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Etude In vitro de Phospholipases mycobactériennes impliquées dans la virulence / In vitro study of micobactérial Phospholipases involved in virulenceBakala n'goma, Jean-claude 26 March 2010 (has links)
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 process, but their exact biochemical properties, mechanism of action and physiological role remain to be elucidated. This lack of data on mycobacterial phospholipases is mainly due to the difficulty to produce and purify these enzymes in large scale.With the aim to better characterise the physiological role of mycobacterial phospholipases, the main challenge of my thesis was to develop an efficient method for expression and purification of recombinant mycobacterial phospholipases C. Since no satisfactory results have been obtained with standard expression systems (E. coli, Pichia pastoris and baculovirus / insect cells), we develop a robust expression technique for these proteins using M. smegmatis as expression system.This allowed us to produce and purify all four PLC (PLC-A, PLC-B, PLC-C and PLC-D) of M. tuberculosis and the PLC of M. abscessus in soluble and active form. For the first time, we have show, that purified proteins have cytotoxic effect on mouse macrophages but have not haemolytic activity. Using radiolabelled lipids, we have confirmed that this first direct evidence that PLC are involved in infection and virulence processes. Another aspect of my thesis work concerned the study of two other secreted proteins of M. tuberculosis belonging to the cutinase family : the Rv 1984c ant the Rv3452. Recombinant proteins obtains in E. coli were found to have distinct substrate specificities and most likely distict physiological role, despite showing 50% amino acids sequence identity. Rv1984c is a lipase and is able to hydrolyse lipids with medium chains lengthn whereas Rv3452 is type A2, phospholipase and i able to induce macrophage lysis.
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Charakterizace Ms1, nově identifikované malé RNA z Mycobacterium smegmatis / Characterization of Ms1, a newly identified small RNA from Mycobacterium smegmatisPospíšil, Jiří January 2014 (has links)
Introduction: In recent years, there has been growing interest in regulation of gene expression by small non-coding RNA (sRNA). The first sRNA discovered in 1960s was 6S RNA from E. coli (length ~184 nt). It took ~ 30 years to obtain meaningful insights into its function. 6S RNA binds during stationary phase to RNA polymerase (RNAP) containing sigma factor 70 (primary sigma factor), thereby preventing transcription from σ70 - dependent promoters. In our laboratory we discovered a small RNA (length ~300 nt) in stationary phase of growht in Mycobacterium smegmatis. This sRNA was named Ms 1. The function of Ms 1 is uknown and preliminary experiments indicated that Ms 1may bind to RNAP that lacks σ factor (σA ). Goals: The aim of this Diploma project is to contribute to the characterization of Ms 1. Approaches: First, by molecular cloning, affinity chromatography and in vitro transcription I prepared the tools for subsequent experiments in vitro: RNAP, σA , Ms 1 and its mutated variants. Next, these tools were used for binding experiments on native gels and for transcription experiments. Results: RNAP, σA , Ms 1 and its variants were prepared. In vitro binding assays showed that wt Ms 1 but not a mutated variant of Ms 1 binds to RNAP. Using this assays were identified areas of Ms 1 that are important...
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Investigation of stability, dynamics and scope of application of mycobacterial porin MspA: a highly versatile biomolecular resourcePerera, Jayaweeralage Ayomi Sheamilka January 1900 (has links)
Doctor of Philosophy / Department of Chemistry / Stefan H. Bossmann / 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”.
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Comparative genomics of drug resistant mycobacterium tuberculosis. / CUHK electronic theses & dissertations collectionJanuary 2012 (has links)
結核病仍是全球疾病和死亡的主要原因。雖然人均新發結核病例自2003年以來一直下降,耐多藥(MDR)和廣泛耐藥(XDR)的結核病例的突然增加為全球疾病控制帶來了新的威脅。結核分枝杆菌(MTB)北京株在過去十年越来越受重視,皆因其席捲亞洲,前蘇聯,和包括美國在內的好幾個地方。北京株在動物實驗中也表現出高毒性和耐多藥的傾向。目前結核菌廣泛耐藥定義為至少對異煙肼和利福平耐藥,再加上任何氟喹諾酮類,和至少一個二線藥物。我們對這種病菌的生物知識仍然有限。在這研究,我們為來自香港和福建五株MTB北京株進行了基因組測序,其中兩株的耐藥性遠超XDR標準 - “全耐藥“(TDR)的表型。五個北京株的比較基因組學為我們提供了在北京株的毒力相關基因的啟示。一個約4 KB大小的片段被找出来了,此片段是所有已知MTB中都没有的。我們討論了此片段對MTB進化的含義。當我們研究在北京耐藥株的獨特基因變化時發現,DNA修復和香葉醇降解有關連。我們還觀察到大的缺失(D)和截斷(T)的事件,顯著高於框移位(F)的突變。此外,在TDR菌株出現的FDT事件更頻繁地涉及到最佳生長和麻風分枝杆菌的基因組中保留的基因。這方面的証據表明,MTB通過缩减進化發展極端耐藥性。適應度的顯著降低也許解釋了TDR菌株的稀缺 。 / Tuberculosis (TB) remains one of the major causes of illness and death globally. Although the number of new TB cases per capita has been falling since 2003, the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) cases of TB poses new threat to the successful worldwide control of the disease (WHO, 2008; Iseman, 2007). The Beijing lineage of Mycobacterium tuberculosis (MTB) has received much attention over the past decade due to its prevalence throughout Asia, parts of the former Soviet Union, and several other geographical locations including the United States. The strain also demonstrated hypervirulence in animal models and an increased likelihood to develop multidrug resistance. The current definition of XDR in TB is defined as resistance to at least isoniazid and rifampicin, any fluoroquinolone, and with at least one of the three second-line drugs. Here we show that our knowledge of the biology of this pathogen is still limited. We performed genome sequencing and reported the complete genomes of five Beijing isolates from Hong Kong and Fujian, of which two were shown to have drug resistance that is far beyond the current XDR standard - a "Totally Drug Resistance" (TDR) phenotype. Comparative genomics of the five Beijing isolates provided us insights into the virulence-related genes in the Beijing family. A distinct region of about 4 kb in size that are absent in all known complete genomes of MTB was also identified. The evolutionary implications of this region were discussed. When we investigated the unique genetic changes in drug resistant Beijing strains, a correlation to DNA repair and geraniol degradation was implicated. We have also observed that the big deletions (D) and truncations (T) events were significant higher when compare with frameshift (F) mutations. Moreover, the FDT events in TDR strains were more frequently found in genes that are involved in growth attenuation and retained in the genome of the Mycobacterium leprae. This evidence suggests that MTB develops its extreme drug resistance through the reductive evolution. The significant decrease in the fitness may explain the rareness of TDR strains. / Detailed summary in vernacular field only. / Leung, Ka Kit. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 93-108). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology - a ubiquitous threat --- p.1 / Chapter 1.2 --- Surviving the Hell --- p.3 / Chapter 1.3 --- Relatives of M. tuberculosis --- p.4 / Chapter 1.4 --- The age of M. tuberculosis --- p.5 / Chapter 1.5 --- Characteristics of Beijing strains --- p.6 / Chapter 1.6 --- Drug resistance --- p.7 / Chapter 1.7 --- Genome sequencing --- p.9 / Chapter 1.7.1 --- Conventional sequencing --- p.9 / Chapter 1.7.2 --- High-throughput sequencing --- p.10 / Chapter 1.8 --- Sequence assembly --- p.11 / Chapter 1.8.1 --- De novo assembly --- p.11 / Chapter 1.8.2 --- Reference mapping --- p.12 / Chapter Chapter 2 --- Materials and Methods --- p.14 / Chapter 2.1 --- Sample preparation --- p.14 / Chapter 2.2 --- DNA extraction and genome sequencing --- p.18 / Chapter 2.3 --- Gap filling and finishing --- p.20 / Chapter 2.3.1 --- In silico gap verification --- p.20 / Chapter 2.3.2 --- Comparison among different reference mapped contigs --- p.24 / Chapter 2.3.3 --- Experimental work --- p.26 / Chapter 2.4 --- Bioinformatics analysis --- p.27 / Chapter 2.4.1 --- Genome annotation --- p.27 / Chapter 2.4.2 --- Phylogeny analysis --- p.27 / Chapter 2.4.3 --- Variation analysis --- p.28 / Chapter 2.4.4 --- In silico functionality analyses --- p.29 / Chapter Chapter 3 --- Results --- p.30 / Chapter 3.1 --- Genome features of M. tuberculosis Beijing genotype strains --- p.30 / Chapter 3.2 --- Phylogeny of M. tuberculosis Beijing genotype strains --- p.36 / Chapter 3.3 --- Evolutionary implications of a 4kb-insertion in Beijing strains --- p.40 / Chapter 3.4 --- Beijing family specific gene variations --- p.48 / Chapter 3.5 --- Drug resistance --- p.52 / Chapter Chapter 4 --- Discussions --- p.75 / Chapter 4.1 --- 4kb insertion, a potential bridge to our knowledge gap --- p.75 / Chapter 4.2 --- Beijing common and Beijing drug resistant specific variations --- p.77 / Chapter 4.3 --- Regions of deletion --- p.79 / Chapter Chapter 5 --- Conclusions --- p.82 / Chapter Chapter 6 --- Future Work --- p.84 / Chapter 6.1 --- Compensatory mutation study --- p.84 / Chapter 6.1.1 --- Database construction for drug resistance compensatory mutations --- p.85 / Chapter 6.2 --- Non-protein coding region study --- p.92
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O papel do colesterol na biossíntese da parede celular de Mycobacterium smegmatisMARINHO, Victor Hugo de Souza 26 February 2015 (has links)
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Previous issue date: 2015-02-26 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Diferentes espécies de micobactérias são agentes causadores de significativas doenças em seres humanos como, por exemplo, a tuberculose. Todas as micobactérias possuem uma complexa e distinta parede celular, conferindo características físico-químicas exclusivas ao gênero Mycobacterium, protegendo-o contra o sistema imune e entrada de muitos antibióticos. Durante a infecção, o bacilo é capaz de se adaptar ao ambiente inóspito, nutrindo-se de fontes lipídicas alternativas, principalmente o colesterol, da própria célula hospedeira (macrófagos). Este perfil nutricional tem sido considerado essencial para a divisão bacilar e consecutivamente progressão da doença. Diante disso, o presente trabalho tem por objetivo avaliar em Mycobacterium smegmatis (espécie saprofítica) a modulação in vitro da biossíntese de constituintes bioativos da parede celular após o consumo de colesterol. Como resultado, verificamos por Cromatografia de Camada Delgada (CCD) que a adaptação do bacilo ao microambiente de escassez nutricional (cultivo em Meio Mínimo – MM) conseguiu manter a biossíntese e o acúmulo dos principais constituintes da parede celular, quando o cultivo ocorre na presença de alguma fonte definida de carbono e energia (glicerol e/ou colesterol). Dentre estes constituintes essenciais sem alterações, verificamos o Trealose de dimicolato (TDM) e os fosfolipídios Fosfatidilinositol (PI), Fosfatidilinositol manosídeos (PIMs), Cardiolipina (CL) e Fosfatidiletanolamina (PE). Diferentemente a esse resultado, o ácido micólico apresentou acúmulo representativo ao cultivo em meio 7H9 somente quando o MM estava igualmente suplementado com glicerol. Este resultado foi confirmado pela marcação álcool-ácido resistente com o marcador fluorescente auroamina, sugerindo alterações nas propriedades físico-químicas da parede celular. Por outro lado, o cultivo em MM favoreceu o acúmulo de Glicopeptídeolipídios (GPLs), independente da suplementação por glicerol e/ou
colesterol. Esta perturbação na biossíntese da parede celular alterou o perfil hidrofóbico do bacilo, independentemente da fonte de carbono e energia, porém não alterou a resistência ou sensibilidade a antibióticos. Estes resultados mostram claramente que a biossíntese da parede celular pode sofre modulações em condições de escassez nutricional, e que a presença ou ausência de colesterol, como ocorrido durante a infecção, não altera significativamente a fisiologia do bacilo a ponto de torna-lo mais vulnerável a ação de antibióticos, sugerindo que tais modificações possam também ocorrer durante a infecção, mantendo o bacilo viável, até o desenvolvimento da doença propriamente dita. / Different Mycobacterium species are causative agents of disease in humans, for example, the tuberculosis. All mycobacteria have a complex cell wall, distinct of others bacteria, conferring specific physic-chemical characteristic to Mycobacterium genus, due to protect against immune system and waterproofing against the intake of much antibiotics. During infection, the bacillus is able to adapter to harsh environment, due to consumption of cholesterol from itself host cell (macrophages) as alternative carbon and energy source. That nutritional aspect has been considered as essential for division of bacilli and consecutive progress of tuberculosis disease. The present study has as objective to evaluate in vitro the modulation of saprophytic Mycobacterium smegmatis cell wall biosynthesis after cholesterol consumption as primordial energy and carbon source. As results, we are found by Thin Layer Chromatography (TLC) that bacillary adaptation to microenvironment with poor nutrient (minimal media – MM) maintained the biosynthesis and accumulation of essential cell wall components, when the growth occurs in presence of someone defined carbon and energy source (glycerol and/or cholesterol). Among them without changes, we analyzed Trehalose Dimicolate (TDM) and the phospholipids (phosphatidylinositol (PI), phosphatidylinositol manosides (PIMs), Cardiolipin (CL) and phosphatidylethanolamine (PE)). Differently of these results, the micolic acid showed representative accumulation, comparing with 7H9 culture, only when the MM was supplemented with glycerol. This result was confirmed by alcohol-acid staining using fluorescent auroamine dye, suggesting some changes in physic-chemistry cell wall properties. On the other hands, the MM culture induced the accumulation of glycopeptidolipids (GPLs), independently of glycerol/cholesterol supplementation. Such disturbance in cell wall biosynthesis also changed the bacillary hydrophobicity in all MM groups, but does not change the resistance and sensibility to antibiotics. Those results clearly show that cell wall biosynthesis might be modulate during nutritional shortage, and such presence or absence of cholesterol, as occurs during infection, does not significantly change the bacillary physiology to become vulnerable for antibiotics. It suggests that such modulations might also occur during infection, maintaining the bacilli available to develop the tuberculosis diseases.
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Identification of rifampin resistant-related genes in Mycobacterium smegmatis. / CUHK electronic theses & dissertations collectionJanuary 2012 (has links)
結核病是由結核桿菌感染而引起的慢性傳染病,它是危害人類健康的主要殺手。根據世界衛生組織的報導,目前在全球範圍內有三分之一的人口感染了結核桿菌,每年約有915 萬人口被確診患有結核病。耐藥結核病尤其是對最有效的一線抗結核藥物異煙阱和利福平產生抗藥的耐多藥結核病的出現,令有效的控制結核病更加棘手。 / 在本研究中,我們首先用利福平誘導得到五株伴有明顯生長緩慢的高水平利褔卒耐藥的恥垢分支桿菌。通過比較基因組學研究發現,在編碼區有四個突變,其中兩個位於中rpoB 基因(N484T and 1488F) ,一個位於MSMEG_0436 (V49M) ,一個位於MSMEG_6872 (V181L)。rpoB 基因突變是該恥垢分支桿菌利福平耐藥的主要原因。而生長緩慢主要源於MSMEG_6872基因的影響。更為有趣的是,我們發現一個與MSMEG_6872具有相同的蛋白模序的結核分支桿菌蛋白質Rv1367 在不間的結核分支桿菌菌株之間存在I193V 多態性。193V 主要存在于北京株或者在耐藥的非北京株上。進一步的研究發現,過量表達MSMEG_6872或者Rv1367c 的恥垢分支桿菌形態上呈現為細長棒狀,而他們的親代則為短棒狀。 / 為獲得耐藥性,以及在高濃度的抗生素環境下生存,細菌必須付出一定的生物學代價。本研究中,恥垢分支桿菌以生長缺陷為代價獲得了對利褔平的耐藥,而這個代價可能是由於MSMEG_6872 基因的突變或者過量表達打破了細胞壁延長和分裂的平衡引起。 / Mycobacterium tuberculosis (MTB), which is the pathogen of tuberculosis (TB), remains a major human public health problem throughout the world. According to the report from the World Health Organization, currently about one third of the world's population was infected by MTB and there is globally 9.15 million recorded cases of TB annually. The occurrence of resistance to drugs used to combat TB, particularly multi-drug resistant TB (MDR-TB), defined as resistance to at least isoniazid and rifampin (RIF), has become a significant public health problem in a number of countries and an obstacle to effective global TB control. / In this project, we firstly obtained high level RIF resistant Mycobacterium smegmatis (MSM) strains with obviously growth retardation by repeated drug selection. Comparative analysis of genomic sequences revealed 4 mutations in coding sequences, including two in rpoB (N484T and I488F), one in MSMEG 0436 (y 49M), and one in MSMEG 6872 (y181L). Characterization of these four mutations showed that the two mutations in rpoB were correlated to RIF resistance. The one in MSMEG_6872 can render obviously growth retardation when MSMEG_6872 is over-expressed. Interestingly, we found an MTB protein, Rv1367c, which has the same motif with MSMEG_6872, had an I193V polymorphism in different MTB strains. The 193V variant was mainly found in Beijing/W or drug resistant non-Beijing/W family strains. The transformants, no matter MSMEG_6872 or Rv 1367 c, all exhibited slim and long rod shape compared to stocky and short rod appearance of the parental strain. / Mycobacterial cells must pay biological cost in order to obtain RIF resistance and survive in the high concentration of RIF. In our case, the growth arrest may be due to the mutation of MSMEG_6872 which disrupts the balance of cell wall elongation and cell division. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Guan, Bing. / "November 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 139-143). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.I / Abstract --- p.II / Abstract in Chinese --- p.IV / List of Abbreviations --- p.V / List of Tables --- p.VI / List of Figures --- p.VII / Contents --- p.IX / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Overview of Tuberculosis --- p.1 / Chapter 1.1.1 --- Pathogens --- p.2 / Chapter 1.1.2 --- Syndromes --- p.2 / Chapter 1.1.3 --- Transmission --- p.3 / Chapter 1.1.4 --- Diagnosis --- p.4 / Chapter 1.1.5 --- Epidemiology --- p.6 / Chapter 1.1.6 --- Mortality --- p.8 / Chapter 1.2 --- The Anti-TB Strategies --- p.8 / Chapter 1.2.1 --- Chemotherapy Treatment for MTB --- p.8 / Chapter 1.2.2 --- Vaccine Development for MTB --- p.9 / Chapter 1.3 --- Genome Sequencing of MTB Isolates --- p.9 / Chapter 1.4 --- Drug Resistance of MTB --- p.13 / Chapter 1.4.1 --- MDR-TB and XDR-TB --- p.15 / Chapter 1.4.2 --- Mechanism of Drug Resistance --- p.18 / Chapter 1.4.2.1 --- Intrinsic Resistance of Mycobacterium Species --- p.20 / Chapter 1.4.2.2 --- Acquired Resistance of Mycobacterium Species --- p.22 / Chapter 1.4.3 --- RIF Resistant MTB --- p.24 / Chapter 1.5 --- Useful tool for MTB Research --- p.26 / Chapter 1.6 --- The Biological Cost of Antibiotic Resistance in MTB --- p.27 / Chapter 1.6.1 --- The meaning of Biological Cost --- p.27 / Chapter 1.6.2 --- Factors Involved in Biological Cost of Mycobacterium Species --- p.29 / Chapter 1.17 --- Objectives of the Project and Experimental Strategies --- p.30 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Selection of RIF Resistant MSM mc²155 Strains --- p.31 / Chapter 2.1.1 --- Bacterial Strains, Media, and Growth Conditions --- p.31 / Chapter 2.1.2 --- Selection of RIF Resistant Strain --- p.31 / Chapter 2.2 --- Minimum-Inhibitory-Concentration (MIC) Assay --- p.34 / Chapter 2.3 --- Detection of Mutations in the rpoB Gene of RIF Resistance Strains --- p.36 / Chapter 2.3.1 --- Primers Design --- p.36 / Chapter 2.3.2 --- PCR and Direct Sequencing --- p.36 / Chapter 2.4 --- Characterization of the RpoB Gene --- p.38 / Chapter 2.4.1 --- Construction of Recombinant Clones --- p.38 / Chapter 2.4.2 --- Preparation of MSM competent cell. --- p.38 / Chapter 2.4.3 --- Electroporation of plasmid into MSM competent cells --- p.39 / Chapter 2.4.4 --- Site-directed Mutagenesis of the RpoB Clone --- p.39 / Chapter 2.5 --- Whole Genome Sequencing of Parental and Drug --- p.43 / Chapter 2.5.1 --- MSM Genomic DNA Extraction --- p.43 / Chapter 2.5.2 --- Genomic Sequencing --- p.44 / Chapter 2.5.3 --- Data Analysis and SNPs Identification --- p.45 / Chapter 2.6 --- Validation of Mutations by PCR and Direct Sequencing --- p.46 / Chapter 2.6.1 --- PCR Primers Design --- p.46 / Chapter 2.6.2 --- PCR and Direct Sequencing --- p.46 / Chapter 2.7 --- Characterization of MSMEG 0436 and MSMEG 6872 --- p.47 / Chapter 2.7.1 --- Construction of the recombinant clones --- p.47 / Chapter 2.8 --- Assay of Ethidium Bromide in Intact Cells --- p.48 / Chapter 2.9 --- Quantitative Real-time PCR to Expression of the Measure the ATP-binding Cassette (ABC) Superfamily Efflux Pumps --- p.49 / Chapter 2.9.1 --- RNA Extraction --- p.49 / Chapter 2.9.2 --- Synthesis of Double-stranded cDNA from Total RNA --- p.49 / Chapter 2.9.3 --- Real-time PCR to Quantify the Efflux Pump Gene Expression Level --- p.49 / Chapter 2.10 --- The construction of the Growth Curve --- p.53 / Chapter 2.11 --- Generation of ΔMSMEG_6872 Mutant Strain --- p.54 / Chapter 2.11.1 --- Preparation of Recombination Strain Stocks --- p.54 / Chapter 2.11.2 --- Preparation of the Electrocompetent Cells of the Recombination Strain --- p.54 / Chapter 2.11.3 --- Preparation of Allelic Exchange Substrate (AES) for Generating Gene Replacement Mutants --- p.55 / Chapter 2.12 --- Validation of Rv1367c (MT1414) in MTB --- p.60 / Chapter 2.12.1 --- Primer Design --- p.60 / Chapter 2.12.2 --- Strain Selection --- p.60 / Chapter 2.12.3 --- PCR and Direct Sequencing --- p.60 / Chapter 2.12.4 --- Alignment the Gene Sequence of Rv1367c of Different MTB Strains --- p.61 / Chapter 2.13 --- Model building of the RpoB protein --- p.62 / Chapter 2.14 --- MSM staining method --- p.63 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- dentification of RIF Resistant Related-genes Using Induced RIF Resistant MSM Model --- p.64 / Chapter 3.1.1 --- Emergence ofRIF Resistant Strains after the Prolonged Drug Exposure --- p.64 / Chapter 3.1.2 --- Induced RIF Resistance Were Stable In the Absence of Selection --- p.66 / Chapter 3.1.3 --- The Growth State of 5 RIF Resistance MSM mc²155 Strain --- p.68 / Chapter 3.1.4 --- Involvement of RpoB in the Mechanisms of the Emergence of RIF Resistance in MSM --- p.71 / Chapter 3.1.4.1 --- Mutations in the RpoB Gene --- p.71 / Chapter 3.1.4.2 --- Identical Mutations of RpoB Gene in Different RIF Resistance Isolates from Different Generation --- p.74 / Chapter 3.1.4.3 --- Characterization of RpoB in MSM strains --- p.76 / Chapter 3.1.4.4 --- Rifampin-Binding Pockets of RpoB Protein Model --- p.80 / Chapter 3.1.5 --- Other Genetic Alternations possibly Involved in RIF Resistance --- p.83 / Chapter 3.1.5.1 --- Whole Genome Sequencing of the Patental and P5 MSM mc²155 Strains --- p.83 / Chapter 3.1.5.2 --- Validation of the 32 Selected Alterations --- p.88 / Chapter 3.1.5.3 --- Characterization of MSMEG_0436 and MSMEG_6872 in RIF Resistance --- p.91 / Chapter 3.1.5.4 --- Characterization of MSMEG_0436 in the Growth Rate of MSM --- p.93 / Chapter 3.1.5.5 --- Characterization of MSMEG_6872 in the Growth Rate of MSM --- p.95 / Chapter 3.1.5.6 --- MSMEG_6872 Knock-out Strain Exhibited Normal Phenotype as its Parent --- p.98 / Chapter 3.1.5.7 --- Identification of Mutations in the Beta-Iactamase Gene of Mycobacterium Tuberculosis (MTB) --- p.101 / Chapter 3.1.5.8 --- Characterization of Rv 1367 c in Mycobacterium Growth Rate --- p.108 / Chapter 3.1.5.9 --- Morphology Changes of the Rv1367c and MSMEG_6872 Transformants --- p.110 / Chapter 3.2 --- Genetic Alterations in Non-coding Sequence --- p.112 / Chapter 3.2.1 --- ATP-binding Cassette (ABC) Superfamily Efflux Pumps Up-regulated in Drug Resistant M Smegmatis Strain --- p.112 / Chapter 3.2.2 --- RIF Resistant M smegmatis mc²155 Strain exhibited Low Level Cross-drug Resistance to INH --- p.115 / Chapter 3.2.3 --- RIF Resistant M smegmatis mc²155 Strain Showed Low level Accumulation of Ethidium Bromide --- p.117 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- The Protocol for the Preparation RIF Resistant Strains --- p.121 / Chapter 4.2 --- RIF Induced Stable Chromosomal Mutations in RIF Resistant MSM Strains --- p.123 / Chapter 4.3 --- MIC Levels of the RIF Resistant Strains --- p.125 / Chapter 4.4 --- Factors May involved in RIF Resistant MSM Strains --- p.128 / Chapter 4.5 --- Cell Shape and Growth Regulation --- p.129 / Chapter 4.6 --- MSMEG _6872 and Twin-Arginine Translocase (TAT) Secretion System --- p.135 / Chapter 4.7 --- Conclusion --- p.137 / Chapter 4.8 --- Future Perspectives --- p.138 / REFERENCES --- p.139
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Genetic and Biochemical Insights into the Mycobacterial PrrAB System as a Regulator of Respiration and Central MetabolismJanuary 2019 (has links)
abstract: 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. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2019
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Screening, isolation and purification of bioactive compounds with antibacterial activity against mycobacterium smegmatisMmushi, Tshepo Joseph January 2011 (has links)
Thesis (M.Sc. (Microbiology)) --University of Limpopo, 2011 / The leaves of fifteen plant species were collected from the Lowveld Botanical Garden in Nelspruit, Mpumalanga Province, South Africa. The collection was based on a list of plants and their ethnopharmacological information provided by the Phytomedicine Programme at the University of Pretoria. The dried leaves of the plants were powdered and extracted using hexane, dichloromethane, acetone and methanol. The extracts were screened for antibacterial activity against Mycobacterium smegmatis and Rhodococcus erythropolis. The acetone extract of Milletia stulhimannii was the most active, showing activity against Mycobacterium smegmatis and Rhodococcus erythropolis with MIC values 0.13 and 0.08 mg/ml, respectively. Acetone extracts for all plants had the lowest MIC values ranging between 0.11-1.25 mg/ml and 0.08-1.25 mg/ml for M. smegmatis and R. erythropolis, respectively. Milletia stulhimannii, Albizia gummifera, Xanthocercis zambesiaca and Barringtonia racemosa extracts have shown the greatest potential for anti-tubercolosis agents. These were all active against M. smegmatis with an average MIC value of acetone extracts of 0.13 mg/ml. Apodytes dimidiata was selected for the isolation of active compounds since its activity on qualitative antibacterial activity assays was highly prominent on TLC plates in comparison to the other plant extracts. Two compounds were isolated from A. dimidiata but after purification, their MICs were above 2.5 mg/ml indicating a possible loss of activity during purification. The preliminary NMR spectra analysis suggested that the compounds were a long fatty acid and a triterpene. Future work is required to elucidate the chemical structures of the latter compounds and to test the activity of these compounds against Mycobacterium tuberculosis. / Department of Water Affairs, and University of Limpopo Research Development and Administration Office
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The Phn and Pst systems of Mycobacterium smegmatis : phosphate transport and gene regulationGebhard, Susanne, n/a January 2006 (has links)
Phosphate is an essential but often growth-limiting nutrient for bacteria. At low concentrations of phosphate in the growth medium, bacteria induce high-affinity uptake systems for phosphate, and this is usually the ABC-type phosphate specific transport system Pst. In the fully sequenced genomes of pathogenic species of mycobacteria, several copies of the genes encoding for the Pst system (pstSCAB) have been identified and some of these genes have been shown to be virulence factors. The reasons for the presence of multiple copies of pst genes in pathogenic mycobacteria are not understood, and phosphate transport by these bacteria, as well as the gene regulation involved, is poorly characterised. The fast-growing M. smegmatis contains only a single copy of the pst operon, but we recently identified a gene locus containing three genes, phnDCE, which encode for a putative ABC-type phosphate/phosphonate transport system, and a gene, phnF, which encodes for a putative transcriptional regulator of the HutC subfamily of GntR like regulators.
To identify a function for the PhnDCE transport system and to characterise high-affinity phosphate transport in M. smegmatis, we created allelic exchange mutants in phnD and pstS, as well as a phnD pstS double deletion mutant. All three mutants failed to grow in minimal medium containing 10 mM phosphate, while the wildtype was able to grow in the presence of micromolar phosphate concentrations. No differences were observed in complex growth medium. Steady-state levels of [��P]-phosphate uptake were approximately 25% lower in all mutant strains as compared to the wildtype. Kinetics of phosphate uptake in the wildtype strain when grown at low phosphate concentrations (50 [mu]M P[i]) were biphasic, suggesting the presence of two inducible transport systems with apparent K[m] values of 16 [mu]M P[i] and 64 [mu]M P[i], respectively. Analysis of the kinetics of phosphate transport in the mutant strains led us to the proposition that the Pst system has an apparent Km value of ca. 16 [mu]M P[i], and the Phn system has an apparent Km of ca. 60 [mu]M P[i]. A third inducible phosphate transport system, which was active in the double mutant strain, had an apparent K[m] of ca. 90 [mu]M P[i]. Uptake of phosphate in all strains was not inhibited by the presence of excess phosphonates or phosphite, suggesting that all three transport systems were specific for phosphate. The study of phosphate transport in the presence of various metabolic inhibitors revealed that uptake by the Phn and Pst systems is driven by ATP-hydrolysis, consistent with ABC-type transport, while the third, unidentified transport system may be driven by the proton motive force.
We showed that phnDCE formed an operon, and that the promoter area of the operon lies within 200 bp of the start of phnD. To investigate the regulation of the phn and pst genes, β-galacosidase activities of strains carrying transcriptional lacZ-fusions of the pstSCAB, phnDCE and phnF promoter areas, and levels of mRNA of the phn and pst genes were studied. All genes were induced when phosphate concentrations fell below a threshold value of 30 [mu]M, which coincided with a shift in the growth characteristics of M. smegmatis. Expression of the pst operon appeared to be controlled directly by the PhoPR two-component regulatory system, while the phn operon may be under direct or indirect control by PhoPR.
To identify a role for PhnF in the regulation of phn gene expression, we created a phnF deletion mutant. PhnF appeared to repress transcription of phnDCE and phnF under phosphate-replete conditions. We identified two putative binding sequences for PhnF in the intergenic region between phnD and phnF with the sequence TGGTATAGACCA, which is similar to the proposed recognition consensus for HutC-like transcriptional regulators. Using site-directed mutagenesis of these sequences, we demonstrated that they are required for the repression of phnDCE and phnF. To prove PhnF binding to these potential binding sites, we attempted to express the M. smegmatis PhnF protein in E. coli, but could not obtain soluble recombinant protein. Electrophoretic mobility shift assays of the phnDCE promoter fragment using cell-free crude extracts of M. smegmatis were not successful.
We propose that Pst and Phn both constitute high-affinity phosphate specific transport systems of M. smegmatis, and that a third inducible phosphate transport system is present in this bacterium. PhnF is required for repression of phnDCE and phnF transcription under phosphate-replete conditions, while induction of the pst operon, and possibly the phn operon, under phosphate-limited conditions involves the PhoPR system.
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Transcriptional Analysis Of The Principal Cell Division Gene ftsZ Of Mycobacterium Tuberculosis And Mycobacterium SmegmatisRoy, Sougata 06 1900 (has links)
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) for the gene. Among them, five transcripts (T1, T2, T3, T4, and T6) were detected in M. tuberculosis cells at exponential phase of growth. T5 could be detected only in M. smegmatis transformants containing 1.117 kb ftsZMt promoter upstream of mycgfp2+ reporter gene. Transcript T1 and T2 originate in the ftsQ-ftsZ intergenic region, while T3, T4, and T6 start in the ftsQ ORF. Analysis of sequence in the –10 and –35 regions of the corresponding promoters for the individual transcripts identified high GC content of the regions, which is characteristic of promoters of M. tuberculosis. All of the individual promoter sequences were independently cloned in a promoter probe vector and confirmed that they are true
promoters, active in M. smegmatis cells, and that the T1-T6 transcripts were not products
of RNA processing.
Differential expression from the multiple ftsZMt promoters
In order to study the activity and regulation of ftsZMt promoters in M. tuberculosis cells, which is a slow grower and also asymptomatically survives as dormant bacteria for decades in human granuloma, a stably genome-integrated plasmid was required where activity of the promoters can be studied by means of stable and enhanced gfp expression. For that purpose, an L5-mycobacteriophage attP (attachment site)-specific integration
proficient promoter probe vector, which contains a stable gfp gene (mycgfp2+) whose codon has been optimized for mycobacterial expression, was generated. Using the vector, all the six promoter regions (P1-P6) were studied in M. smegmatis and M. tuberculosis cells. Flow cytometric and semi-quantitative RT-PCR analyses showed that promoter P5 is unable to elicit activity in M. tuberculosis cells, unlike in M. smegmatis transformants.
Semi-quantitative RT-PCR analyses showed that expression of P3 is only 10-20% of the
total promoter activity. Promoters P1, P2, P4 and P6 showed 50-80% activity of the total promoter activity and their activity were comparable in M. smegmatis and M. tuberculosis.
The presence of multiple promoters reflects the requirement to maintain high
basal level of, or to differentially regulate a critical level of, FtsZ expression during different pathogenic stages of tubercle bacilli. In order to investigate the role of multiple
promoters, we verified the levels of expression of the five transcripts from the five ftsZ promoters in M. tuberculosis cells under conditions of growth inside mouse macrophage cell line and also under various stress conditions mimicking those that exist in the granuloma environment, like conditions of nonreplicating persistence, gradual nutrient depletion stress, oxidative stress, surface tension stress, acidic stress, heat shock, DNA damaging conditions and osmotic stress. For this purpose, individual promoter regions
were cloned into a stably inheritable gfp reporter plasmid vector, and into an L5
mycobacteriophage attP (attachment site)-specific integration-proficient variant of the same vector, for the expression of the promoters from the chromosomal locus in M. smegmatis and M. tuberculosis cells. Quantitative primer extension analyses, semiquantitative RT-PCR analyses on RNA from M. tuberculosis cells grown under these
different conditions, and quantitative GFP fluorescence analyses in these cells showed differential activation of the five promoters under different conditions of growth. Under hypoxic and nutrient-depleted stationary phase of growth, two new promoters, Tdor and Ts, in the ftsQ ORF were identified, and these promoters showed maximal activity only under those specific conditions of growth. None of the ftsZ promoters were found to be responsive to stringent response mediated by overexpression of M. tuberculosis RelA.
None of the promoters were also found to be responsive of overexpression of heat-shock
sigma factor SigH in M. tuberculosis, implicating new pathway of regulation of ftsZ promoters.
Multiple promoters driving expression of ftsZ gene of M. smegmatis
Similar studies, which were carried out on the identification, structural and
functional characterization, regulation of the promoters of cell division gene ftsZ in the fast growing saprophyte M. smegmatis cells, showed the presence of four ftsZ promoters, three of which originates from the 249 bp ftsQ-ftsZ intergenic region and one from the ftsQ ORF. RT-PCR analysis showed that both ftsQ and ftsZ are co-transcribed. Cloning and expression analysis of the individual promoters mapped by primer extension in a GFP based reporter plasmid showed that all the four putative regions are true promoters.
Quantitative primer extension on RNA from a synchronously grown culture identified P2
promoter to be responsive to either initiation of cell division or stress, although expression of P1, P3, and P4 did not vary with respect to synchronous division. Quantitative primer extension analysis and semi-quantitative RT-PCR analysis on the RNA from M. smegmatis cells showed that under various stress conditions, P2 activity goes down significantly. Under nutrient depleted stationary phase and hypoxic nonreplicating persistence stage-2, the levels of P2 and P3 activity could hardly be detected, whereas, expression from P1 and P4 goes down only in hypoxia. Level of total ftsZ mRNA remains almost the same under various stress conditions, although upon hypoxia and stationary phase the level goes down almost two fold. Thus, in fast growing
M. smegmatis too, multiple ftsZ promoters are differentially regulated under various stress conditions and a critical level of ftsZ mRNA is maintained.
Taken together, the study of ftsZ promoters of a slow-growing pathogenic
mycobacterium and a fast growing non-pathogenic mycobacterium indicate that
differential expression of the multiple promoters, along with conditional activation of stage specific promoters like Pdor or Ps, is one of the mechanisms through which the bacilli probably maintain required levels of FtsZ protein that are crucial for the cell survival, probably through cytoskeletal maintenance, and cell division.
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