However, with respect to hydrolysis of 3’, 5’-cAMP, ML2210 was catalytically more efficient than Rv0805, suggesting that in spite of being orthologs, these enzymes have evolved distinct specificities at their active site. A parallel of monoclonal antibodies raised to Rv0805 was also used understand the differences in the biochemical properties of Rv0805 and ML2210 better. It was observed that only one monoclonal antibody was able to recognize ML2210 by ELISA and not by Western blot analysis. These results revealed that conformational differences between ML2210 and Rv0805 exist.
Over-expression of ML2210 in M.
smegmatis resulted in a modest decrease in intracellular cAMP levels. Despite the absence of a predicted transmembrane region or a membrane-targeting signal, ML2210 localized to the cell envelop fraction upon over expression in M.
smegmatis. Moreover, like Rv0805, over-expression of ML2210 also resulted in perturbation of the cell wall of…
Advisors/Committee Members: Visweswariah, Sandhya S.
Subjects/Keywords: Metallophosphoesterase; Mycobacterial Physiology; Mycobacterial Phosphodiesterases; Cyclic Nucleotide Signaling; Mycobacteria - Pathogenesis; Mycobacterium Leprae; Mycobacterium Smegmatis; Cyclic Adenosine Phosphate Signaling; Cyclic Nucleotide Phosphodiesterases; M. lepre; M. Smegmatis; Bacteriology
Record Details
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Record Details
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Mattoo, R. (2012). Metallophosphoesterases In Mycobacteria Enigmatic Roles In Regulating Mycobacterial Physiology. (Thesis). Indian Institute of Science. Retrieved from http://hdl.handle.net/2005/2305
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Mattoo, Rohini. “Metallophosphoesterases In Mycobacteria Enigmatic Roles In Regulating Mycobacterial Physiology.” 2012. Thesis, Indian Institute of Science. Accessed December 10, 2019.
http://hdl.handle.net/2005/2305.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Mattoo, Rohini. “Metallophosphoesterases In Mycobacteria Enigmatic Roles In Regulating Mycobacterial Physiology.” 2012. Web. 10 Dec 2019.
Vancouver:
Mattoo R. Metallophosphoesterases In Mycobacteria Enigmatic Roles In Regulating Mycobacterial Physiology. [Internet] [Thesis]. Indian Institute of Science; 2012. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/2005/2305.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Mattoo R. Metallophosphoesterases In Mycobacteria Enigmatic Roles In Regulating Mycobacterial Physiology. [Thesis]. Indian Institute of Science; 2012. Available from: http://hdl.handle.net/2005/2305
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Indian Institute of Science
24.
Mattoo, Rohini.
Metallophosphoesterases In Mycobacteria Enigmatic Roles In Regulating Mycobacterial Physiology.
Degree: 2012, Indian Institute of Science
URL: http://etd.iisc.ernet.in/handle/2005/2305
;
http://etd.ncsi.iisc.ernet.in/abstracts/2965/G25484-Abs.pdf
► Pathogenic bacteria such as M.tuberculosis have evolved several mechanisms to aid their intracellular survival and subvert host defenses. One of the contributing factors is thought…
(more)
▼ Pathogenic bacteria such as M.tuberculosis have evolved several mechanisms to aid their intracellular survival and subvert host defenses. One of the contributing factors is thought to be the production and secretion of large amount of cAMP, Mycobacterial genomes encode a large number of adenylyl cyclases distinct in their structure and regulatory mechanisms. The roles of these enzymes in the physiology and pathogenesis of virulent mycobacteria are only now being elucidated. The roles of phosphodiesterases (PDEs), which serve to lower cAMP levels through degradation are, however, relatively unexplored.
The Rv0805 gene was previously shown to code for an active phosphodiesterase from
Mycobacterium tuberculosis. Bioinformatics analysis revealed that orthologs of Rv0805 were found even in eukaryotes. Biochemical and structural characterization of Rv0805 revealed that it was a class III cAMP phosphodiesterase. Comparative genomics identified a close ortholog of Rv0805 in M. leprae (ML2210). The genome of M. leprae
Encodes only 1,604 predicted proteins and possesses the highest number of pseudogenes, 1,116. The retention of a functional PDE, the ortholog of Rv0805, in the minimal genome of M. leprae is indicative of its importance in cellular physiology. Biochemical characterization of proteins from M. leprae and use of heterologous hosts will help understand this human pathogen better, since there are no tools currently available to genetically manipulate this bacterium.
Sequence analysis of ML2210 revealed the presence of conserved motifs and residues known to be critical for catalysis and unique to class III phosphodiesterases. ML2210 shares 83% sequence identity with Rv0805 and 24% sequence identity with the phosphodiesterase from E. coli (cpdA). In vitro biochemical characterization of ML2210 using non-nucleotide colorigenic and cyclic nucleotide substrates revealed that it was an enzymatically active phosphodiesterase. Kinetic parameters of ML2210 with respect ot colorigenic substrates revealed that its catalytic properties were similar to that of Rv0805. However, with respect to hydrolysis of 3’, 5’-cAMP, ML2210 was catalytically more efficient than Rv0805, suggesting that in spite of being orthologs, these enzymes have evolved distinct specificities at their active site. A parallel of monoclonal antibodies raised to Rv0805 was also used understand the differences in the biochemical properties of Rv0805 and ML2210 better. It was observed that only one monoclonal antibody was able to recognize ML2210 by ELISA and not by Western blot analysis. These results revealed that conformational differences between ML2210 and Rv0805 exist.
Over-expression of ML2210 in M.
smegmatis resulted in a modest decrease in intracellular cAMP levels. Despite the absence of a predicted transmembrane region or a membrane-targeting signal, ML2210 localized to the cell envelop fraction upon over expression in M.
smegmatis. Moreover, like Rv0805, over-expression of ML2210 also resulted in perturbation of the cell wall of…
Advisors/Committee Members: Visweswariah, Sandhya S.
Subjects/Keywords: Metallophosphoesterase; Mycobacterial Physiology; Mycobacterial Phosphodiesterases; Cyclic Nucleotide Signaling; Mycobacteria - Pathogenesis; Mycobacterium Leprae; Mycobacterium Smegmatis; Cyclic Adenosine Phosphate Signaling; Cyclic Nucleotide Phosphodiesterases; M. lepre; M. Smegmatis; Bacteriology
Record Details
Similar Records
Cite
Share »
Record Details
Similar Records
Cite
« Share
❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Mattoo, R. (2012). Metallophosphoesterases In Mycobacteria Enigmatic Roles In Regulating Mycobacterial Physiology. (Thesis). Indian Institute of Science. Retrieved from http://etd.iisc.ernet.in/handle/2005/2305 ; http://etd.ncsi.iisc.ernet.in/abstracts/2965/G25484-Abs.pdf
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Mattoo, Rohini. “Metallophosphoesterases In Mycobacteria Enigmatic Roles In Regulating Mycobacterial Physiology.” 2012. Thesis, Indian Institute of Science. Accessed December 10, 2019.
http://etd.iisc.ernet.in/handle/2005/2305 ; http://etd.ncsi.iisc.ernet.in/abstracts/2965/G25484-Abs.pdf.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Mattoo, Rohini. “Metallophosphoesterases In Mycobacteria Enigmatic Roles In Regulating Mycobacterial Physiology.” 2012. Web. 10 Dec 2019.
Vancouver:
Mattoo R. Metallophosphoesterases In Mycobacteria Enigmatic Roles In Regulating Mycobacterial Physiology. [Internet] [Thesis]. Indian Institute of Science; 2012. [cited 2019 Dec 10].
Available from: http://etd.iisc.ernet.in/handle/2005/2305 ; http://etd.ncsi.iisc.ernet.in/abstracts/2965/G25484-Abs.pdf.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Mattoo R. Metallophosphoesterases In Mycobacteria Enigmatic Roles In Regulating Mycobacterial Physiology. [Thesis]. Indian Institute of Science; 2012. Available from: http://etd.iisc.ernet.in/handle/2005/2305 ; http://etd.ncsi.iisc.ernet.in/abstracts/2965/G25484-Abs.pdf
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Universidade de Lisboa
25.
Diogo, Joana Patrícia Baptista, 1982-.
Utilização de transportadores coloidais submicrométricos como estratégia para promover a fagocitose e o tropismo para as vias endocíticas em macrófagos.
Degree: 2009, Universidade de Lisboa
URL: http://www.rcaap.pt/detail.jsp?id=oai:repositorio.ul.pt:10451/1951
► Tese de mestrado, Microbiologia Clínica, Faculdade de Medicina, Universidade de Lisboa, 2010
O Mycobacterium tuberculosis é o agente patogénico da Tuberculose. Uma das suas principais…
(more)
▼ Tese de mestrado, Microbiologia Clínica, Faculdade de Medicina, Universidade de Lisboa, 2010
O Mycobacterium tuberculosis é o agente patogénico da Tuberculose. Uma das suas
principais características está relacionada com a sua capacidade de sobrevivência no interior
dos macrófagos. Muito embora a eficácia e tolerância dos fármacos disponíveis não esteja em
questão, o desenvolvimento de resistências e a falta de selectividade para as células-alvo
revela-se cada vez mais preocupante. A terapêutica actual prolonga-se por longos períodos de
tempo e requer a administração contínua e repetida de doses elevadas de fármacos. Este facto,
em conjunto com os efeitos tóxicos, relacionados sobretudo com a administração sistémica,
contribui para a falta de adesão dos doentes à terapêutica. É urgente o desenvolvimento de
novos fármacos ou o desenvolvimento de sistemas que permitam a libertação modificada dos
mesmos. As nanopartículas lipídicas (nsLp) são formulações farmacêuticas com um elevado
potencial para direccionar e manter a concentração farmacológica nos tecidos-alvo, reduzindo
desta forma os efeitos sistémicos dos fármacos incorporados.
Neste trabalho desenvolveu-se um sistema lipídico-coloidal (nsLp), como
transportador da proteína LysB, uma proteína obtida por indução da sua expressão em E. coli
recombinante (pMP302), que demonstrou ser eficaz na inibição do crescimento bacteriano de
culturas de Mycobacterium smegmatis. A investigação realizada em culturas de
Mycobacterium smegmatis e em macrófagos infectados com o mesmo agente bacteriano
permitiram avaliar o efeito da incorporação da LysB em nanopartículas lipídicas,
nomeadamente, na sua estrutura e actividade antimicrobiana.
Os resultados obtidos demonstraram que o sistema nsLp_LysB é mais eficaz que a
proteína na sua forma livre para a inibição do crescimento bacteriano de Mycobacterium
smegmatis, o que pode estar relacionado com a internalização celular facilitada das partículas endocitáveis. O sistema nsLp_LysB funciona assim como um transportador eficaz da proteína
para células fagocitárias.
A utilização de proteínas fágicas com propriedades lipolíticas como agentes
terapêuticos da Tuberculose revela-se uma hipótese interessante. No entanto é necessário
garantir a vectorização destas biomoléculas à sua célula-alvo. A utilização das nsLp com este
objectivo terapêutico revela-se uma inovação tecnológica de sucesso, uma vez que estas têm
sido descritas como transportadores selectivos de fármacos para o tratamento de doenças pulmonares.
The pathogen of tuberculosis is Mycobacterium tuberculosis. One of its main
characteristics is related to the ability of survival within macrophages. Although the drugs
currently available are relatively well tolerated and effective, the development of resistance
and difficulty in reaching the target cells (alveolar macrophages) is increasingly worrying.
The current treatment extends over long periods of time and requires the continuous and
repeated administration of high doses of drugs. Furthermore, the toxic side effects related…
Advisors/Committee Members: Videira, Mafalda de Castro Ascensão Marques, 1963-, Pimentel, Madalena Maria Vilela, 1961-.
Subjects/Keywords: Microbiologia; Mycobacterium Smegmatis; Sistemas de liberação de medicamentos; Lípidos; Nanopartículas; Terapêutica; Teses de mestrado - 2010
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APA (6th Edition):
Diogo, Joana Patrícia Baptista, 1. (2009). Utilização de transportadores coloidais submicrométricos como estratégia para promover a fagocitose e o tropismo para as vias endocíticas em macrófagos. (Thesis). Universidade de Lisboa. Retrieved from http://www.rcaap.pt/detail.jsp?id=oai:repositorio.ul.pt:10451/1951
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Diogo, Joana Patrícia Baptista, 1982-. “Utilização de transportadores coloidais submicrométricos como estratégia para promover a fagocitose e o tropismo para as vias endocíticas em macrófagos.” 2009. Thesis, Universidade de Lisboa. Accessed December 10, 2019.
http://www.rcaap.pt/detail.jsp?id=oai:repositorio.ul.pt:10451/1951.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Diogo, Joana Patrícia Baptista, 1982-. “Utilização de transportadores coloidais submicrométricos como estratégia para promover a fagocitose e o tropismo para as vias endocíticas em macrófagos.” 2009. Web. 10 Dec 2019.
Vancouver:
Diogo, Joana Patrícia Baptista 1. Utilização de transportadores coloidais submicrométricos como estratégia para promover a fagocitose e o tropismo para as vias endocíticas em macrófagos. [Internet] [Thesis]. Universidade de Lisboa; 2009. [cited 2019 Dec 10].
Available from: http://www.rcaap.pt/detail.jsp?id=oai:repositorio.ul.pt:10451/1951.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Diogo, Joana Patrícia Baptista 1. Utilização de transportadores coloidais submicrométricos como estratégia para promover a fagocitose e o tropismo para as vias endocíticas em macrófagos. [Thesis]. Universidade de Lisboa; 2009. Available from: http://www.rcaap.pt/detail.jsp?id=oai:repositorio.ul.pt:10451/1951
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Indian Institute of Science
26.
Ghosh, Subho.
Biochemical, Biophysical and Evolutionary Perspectives of Zinc Finger Proteins in Mycobacterium smegmatis.
Degree: 2017, Indian Institute of Science
URL: http://etd.iisc.ernet.in/2005/3567
;
http://etd.iisc.ernet.in/abstracts/4435/G28419-Abs.pdf
► Transcription is a major step in expression of genes of a given organism. Due to environmental constrains this step must be regulated in the favour…
(more)
▼ Transcription is a major step in expression of genes of a given organism. Due to environmental constrains this step must be regulated in the favour of the sustenance and growth of the organism. Here comes the relevance of transcription factors, mostly proteins which regulate transcription. One such important group of transcription factors is the zinc finger proteins.
It is well known that in eukaryotes the C2H2 zinc finger domain containing proteins are the largest group of transcription factors while in prokaryotes the largest group of transcription factors are represented by helix-turn-helix motif containing proteins. Till now only two C2H2 zinc finger domain proteins-Ros and Muc have been found in alpha proteobacteria which are also transcription factors. In eukaryotes the second largest group of zinc finger proteins have their zinc ion coordinated by four cysteine residues- the C4 zinc finger proteins. They make the nuclear hormone receptor superfamily of proteins. They have also been shown to act as transcription factors. But in eubacteria no such proteins have been described in details except an isolated report of crystal structure of a C-terminal zinc finger domain protein- Jann_2411 from Jannaschia sp.
Though a lot of transcription factors have been described in mechanistic details in Escherichia coli and Bacillus subtilis, the list of well described mycobacterial transcription factors is short. Given this fact and the lack of any known zinc finger domain transcription factor in actinobacteria we wanted to see whether M.
smegmatis genome also encode any homologue of Jann_2411 and if does whether they have ability to modulate transcription.
To meet our aim we did BLASTP search against the genome of M.
smegmatis using Jann_2411 as query. We found four C-terminal zinc finger domain proteins –Msmeg_0118. Msmeg_3613, Msmeg_3408 and Msmeg_1531, which we named as Mycobacterial single zinc finger protein (Mszfp) and numbered- Mszfp1, Mszfp2, Mszfp3 and Mszfp4, respectively. Mszfp1 and Mszfp2 were chosen for study as they were the top most hits.
In this thesis:-
Chapter1 introduces zinc finger proteins, transcription and several levels of control of transcription process in eubacteria.
In chapter2 we characterised Mszfp1 biophysically and probed its secondary structure content and oligomeric state in the native and demetallated conditions. We have also shown that this conserved hypothetical protein is expressed throughout the growth phase of M.
smegmatis, regulated by SigA and SigB. We have also showed that Mszfp1 is a DNA binding protein in the native state and the demetallated protein has altered DNA binding ability. It was noted that on over expression Mszfp1 affects colony morphology and biofilm forming ability, of M.
smegmatis.
In chapter3 the ability of Mszfp1 to bind to RNA polymerase of M.
smegmatis has been explored. It was found that Mszfp1 can activate transcription by interacting with CTD/NTD of α subunit and domain 4 of σA like CRP on type II CRP activated promoter.
In chapter4 similar to Mszfp1…
Advisors/Committee Members: Chatterji, Dipankar.
Subjects/Keywords: RNA Polymerase (RNAP); Mszfp2; Mycobacteria; Mycobacterium Smegmatis; Zinc Finger Protein; Mszfp1; Molecular Biophysics
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APA (6th Edition):
Ghosh, S. (2017). Biochemical, Biophysical and Evolutionary Perspectives of Zinc Finger Proteins in Mycobacterium smegmatis. (Thesis). Indian Institute of Science. Retrieved from http://etd.iisc.ernet.in/2005/3567 ; http://etd.iisc.ernet.in/abstracts/4435/G28419-Abs.pdf
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Ghosh, Subho. “Biochemical, Biophysical and Evolutionary Perspectives of Zinc Finger Proteins in Mycobacterium smegmatis.” 2017. Thesis, Indian Institute of Science. Accessed December 10, 2019.
http://etd.iisc.ernet.in/2005/3567 ; http://etd.iisc.ernet.in/abstracts/4435/G28419-Abs.pdf.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Ghosh, Subho. “Biochemical, Biophysical and Evolutionary Perspectives of Zinc Finger Proteins in Mycobacterium smegmatis.” 2017. Web. 10 Dec 2019.
Vancouver:
Ghosh S. Biochemical, Biophysical and Evolutionary Perspectives of Zinc Finger Proteins in Mycobacterium smegmatis. [Internet] [Thesis]. Indian Institute of Science; 2017. [cited 2019 Dec 10].
Available from: http://etd.iisc.ernet.in/2005/3567 ; http://etd.iisc.ernet.in/abstracts/4435/G28419-Abs.pdf.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Ghosh S. Biochemical, Biophysical and Evolutionary Perspectives of Zinc Finger Proteins in Mycobacterium smegmatis. [Thesis]. Indian Institute of Science; 2017. Available from: http://etd.iisc.ernet.in/2005/3567 ; http://etd.iisc.ernet.in/abstracts/4435/G28419-Abs.pdf
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Stellenbosch University
27.
Steyn, Nastassja Lise.
Investigating the localisation of the ESX-3 secretion system in Mycobacterium smegmatis.
Degree: MScMedSc, 2012, Stellenbosch University
URL: http://hdl.handle.net/10019.1/71959
► ENGLISH ABSTRACT: Mycobacterium tuberculosis is a pathogenic organism that infects a third of the world’s population and causes approximately 2 million deaths per year. Extensive…
(more)
▼ ENGLISH ABSTRACT: Mycobacterium tuberculosis is a pathogenic organism that infects a third of the world’s population and
causes approximately 2 million deaths per year. Extensive research has been done on this pathogen,
however our knowledge of the mechanisms of pathogenicity remain limited. The M. tuberculosis genome
contains five ESAT-6 gene cluster regions, ESX-1 to 5, which encode specialized type VII secretion
systems. These secretion systems are known to secrete members of the ESAT-6/CFP-10 and PE/PPE
protein families, some of which contribute to the pathogenicity and phagosomal escape of the pathogen.
ESX-3 has been shown to be essential for in vitro growth and survival of M. tuberculosis. The expression
of ESX-3 in M. tuberculosis is regulated by IdeR and Zur, in response to intracellular iron and zinc
concentrations, respectively. Interestingly, ESX-3 is not essential for the growth and survival of the
saprophytic organism M. smegmatis.
In this study, we aimed to identify the subcellular localisation of the individual components of the ESX-3
secretion system in the non-pathogenic, fast-growing organism M. smegmatis. The esx conserved
component (ecc) genes from ESX-3 were expressed from the episomal expression vector pDMNI as
fusion proteins with green fluorescent protein (GFP). MSMEG_0615 (eccA3), MSMEG_0616 (eccB3),
MSMEG_0623 (eccD3) and MSMEG_0626 (eccE3) were successfully cloned into pDMNI and expression
of fusion proteins was confirmed by Western blotting for MSMEG_0615-GFP, MSMEG_0616-GFP and
MSMEG_0626-GFP in M. smegmatis. In the M. smegmatis ESX-3 knock-out (with MSMEG_0615 to
MSMEG_0626 deleted) expression was confirmed for MSMEG_0615-GFP and MSMEG0626-GFP.
Fluorescent microscopy determined that MSMEG_0615-GFP localised to a single mycobacterial pole in
both strains. MSMEG_0616-GFP and MSMEG_0626-GFP were found to be membrane associated in M.
smegmatis, while MSMEG_0626-GFP was found to be membrane associated in the M. smegmatis ESX-3
knock-out. The unipolar localisation of MSMEG_0615-GFP suggests that the assembled ESX-3 secretion system
apparatus is situated at a single pole in M. smegmatis. Therefore, we hypothesize that MSMEG_0615
might act as a recruiter protein that is involved in the assembly of ESX-3 at the mycobacterial pole.
AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis is ‘n patogene organisme wat ‘n derde van die wêreld se bevolking infekteer
en eis jaarliks 2 miljoen lewens deur tuberkulose. Ten spyte van uitgebreide navorsing, is daar min kennis
oor die meganismes van patogenisiteit van hierdie organisme. Die M. tuberculosis genoom bevat vyf
duplikasies van die ESAT-6 geen groep gebiede, ESX-1 tot 5, wat kodeer vir gespesialiseerde Tipe VII
sekresie sisteme. Hierdie sekresie sisteme is bekend vir die sekresie van lede van die ESAT-6/CFP-10
en PE/PPE proteïen families, waarvan sommige bydra tot die patogenisieit en fagosomale ontsnapping
van hierdie organisme. ESX-3 is noodsaaklik vir die in vitro groei en oorlewing van M. tuberculosis. Die
…
Advisors/Committee Members: Gey van Pittius, N. C., Warren, R. M., Stellenbosch University. Faculty of Medicine and Health Sciences. Department of Biomedical Sciences..
Subjects/Keywords: Molecular biology and human genetics; Dissertations – Molecular biology and human genetics; Mycobacterium smegmatis
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APA ·
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APA (6th Edition):
Steyn, N. L. (2012). Investigating the localisation of the ESX-3 secretion system in Mycobacterium smegmatis. (Masters Thesis). Stellenbosch University. Retrieved from http://hdl.handle.net/10019.1/71959
Chicago Manual of Style (16th Edition):
Steyn, Nastassja Lise. “Investigating the localisation of the ESX-3 secretion system in Mycobacterium smegmatis.” 2012. Masters Thesis, Stellenbosch University. Accessed December 10, 2019.
http://hdl.handle.net/10019.1/71959.
MLA Handbook (7th Edition):
Steyn, Nastassja Lise. “Investigating the localisation of the ESX-3 secretion system in Mycobacterium smegmatis.” 2012. Web. 10 Dec 2019.
Vancouver:
Steyn NL. Investigating the localisation of the ESX-3 secretion system in Mycobacterium smegmatis. [Internet] [Masters thesis]. Stellenbosch University; 2012. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/10019.1/71959.
Council of Science Editors:
Steyn NL. Investigating the localisation of the ESX-3 secretion system in Mycobacterium smegmatis. [Masters Thesis]. Stellenbosch University; 2012. Available from: http://hdl.handle.net/10019.1/71959
Kansas State University
28.
Perera, Jayaweeralage Ayomi Sheamilka.
Investigation
of stability, dynamics and scope of application of mycobacterial
porin MspA: a highly versatile biomolecular resource.
Degree: PhD, Department of
Chemistry, 2012, Kansas State University
URL: http://hdl.handle.net/2097/14845
► Porin A from Mycobacterial smegmatis (MspA) is an octameric trans-membrane channel protein and is one of the most stable porins known to date. MspA has…
(more)
▼ Porin A from Mycobacterial
smegmatis (MspA) is an
octameric trans-membrane channel protein and is one of the most
stable porins known to date. MspA has been successfully isolated
and purified to obtain liquid extracts and crystals using a
modified extraction procedure. A full analytical assessment has
been carried out to authenticate its’ structure, including gel
electrophoresis, spectroscopy (fluorescence, UV, FTIR, NMR), HPLC,
Bradford protein assay, dynamic light scattering and X-ray
crystallography. Nanoscopic vesicle formation of MspA molecules in
aqueous media has been thoughroughly investigated. Temperature
dependent dynamic light scattering experiments reveal that size of
such vesicles is dependent on temperature but is independent of
ionic strength of the medium. Zeta potential measurements reveal a
steady build up of positive charge on the vesicle surface with
increasing temperature.
For the first time, wild type (WT) MspA
has been utilized as a channel forming agent. This phenomenon has
future potential in DNA sequencing and the development of
antimycobacterial drugs. Channel activity of WT MspA and mutant
A96C MspA has been investigated and has shown to form stable
channels across DPhPC lipid bilayers. Blocking of the channel
current via external molecules (i.e. channel blocking) is an
extremely important process, which helps to evaluate the biosensor
ability of the pore. In this regard, two Ruthenium based compounds,
Ru(QP-C2)38+ (i.e. RuC2) and Ru(bpy)32+have been successfully
employed as channel blocking agents. Both compounds show evidence
for channel blocking of WT MspA. However, these results are not
reproducible.
Three dimensional aggregation behavior of RuC2-MspA
vesicles have been thoughroughly investigated. It is evident that
addition of RuC2 significantly increases vesicle size and
polydispersity of MspA aggregates in solution. The results provide
explanations onto the lack of channel blocking ability of MspA by
RuC2.
Development of a ‘greener’ dye sensitized solar cell with
the use of MspA as an electron carrier is investigated for the
first time. A series of Ru(II)-phenanthroline-based dyes have been
synthesized as non-toxic dyes in this regard. Chemical binding
between the dyes and MspA has been achieved successfully. Two types
of solar cell prototypes, i.e. TiO2-based (Grätzel type) and
FTO-based have been developed and tested. Significant current
generation and conversion efficiencies have been achieved for both
cell types. This marks the first development of a protein-based
photovoltaic device, which has the potential to be developed as a
new class of “hybrid soft solar cells”.
Advisors/Committee Members: Stefan H. Bossmann.
Subjects/Keywords: Mycobacterium smegmatis;
MspA; Protein
solar cell; Alternative Energy (0363); Biochemistry (0487); Chemistry (0485)
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APA ·
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Export
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Manager
APA (6th Edition):
Perera, J. A. S. (2012). Investigation
of stability, dynamics and scope of application of mycobacterial
porin MspA: a highly versatile biomolecular resource. (Doctoral Dissertation). Kansas State University. Retrieved from http://hdl.handle.net/2097/14845
Chicago Manual of Style (16th Edition):
Perera, Jayaweeralage Ayomi Sheamilka. “Investigation
of stability, dynamics and scope of application of mycobacterial
porin MspA: a highly versatile biomolecular resource.” 2012. Doctoral Dissertation, Kansas State University. Accessed December 10, 2019.
http://hdl.handle.net/2097/14845.
MLA Handbook (7th Edition):
Perera, Jayaweeralage Ayomi Sheamilka. “Investigation
of stability, dynamics and scope of application of mycobacterial
porin MspA: a highly versatile biomolecular resource.” 2012. Web. 10 Dec 2019.
Vancouver:
Perera JAS. Investigation
of stability, dynamics and scope of application of mycobacterial
porin MspA: a highly versatile biomolecular resource. [Internet] [Doctoral dissertation]. Kansas State University; 2012. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/2097/14845.
Council of Science Editors:
Perera JAS. Investigation
of stability, dynamics and scope of application of mycobacterial
porin MspA: a highly versatile biomolecular resource. [Doctoral Dissertation]. Kansas State University; 2012. Available from: http://hdl.handle.net/2097/14845
Indian Institute of Science
29.
Chandran, Anu V.
Structural and Related Studies on Mycobacterial RecA and LexA.
Degree: 2016, Indian Institute of Science
URL: http://etd.iisc.ernet.in/2005/3718
;
http://etd.iisc.ernet.in/abstracts/4588/G27769-Abs.pdf
► Genetic material of bacteria is subject to damage due to multitudinous factors, both extrinsic and intrinsic in origin. Mechanisms for the maintenance of genomic integrity…
(more)
▼ Genetic material of bacteria is
subject to damage due to multitudinous factors, both extrinsic and intrinsic in origin. Mechanisms for the maintenance of genomic integrity are thus essential for a bacterium to survive. Bacterium also requires appropriate minor changes in the genetic material so as to adapt to the changing environments. Structural and related studies of two proteins from mycobacteria, one involved in recombinational DNA repair (RecA) and the other involved in SOS response which helps in adaptation to stress (LexA) form the
subject matter of the thesis.
The available literature on structural and related studies on RecA and LexA are reviewed in the introductory chapter. The action of RecA involves transition to an active filament formed in association with DNA and ATP, from an inactive filament in the absence of DNA. The structure of the inactive filament was first established in E. coli RecA (EcRecA). The interaction of RecA with non-hydrolysable ATP analogues and ADP were thoroughly characterised and the DNA binding loops were visualised in this laboratory using the crystal structures involving the proteins from
Mycobacterium tuberculosis (MtRecA) and
Mycobacterium smegmatis (MsRecA). A switch residue, which triggers the transformation of the information on ATP binding to the DNA binding regions, was identified. The 20 residue C-terminal stretch of RecA, which is disordered in all other relevant crystal structures, was defined in an MsRecA-dATP complex. The ordering of the stretch is accompanied by the generation of a new nucleotide binding site which can communicate with the original nucleotide binding site of an adjacent molecule in the filament. The plasticity of MsRecA and its mutants involving the switch residue was explored by studying crystals grown under different conditions at two different temperatures and, in one instance, at low humidity. The structures of these crystals and those of EcRecA and Deinococcus radiodurans RecA (DrRecA) provide information on correlated movements involving different regions of the molecule. MtRecA has an additional importance as an adjuvant drug target in
Mycobacterium tuberculosis. Apart from recombination, another important property of RecA is its coprotease activity whereby it stimulates the inherent cleavage of a certain class of proteins. One of the substrates for the coprotease activity of RecA is LexA. LexA is a transcriptional repressor involved in SOS response in bacteria. LexA performs its function through an autoproteolysis stimulated by RecA, resulting in the derepression of the genes under its control. Structural studies on LexA from E. coli have shown that it has an N-terminal domain involved in binding to DNA and a C-terminal domain involved in catalysis and dimerisation. LexA mediated SOS response in bacteria has been shown in many cases to be responsible for the resistance gained by bacteria on treatment with antibiotics. In that respect, LexA is considered to be a potential drug target in
Mycobacterium tuberculosis.
Structures of crystals of…
Advisors/Committee Members: Vijayan, M, Murthy, M R N, Gopal, B.
Subjects/Keywords: Mycobacterial RecA; Mycobacterial LexA; Mycobacterium Tuberculosis LexA; LexA Family of Proteins; Molecule - Plasticity; MtRECA; MtLEXA; EcLexA; Mycobacterium smegmatis; Molecular Biophysics
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APA (6th Edition):
Chandran, A. V. (2016). Structural and Related Studies on Mycobacterial RecA and LexA. (Thesis). Indian Institute of Science. Retrieved from http://etd.iisc.ernet.in/2005/3718 ; http://etd.iisc.ernet.in/abstracts/4588/G27769-Abs.pdf
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Chandran, Anu V. “Structural and Related Studies on Mycobacterial RecA and LexA.” 2016. Thesis, Indian Institute of Science. Accessed December 10, 2019.
http://etd.iisc.ernet.in/2005/3718 ; http://etd.iisc.ernet.in/abstracts/4588/G27769-Abs.pdf.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Chandran, Anu V. “Structural and Related Studies on Mycobacterial RecA and LexA.” 2016. Web. 10 Dec 2019.
Vancouver:
Chandran AV. Structural and Related Studies on Mycobacterial RecA and LexA. [Internet] [Thesis]. Indian Institute of Science; 2016. [cited 2019 Dec 10].
Available from: http://etd.iisc.ernet.in/2005/3718 ; http://etd.iisc.ernet.in/abstracts/4588/G27769-Abs.pdf.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Chandran AV. Structural and Related Studies on Mycobacterial RecA and LexA. [Thesis]. Indian Institute of Science; 2016. Available from: http://etd.iisc.ernet.in/2005/3718 ; http://etd.iisc.ernet.in/abstracts/4588/G27769-Abs.pdf
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Indian Institute of Science
30.
Murdeshwar, Maya S.
Expanding The Horizon Of Mycobacterial Stress Response : Discovery Of A Second (P)PPGPP Synthetase In Mycobacterium Smegmatis.
Degree: 2012, Indian Institute of Science
URL: http://hdl.handle.net/2005/2499
► The stringent response is a highly conserved physiological response mounted by bacteria under stress (Ojha and Chatterji, 2001; Magnusson et al., 2005; Srivatsan and Wang,…
(more)
▼ The stringent response is a highly conserved physiological response mounted by bacteria under stress (Ojha and Chatterji, 2001; Magnusson et al., 2005; Srivatsan and Wang, 2007; Potrykus and Cashel, 2008). Until recently, the only known players in this pathway were the (p)ppGpp synthesizing and hydrolyzing long RSH enzymes (Mittenhuber, 2001; Atkinson et al., 2011) - RelA and SpoT in Gram negative bacteria and the bifunctional Rel in Gram positive bacteria including mycobacteria. The existence of Short Alarmone Synthetases (SAS) (Lemos et al., 2007, Nanamiya et al., 2008; Das et al., 2009; Atkinson et al., 2011) and Short Alarmone Hydrolases (SAH) (Sun et al., 2010, Atkinson et al., 2011), small proteins possessing a single functional (p)ppGpp synthetase or hydrolase domain respectively, is a recent discovery that has modified this paradigm. Around the same time that the presence of the SAS proteins was reported, we chanced upon such small (p)ppGpp synthetases in the genus
Mycobacterium. The stringent response in the soil saprophyte
Mycobacterium smegmatis was first reported by Ojha and co-workers (Ojha et al., 2000), and the bifunctional RSH, RelMsm, responsible for mounting the stringent response in this bacterium, has been characterized in detail (Jain et al., 2006 and 2007). RelMsm was the only known RSH enzyme present in M.
smegmatis, and consequently, a strain of M.
smegmatis deleted for the relMsm gene (ΔrelMsm) (Mathew et al., 2004), was expected to show a null phenotype for (p)ppGpp production. In this body of work, we report the surprising observation that the M.
smegmatis ΔrelMsm strain is capable of synthesizing (p)ppGpp in vivo. This unexpected turn of events led us to the discovery of a second (p)ppGpp synthetase in this bacterium. The novel protein was found to possess two functional domains – an RNase HII domain at the amino-terminus, and a (p)ppGpp synthetase or RSD domain at the carboxy-terminus. We have therefore named this protein ‘MS_RHII-RSD’, indicating the two activities present and identifying the organism from which it is isolated. Orthologs of this novel SAS protein occur in other species of mycobacteria, both pathogenic and non-pathogenic. In this study, we report the cloning, purification and in-depth functional characterization of MS_RHII-RSD, and speculate on its in vivo role in M.
smegmatis.
Chapter 1 reviews the available literature in the field of stringent response research and lays the background to this study. A historical perspective is provided,
starting with the discovery of the stringent response in bacteria in the early 1960s, highlighting the development in this area till date. The roles played by the long and short RSH enzymes, ‘Magic Spot’ (p)ppGpp, the RNA polymerase enzyme complex, and a few other RNA and proteins are described, briefly outlining the inferences drawn from recent global gene expression and proteomics studies. The chapter concludes with a description of the motivation behind, and the scope of the present study.
Chapter 2 discusses the in vivo and in…
Advisors/Committee Members: Chatterji, Dipankar.
Subjects/Keywords: Mycobacterium Smegmatis; Mycobacterium Smegmatis - (p)ppGpp Synthetase; Mycobacteria - Stress Response; Bacterial Proteins; RelA-SpoT Homolog (RSH) Proteins; RNA Polymerase; (p)ppGpp Synthetase; MS_RHII_RSD (Rnase HII-(p)ppGpp) Synthetase; Glycopeptidolipids; Mycobacterim Smegmatis Biofilm Cultures; Mycobacterial Stress; RSH Proteins; RSH Enzymes; Biochemistry
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APA ·
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APA (6th Edition):
Murdeshwar, M. S. (2012). Expanding The Horizon Of Mycobacterial Stress Response : Discovery Of A Second (P)PPGPP Synthetase In Mycobacterium Smegmatis. (Thesis). Indian Institute of Science. Retrieved from http://hdl.handle.net/2005/2499
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Murdeshwar, Maya S. “Expanding The Horizon Of Mycobacterial Stress Response : Discovery Of A Second (P)PPGPP Synthetase In Mycobacterium Smegmatis.” 2012. Thesis, Indian Institute of Science. Accessed December 10, 2019.
http://hdl.handle.net/2005/2499.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Murdeshwar, Maya S. “Expanding The Horizon Of Mycobacterial Stress Response : Discovery Of A Second (P)PPGPP Synthetase In Mycobacterium Smegmatis.” 2012. Web. 10 Dec 2019.
Vancouver:
Murdeshwar MS. Expanding The Horizon Of Mycobacterial Stress Response : Discovery Of A Second (P)PPGPP Synthetase In Mycobacterium Smegmatis. [Internet] [Thesis]. Indian Institute of Science; 2012. [cited 2019 Dec 10].
Available from: http://hdl.handle.net/2005/2499.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Murdeshwar MS. Expanding The Horizon Of Mycobacterial Stress Response : Discovery Of A Second (P)PPGPP Synthetase In Mycobacterium Smegmatis. [Thesis]. Indian Institute of Science; 2012. Available from: http://hdl.handle.net/2005/2499
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
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Open Access Theses and Dissertations