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Dissertations / Theses on the topic 'Mycobacterium smegmatis'
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Vorwieger, Stefan [Verfasser], and Dirk [Akademischer Betreuer] Wagner. "Efflux-Inhibition bei Mycobacterium smegmatis und Mycobacterium avium." Freiburg : Universität, 2012. http://d-nb.info/1123467102/34.
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Mahenthiralingam, Eshwar. "The amidase promotor of Mycobacterium smegmatis." Thesis, Open University, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.278899.
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Bruell, Christian M. "Mechanism of protein synthesis in Mycobacterium smegmatis /." Zürich : ETH, 2008. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17733.
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Trower, Carolyn Joy 1975. "A preliminary investigation of a sialidase activity associated with M. smegmatis." Monash University, Dept. of Medicinal Chemistry, 2003. http://arrow.monash.edu.au/hdl/1959.1/7646.
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Smeulders, Maria Jeanne. "The stationary phase survival response of Mycobacterium smegmatis." Thesis, Imperial College London, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.287866.
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Sikder, Mahmudul Hasan. "Characterisation of the Mycobacterium smegmatis transcriptional regulator MSMEG_5424." Thesis, Royal Veterinary College (University of London), 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.618297.
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Rao, Tara. "Analysis of the multiple chaperonins of Mycobacterium smegmatis." Thesis, University of Birmingham, 2010. http://etheses.bham.ac.uk//id/eprint/1006/.
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Although most bacteria contain a single gene for the essential oligomeric chaperonin Cpn60, many contain two or more cpn60 genes. The non-pathogenic Mycobacterium smegmatis has three cpn60 homologues, while the pathogenic Mycobacterium tuberculosis has two. This study is a functional characterisation of the chaperonins of M. smegmatis. Expression of cpn60.1, cpn60.2 and cpn10, but not cpn60.3, was found to be induced under stress conditions, particularly heat shock. Studies of the cpn10-cpn60.1 operon concluded that transcription is from a single promoter, with a subsequent post-transcriptional cleavage of the mRNA between the two genes. Cpn60.1 in M. smegmatis is required for biofilm maturation. Using this assay, we intended to test various Cpn60 homologues for their ability to function in M. smegmatis. Preliminary results obtained revealed that only the M. tuberculosis Cpn60.1 can fully complement for loss of the M. smegmatis Cpn60.1. While E. coli GroEL and Cpn60.3 appear to only partially complement, Cpn60.2 shows no complementing ability. Cpn60.2 expresses well and complements for loss of the E. coli Cpn60 homologue GroEL even at higher temperatures (42°C). Using native gels and analytical ultracentrifugation, purified Cpn60.2 did not oligomerise under normal conditions, however in the presence of nucleotide and high concentrations of salt, the formation of large oligomers was observed. Neither Cpn60.1 nor Cpn60.3 complemented for loss of GroEL.
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Ogwang, Sam. "Intrinsic Antifolate Resistance in Mycobacterium smegmatis." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1270147885.
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Berger, Sven. "Expression der Poren bildenden Hämolysine Listeriolysin und TlyA in Mycobacterium smegmatis." [S.l. : s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=964920824.
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Faller, Michael. "Kristallstruktur eines mycobacteriellen Porins." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972775730.
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Whiteford, Danelle. "Stress survival in Mycobacterium tuberculosis and Mycobacterium bovis and the role of hup in Mycobacterium smegmatis." Pullman, Wash. : Washington State University, 2008. http://www.dissertations.wsu.edu/Dissertations/Fall2008/D_Whiteford_100908.pdf.
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Mpongoshe, Vuyiseka. "Gene expression changes in macrophages infected with pathogenic M. tuberculosis and non-pathogenic M. smegmatis and M. bovis BCG." Thesis, Stellenbosch : Stellenbosch University, 2014. http://hdl.handle.net/10019.1/86729.
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Thesis (MScMedSc)--Stellenbosch University, 2014.ENGLISH ABSTRACT: The current anti-TB drugs have had success in decreasing the number of deaths caused by TB, however, this success is limited by the emergence of drug resistant TB strains. Therefore, a novel TB therapy that limits the development of resistance has become necessary in an attempt to effectively control TB. The anti-TB drugs directly target mycobacterial enzymes, and potentiate the development of this resistance, and have therefore provided the rationale for this study. The aim was therefore to identify host macrophage genes that affect M. tb intracellular survival. The proposed alternative anti-TB therapy potentially involves the application of RNA interference (RNAi) and RNA activation (RNAa) biological processes that will target host genes, thereby inducing an indirect bactericidal effect. We hypothesized that macrophage genes that are differentially expressed by pathogenic and non-pathogenic mycobacterial species may be important in the regulation of M. tb intracellular survival. The lipid-rich mycobacterial cell wall is implicated in the excessive clumping of the mycobacterial cells in liquid culture. In order to minimize this, Tween 80 detergent was supplemented (mycobacteriaT). However, due to substantial evidence emphasising the detrimental effects of Tween 80 on the mycobacterial cell wall, mycobacteria were also cultured without Tween 80 (mycobacteriaNT), in order to investigate if the perturbed mycobacterial cell wall induced by Tween 80 affects the transcriptional response of macrophages. We endeavoured to develop a new method to culture mycobacteria without Tween 80 that will still generate single cells. We further hypothesized that the macrophage gene expression profile induced by mycobateriaNT differs from the response induced by mycobacteriaT. Differentiated THP-1 (dTHP-1) cells were infected with pathogenic and non-pathogenic mycobacteria (for 3 h, 24 h and 48 h with M. tb and M. bovis BCG, and 3 h and 8 h with M. smegmatis) cultured in the presence or absence of Tween 80. The expression of 12 macrophage genes, selected based on their involvement in the phagocytic pathway and autophagy, as well as their general involvement in the immune response, was determined by qRT-PCR and further analysed on the REST programme. The expression of each target gene was normalised relative to the expression of the reference gene (Beta actin). We observed that out of the 12 genes, TLR7 and VAMP7 were consistently downregulated in dTHP-1 cells infected with M. tbNT and upregulated in dTHP-1 cells infected with M. smegmatisNT. Their response to M. bovis BCG was inconsistent and not significantly different, and therefore could not be interpreted. Furthermore, CCL1 was upregulated by all the mycobacterial species. However, its expression was more pronounced in response to mycobacteriaNT, when compared to mycobacteriaT. Differential gene expression of TLR7 and VAMP7 in response to pathogenic and non-pathogenic mycobacteriaNT suggests that these 2 genes may be potential targets for RNAa-based anti-TB therapy, even though we could not conclude whether their response was specific to macrophages. In addition, the observed difference in the expression of CCL1 induced by mycobacteriaNT, compared to mycobacteriaT suggests that the perturbation caused by Tween 80 on the mycobacterial cell wall most likely affected the response of macrophages to infection with mycobacteria. Furthermore, this study has demonstrated a feasible method by filtration to generate single cells from mycobacteriaNT, which should be considered for future mycobacterial infection studies.
AFRIKAANSE OPSOMMING: Die huidige anti-tuberkulose middels se sukses lê daarin dat dit die aantal sterftes verminder maar hierdie sukses word weer beperk met die ontstaan van middel-weerstandige M.tb stamme. Daarom is nuwe middels nodig wat die ontwikkeling van middel-weerstandigheid beperk in ʼn poging om effektiewe TB behandeling te bewerkstellig. Anti-tuberkulose middels teiken hoofsaaklik mycobakteriële ensiemsisteme en ontlok sodoende weerstandigheid in M.tb stamme en dit vorm die rasionale vir hierdie studie. Die doel was om gasheer makrofaag gene te identifiseer wat M.tb oorlewing intrasellulêr bewerkstellig. Die voorgestelde alternatiewe anti-TB behandeling sal dan behels die toepassing van RNA intervensie (RNAi) en RNA aktivering (RNAa) tegnologie wat gasheer selgene teiken (inaktiveer) en sodoende ʼn bakterisidiese respons induseer. Die kanse is skraal dat mycobakterieë weerstandigheid sal kan ontwikkel onder hierdie omstandighede. Ons hipotetiseer dus dat makrofaag gene wat differensieel uitgedruk word deur patogeniese en nie-patologiese mycobakteriële spesies belangrik mag wees vir die oorlewing van M.tb intrasellulêr. Die lipiedryke selwand van mycobakterieë word geïmpliseer in die oormatige sameklomping van die bakterieë in vloeistofkulture. Om hierdie effek te minimaliseer word Tween 80 normaalweg tot die medium gevoeg (mycobakterieëT). Maar weens genoegsame bewyse dat Tween-80 die selwand van bakterieë nadelig beïnvloed, is mycobakterieë ook in die afwesigheid van Tween 80 gekultureer (mycobakterieëNT) om te bepaal of die nadelige effek van Tween 80 op die selwand die transkripsionele respons in makrofage beïnvloed post-infeksie. Dit was daarom ook ons doelstelling om ʼn nuwe tegniek te ontwikkel om mycobakterieë te kultureer in die afwesigheid van Tween 80 wat ook enkelselle sal genereer vir beter gekontroleerde makrofaag infeksie. Ons hipotetiseer ook verder dat makrofaag geenuitdrukking-profiele verskil afhangende of infeksie gedoen is met mycobakterieë wat in die afwesigheid of teenwoordigheid van Tween 80 gekultureer is. Gedifferensieerde THP-1 (dTHP-1) was geïnfekteer met patogeniese en nie-patogeniese mycobakterieë (vir 3 h, 24 h en 48 h met M.tb en M.bovis BCG, en 3 h en 8 h met M.smegmatis) gekultureer in die teenwoordigheid en afwesigheid van Tween 80. Die uitdrukking van 12 makrofaag gene, geselekteer op grond van hul betrokkenheid in die fagositose meganisme en in outofagie asook hul betrokkenheid in die immuunrespons, is gekwantifiseer met qRT-PCR en daaropvolgens geanaliseer met die REST-program. Die uitdrukking van elke geen is genormaliseer relatief tot die uitdrukking van die verwysingsgeen (Beta actin). Daar is bevind dat van die 12 gene, TLR7 en VAMP7 deurlopend afgereguleer was in dTHP-1 selle geïnfekteer met M.tbNT en opgereguleer was in dTHP selle geïnfekteer met M.smegmatisNT. Selrespons met M.bovis BCG was onbeduidend en derhalwe kon geen gevolgtrekking hier gemaak word nie. Ook, CCL1 was opgereguleer met infeksie deur enige van die mycobakteriële spesies, maar CCL1 se uitdrukking was groter in respons tot mycobakterieëNT wanneer vergelyk word met respons tot mycobakterieëT. Differensiële geenuitdrukking van TLR7 en VAMP7 in respons tot patogeniese en nie-patogeniese mycobakterieëNT impliseer dat hierdie twee gene potensiële teikens kan wees vir RNAa-gebaseerde anti-TB behandeling, alhoewel ons nie kon beslis of hierdie respons spesifiek vir makrofage was nie. Ook, die verskille waargeneem in die uitdrukking van CCL1 geïnduseer deur mycobakterieëNT, vergeleke met mycobakterieëT, impliseer dat die steuring in die selwand veroorsaak deur Tween 80, heelwaarskynlik die respons van die makrofaag beïnvloed het. Hierdie studie beskryf ook ʼn filtrasiemetode om enkele mycobakteriële selle te genereer wat oorweeg moet word by toekomstige mycobakteriële infeksiestudies.
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de, Camargo Bertuso Paula. "Roles of regulation of mRNA cleavage in Mycobacterium smegmatis." Digital WPI, 2016. https://digitalcommons.wpi.edu/etd-theses/776.
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One third of the world's population is infected with Mycobacterium tuberculosis, the bacterium that causes TB. During an infection, bacteria often survive host immune system attacks, which include oxidative stress conditions for bacteria growing inside macrophages. This makes treatment difficult and time-consuming. We hypothesize bacteria can adapt to environmental conditions by changing their mRNA maturation and degradation profiles. Using a model system, Mycobacteruim smegmatis, we focus on how mRNA expression is affected by oxidative stress. After construction and sequencing of RNA expression libraries, preliminary analysis showed that after three hours of H2O2 exposure most upregulated genes were related to DNA repair, while downregulated genes included transport proteins. After six hours of exposure, upregulated genes were similar to three hours and downregulated genes included tRNAs. 5' end mapping libraries were also constructed to access differential cleavage site abundance under oxidative stress conditions. We also investigated the roles RNase J may have in stress response and mRNA processing in Mycobacteria. RNase J and RNase E are thought to be the major RNases in bacteria. While most bacteria only have one of them, mycobacteria encode both in their genome, with RNase J being non-essential. We constructed a set of 4 strains (WT, RNase J overexpression, RNase J deletion, and complemented RNase J deletion) and tested their drug resistance and stress tolerance. Results suggests that RNase J deletion and overexpression alter drug sensitivity. Stress tolerance assays showed that WT is more tolerant to oxidative stress, followed by RNase J deletion strain and overexpression and complemented RNase J deletion strains, with the last two showing no growth when cultured with H2O2. Analysis of the expression profile of these strains was performed to help understand if gene expression differences are responsible for the phenotypes observed. For the complemented RNase J deletion, one operon had almost all its genes upregulated. This operon encodes a hydrogenase (Hyd3), suggesting that redox balance in the strain is perturbed.
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Kirsten, Catriona Jane. "Nitrogen metabolism and the regulation thereof in Mycobacterium smegmatis." Thesis, Stellenbosch : University of Stellenbosch, 2011. http://hdl.handle.net/10019.1/17991.
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Thesis (PhD (Med))--University of Stellenbosch, 2011.ENGLISH ABSTRACT: The nitrogen metabolic pathway is essential for growth and survival of all living organisms including prokaryotes. Certain components of the pathway, such as the enzyme glutamine synthetase (GS), have been studied; however, little information is available regarding the pathway in the mycobacteria. Our in silico studies revealed that many of the components and mechanisms involved in the pathway appear to be conserved between closely related Actinomycetales. Therefore, we investigated three aspects of nitrogen metabolic control in Mycobacterium smegmatis; namely, transcriptional regulation of nitrogen metabolism-related genes, control of enzyme activity and the signalling cascade governing the nitrogen metabolic response. At the transcriptional level, it was found that nitrogen metabolism-related genes were regulated in response to ammonium availability. Two possible transcriptional regulators, AmtR and GlnR, which are the regulators responsible for control of nitrogen-related gene transcription in Streptomyces coelicolor and Corynebacterium glutamicum respectively, were identified in M. smegmatis. Through generation of amtR and glnR deletion mutants, we found that both potential regulators played a role in the control of nitrogen-related gene expression in M. smegmatis. GlnR acted as both an activator and repressor of gene transcription whilst AmtR appeared to activate gene expression which is different to the role its homolog plays in C. glutamicum. On a protein level we found that both GS and glutamate dehydrogenase (GDH) were responsible for ammonium assimilation in M. smegmatis and were regulated in response to ammonium availability. Two GDH isoforms (NAD+- and NADP+-specific) were identified in M. smegmatis and whereas only an NAD+-GDH was detected in M. tuberculosis. The M. tuberculosis GDH also played a largely anabolic role with regard to ammonium assimilation which is in contrast to the belief that ammonium can only be assimilated via GS in this pathogen. The signaling cascade was investigated through generation of a glnD deletion mutant in M. smegmatis. We were able to show that this pivotal protein (GlnD) was able to relay the cellular nitrogen status to the transcriptional machinery as well as to GS. The data presented in this study has advanced our understanding of the nitrogen metabolic pathway in the mycobacteria. Through elucidation of such pathways, our knowledge of mycobacterial physiology and thus infection and survival improves, which could ultimately lead to the discovery of novel mechanisms to aid in the eradication of the disease.
AFRIKAANSE OPSOMMING: Stikstof metabolisme is noodsaaklik vir die oorlewing en groei van alle organismes, prokariote ingesluit. Sekere sellulêre komponente, soos die ensiem glutamine sintetase (GS), is al tevore bestudeer, maar baie min verdere inligting is beskikbaar oor stikstof metabolisme in die mycobacteria. Ons in silico studies het gewys dat baie van die komponente en meganismes gekonserveerd gebly het tussen nou-verwante Actinomycetales. Dus het ons drie aspekte in die beheer van stikstof metabolisme ondersoek; naamlik, die transkriptionele regulering van stikstof metabolisme-verwante gene, die beheer van ensiem aktiwiteit en die sein-meganisme wat die reaksie op stikstof konsentrasie reageer. Op transkripsionele vlak het ons gevind dat stikstof metabolisme-verwante gene gereguleer word in reaksie op stikstof beskikbaarheid. AmtR en GlnR is twee moontlike transkripsie reguleerders wat verantwoordelik is vir transkripsionele beheer in onderskeidelik Streptomyces coelicolor en Corynebacterium glutamicum. Beide hierdie proteïene is geïdentifiseer in M. smegmatis. Deur die konstruksie van amtR en glnR mutante, het ons gevind dat beide potensiële reguleerders ‘n rol gespeel het in die beheer van stikstof-verwante transkripsie in M. smegmatis. GlnR het opgetree as beide ‘n aktiveerder en ‘n onderdrukker van transkripsie terwyl AmtR net ‘n aktiverende rol gespeel het. Die funksie van AmtR in M. smegmatis is dus verskillend van sy homoloog in C. glutamicum. Op proteïen-vlak het ons gevind dat beide GS en glutamaat dehidrogenase (GDH) verantwoordelik was vir die assimilasie van ammonium in M. smegmatis en albei was gereguleer in reaksie op ammonium beskikbaarheid. Twee vorme van GDH (NAD+- spesifieke- en NADP+-spesifieke GDH) was geïdentifiseer in M. smegmatis terwyl net ‘n NAD+- spesifieke GDH in M. tuberculosis gevind is. Die M. tuberculosis GDH het ook ‘n anaboliesie rol gespeel met betrekking tot ammonium assimilasie wat in teenstelling is met die huidige opvatting dat ammonium alleenlik deur GS ge-assimileer kan word. Die sein-meganisme is ondersoek deur ‘n glnD M. smegmatis mutant te konstrueer. Ons het bewys dat hierdie deurslaggewende proteïen (GlnD) die sellulêre stikstof status aan die transkripsionele masjinerie, en aan GS kon oordra. Die data wat in hierdie studie voorgelê word, het ons kennis van stikstof metabolisme in die mycobacteria gevorder. Sodanige metaboliese studies verbreed ons kennis van mycobacteriële fisiologie en dus M. tuberculosis infeksie en oorlewing en kan uiteindelik lei tot die ontdekking van unieke teiken meganismes om te help met die beheer van die siekte en nuwe middelontwikkeling.
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Shires, Karen Lesley. "Characterisation of the cold-shock response in Mycobacterium smegmatis." Doctoral thesis, University of Cape Town, 1999. http://hdl.handle.net/11427/25670.
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The response of Mycobacterium smegmatis to a cold shock was investigated in order to gain insight into the stress responses of members of the genus Mycobacterium. Mycobacterium smegmatis cultures were shocked from 37°C to 30°C, 25°C, 15°C, and 10°C and the effects on both growth (ATP concentration, culture turbidity, colony-forming units) and metabolism (incorporation of ¹⁴C-leucine and ³H-uracil) were investigated. The magnitude of the cold-shock response was found to be dependent upon the degree of the cold shock. A cold shock to 10°C had the greatest effect and resulted in a "lag period" of 24 hours in both the growth and metabolism of the culture. The synthesis of proteins was reduced 20-fold during this period, indicating at block in translation. The cold-shock response in Mycobacterium smegmatis was an adaptive response with growth eventually being resumed at the colder temperature, but at a reduced rate. Using the techniques of one-dimensional sodium-dodecyl-sulphate polyacrylamide gel electrophoresis and two-dimensional protein gel electrophoresis, ³⁵S-methiononine-labelled proteins that were synthesised during the cold shock were analysed. At least fourteen radio-labelled proteins were induced during the first 24-hour period and these demonstrated two distinct patterns of cold-shock induced expression: transient and continuous. Depending upon the pattern of expression and size, the cold-shock proteins were classified as "cold-induced proteins", "cold-shock proteins" or "cold-acclimation proteins". CipM, a 27kDa protein, was identified as the major cold-shock protein through one-dimensional protein electrophoresis. From N-terminal sequence data generated from a protein (CipM.1) within this band, a corresponding degenerate DNA probe was used to isolate cipM.1. This gene was cold-inducible, with mRNA levels transiently increasing 5-7 fold after a 37°C to 10°c cold-shock. Homologues of this cold-shock gene are found in the genomes of Mycobacterium tuberculosis and Mycobacterium leprae. The corresponding mycobacterial proteins showed homology at the N-terminus to the HU~ subunit of HU of Escherichia coli and possessed similar C-terminal praline, lysine and alanine degenerate repeats to the mycobacterial heparin-binding hemagglutinin. The response of several mycobacterial cold-shock gene homologues to a cold shock was also investigated, by northern-hybridisation and S1 nuclease analysis. The cspA homologue of Mycobacterium smegmatis demonstrated a 16-24 fold transient induction in mRNA levels following a 37°C to 10°C temperature-shift, while gyrA mRNA levels were maintained at a constant level throughout the cold shock. Although some similarities were demonstrated between the cold-shock response of Escherichia coli and Mycobacterium smegmatis, definite differences occur in the proteins that are involved in the adaptive stages of the response.
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Kirykowicz, Angela Mary. "High-Throughput Determination of Mycobacterium smegmatis Protein Complex Structures." Master's thesis, University of Cape Town, 2018. http://hdl.handle.net/11427/29644.
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Tuberculosis (TB) is an endemic health-crisis, particularly in sub-Saharan Africa. The rise of multiand extensively-drug resistant Mycobacterium tuberculosis (Mtb), the causative agent of TB, has led to further developments in understanding the physiology of Mtb during infection, as well as searching for novel drug targets, in order to combat the disease. Our understanding of cells, both eukaryotic and prokaryotic, has changed substantially in the last 50 years, incorporating the role of stable and transient protein-protein interactions which govern cell function and behaviour. Although there are many in vivo and in vitro methods for studying protein-protein interactions, they suffer from the lack of ability to distinguish physiological interactions from interactions that occur which are not physiologically relevant to the cell. Structure-based methods for determining protein interactions have the benefit of screening out false positives whilst simultaneously assessing the possible biological function of the protein complex in question. This study sought to assess different high-throughput methods for capturing stable, water soluble protein complexes from M. smegmatis (Msm), a close relative of Mtb, for structural characterisation by low-resolution transmission electron microscopy (EM). The use of partial biochemical fractionation was assessed, which produced low-resolution structures of glutamine synthetase I, bacterioferritin, and Encapsulin. These structures were unambiguously identified through a combination of fitting of homologous crystal structures into the low-resolution maps, and information obtained by liquid chromatography mass spectrometry (LC-MS/MS) of bands isolated from native- and SDS-PAGE gels. Since Encapsulin is likely to participate in the Msm oxidative stress response and functions to enclose the target proteins DyP-type peroxidase (DyP) and ferritin-family protein (BrfB), optimal conditions for cryo-EM were tested for further efforts to obtain a high-resolution structure. Furthermore, hypotheses were generated for the function of Mtb and Msm Encapsulin based on the Msm Encapsulin structure obtained with the aid of a crystal structure homologue; these related to the mode of cargo binding and pore selectivity. A single-step purification method was also assessed through grid blotting on blue native (BN) PAGE using GroEL as a test protein. The hydrophobicity and charge of the EM copper grid was tested to find the optimal grid property for particle transfer. This established that particles of GroEL could be transferred from BN-PAGE onto an EM copper grid and a successful negative stain reconstruction was obtained. In summary, the pipeline from purifying protein complexes to generating hypotheses based on structure was successfully investigated in Msm, which will aid in the production of novel drug targets for Mtb as well as in the application to other organisms.
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Mothiba, Maborwa Tebogo. "The effects of clofazimine on mycobacterium smegmatis biofilm formation." Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/31569.
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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.Dissertation (MSc)--University of Pretoria, 2013.
Immunology
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Brooke, Edward W. "Protein-ligand interactions of arylamine N-acetyltransferase from Mycobacterium smegmatis." Thesis, University of Oxford, 2003. http://ora.ox.ac.uk/objects/uuid:f13c3191-b098-4a85-84c3-90590b365d30.
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Tuberculosis is the world's largest cause of death from an infectious agent. Treatment is by an extended period of combination chemotherapy. Drug resistance is an increasing problem in tuberculosis therapy, particularly to the frontline anti-tubercular drug isoniazid (INH). Recombinant arylamine N-acetyltransferase (NAT) of Mycobacterium tuberculosis N-acetylates INH using the cofactor Acetyl Coenzyme A. NAT from M. tuberculosis is a polymorphic enzyme and also acetylates INH in vivo. Acetylated INH is inactive therapeutically against M. tuberculosis both in vivo and in vitro. The acetylation of isoniazid in the mycobacterial cell may compete with the activation of INH by the catalase-peroxidase, katG, and hence contribute to INH resistance in clinical isolates. Inhibition of NAT in M. tuberculosis may thus increase the efficacy of INH therapy. A novel assay based around the detection of free Coenzyme A released during the acetylation reaction was used to determine the substrate specificity of recombinant NAT from the related Mycobacterium M. smegmatis (MSNAT). A relationship was observed between the lipophilicity of simple arylamine substrates and the rate of acetylation by MSNAT. Several MSNAT substrates possess antibacterial activity. The assay could also be used to screen compound libraries for MSNAT inhibitors. Synthesis of seventeen thiazolidinedione sultams in collaboration with Dr.Vickers (Dyson Perrins), identified as weak inhibitors of MSNAT, gave a minimum competitive inhibitory constant of 14μM. Screening a library of 5,074 drug-like compounds for inhibition of MSNAT identified thirteen compounds with semi-maximal inhibition constants (IC50) of below 10μM. Based on this, fifteen maleimides were synthesised and were irreversible inhibitors of MSNAT with submicromolar potency. Similarly, ninety-six aminothiazoles were synthesised by Dr. Vickers and were uncompetitive inhibitors of MSNAT with a minimum IC50 of 1.5μM. The most potent aminothiazole showed no effect on the growth of M. smegmatis or M. bovis BCG or the sensitivity of the bacteria to isoniazid. However the aminothiazoles were shown not to penetrate the cells.
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Burgess, Jeremy Gareth. "Helical reconstruction of Mycobacterium smegmatis Mycothiol S-conjugate amidase filaments." Master's thesis, University of Cape Town, 2017. http://hdl.handle.net/11427/24868.
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The metabolic pathway of mycothiol (MSH) is a major cellular defence against oxidative stress, and several antibiotics for mycobacteria, including Mycobacterium tuberculosis. The central enzyme used in the clearance of electrophilic toxins is Mycothiol S-conjugate amidase (Mca). Mca is similar to a biosynthetic enzyme MshB, which has partial overlapping substrate activity and is the closest homologue to Mca with a known structure. The basis for the substrate specificity differences in Mca and MshB is not well understood. Several regions of low sequence similarity between MshB and Mca are contained within an active site pocket, and these may affect the observed substrate preferences. However, these regions cannot be modelled in Mca with confidence, which makes it essential to obtain a structure of Mca experimentally. Mca is also a potential drug target, and a structure of Mca would enhance the rational design of inhibitors against the enzyme. A search for crystalline forms of MsMca (Mycobacterium smegmatis Mca) led to the discovery of regular filaments, which showed helical order. Helical symmetry was estimated using power spectra from single filaments. The number of potential symmetry solutions was reduced using phase information from Fourier transforms of single filaments. Three possible solutions to the helical symmetry were suggested, two of which converged on the same symmetry parameters using Iterative Helical Real-Space Reconstruction. The first solution had a selection rule of l = 18m + n, and the second l = 20m + n. Reconstructions made from the predicted helical symmetries were compared in their power spectra and through rigid-body fitting with an atomic model of MsMca. The first reconstruction, with a final symmetry of Δφ = 20.05o and Δz = 10.27 Å, better matched the predicted helical symmetry than did the second reconstruction. However, rigid-body fitting did not indicate either reconstruction as being superior. Following this, the second reconstruction was improved using a number of additional techniques to those used in the initial reconstruction. These included the use of the fortuitous 3-fold cyclic symmetry, the removal of double-walled filaments, use of a cut-off filter for images with low correlation to projections of the 3D reconstruction, and use of a layer-line filter to reduce the noise in the images. These were used individually, then in a single reconstruction, to improve the and agreement between the predicted helical symmetry and that obtained from the reconstruction. Several of the improved reconstructions were used via rigid-body fitting to assess the favoured handedness of the filament through examination of the major interfaces between subunits. These suggest that the 3-start helix is right-handed. Future work would be to determine the handedness of the filament using alternative techniques, such as metal-shadowing. This work provides a springboard for high resolution cryo-electron microscopy, to determine a high-resolution structure of MsMca, which will enable rational inhibitor design and give the basis for the different substrate specificity in Mca and MshB.
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Zhang, Tianbu. "Structural and biochemical studies of F₁-ATPase from Mycobacterium smegmatis." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708118.
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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." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2007. http://hdl.handle.net/10183/30206.
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A tuberculose (TB) é uma séria doença infecciosa causada por Mycobacterium tuberculosis, e constitui um importante problema de saúde pública em todo o mundo. Novas drogas e vacinas de segunda geração são urgentemente necessárias para o controle da TB, mas a complexa biologia de M. tuberculosis tem dificultado o desenvolvimento de estratégias terapêuticas inovadoras. A manipulação genética de M. tuberculosis também é complicada, mas, atualmente, técnicas novas e mais eficientes de troca alélica permitem o estudo detalhado de diversos genes micobacterianos. No presente trabalho, os genes da corismato mutase (CM) e fosforilase de nucleosídeos purínicos (PNP) de M. tuberculosis e Mycobacterium smegmatis foram estudados. A CM catalisa a conversão de corismato em prefenato na rota de biossíntese dos aminoácidos aromáticos fenilalanina e tirosina em bactérias, fungos e plantas. Dois genes de CMs monofuncionais (aroQ e *aroQ) foram identificados, clonados, expressos e bioquimicamente caracterizados em ambas as espécies de micobactérias. Esses genes também foram investigados usando uma metodologia de recombinação homóloga e ensaios de atividade promotora. Os resultados indicam que os genes aroQ são provavelmente essenciais ao crescimento in vitro de M. tuberculosis e M. smegmatis, enquanto uma linhagem mutante para o gene *aroQ de M. smegmatis pôde ser obtida. A PNP catalisa a interconversão e reciclagem de bases, nucleosídeos e nucleotídeos purínicos na via de salvamento das purinas. A construção de mutantes para o gene deoD (que codifica a PNP) de M. tuberculosis e M. smegmatis, usando um método eficiente de troca alélica em duas etapas, não foi possível. Assim, sugere-se que, nas condições testadas, o gene deoD seja essencial ao crescimento in vitro dessas micobactérias. A identificação de genes essenciais é de fundamental importância, pois indica tanto a relevância biológica dos mesmos como, no caso de M. tuberculosis, que seus produtos constituem alvos moleculares interessantes para o desenvolvimento de novas drogas antimicobacterianas.Tuberculosis (TB), a serious infectious disease caused by Mycobacterium tuberculosis, still remains a public health problem in the world. New drugs and second generation vaccines are urgently required to control TB, but the complex biology of M. tuberculosis has hindered the development of novel therapeutic tools. Genetic manipulation of M. tuberculosis is also difficult, but currently new, more efficient gene replacement techniques have allowed detailed studies of many mycobacterial genes. In the present work, the chorismate mutase (CM) and purine nucleoside phosphorylase (PNP) genes from M. tuberculosis and Mycobacterium smegmatis were studied. CM catalyzes the rearrangement of chorismate to prephenate in the biosynthetic pathway that forms phenylalanine and tyrosine in bacteria, fungi, and plants. Two monofunctional CM genes (aroQ and *aroQ) were identified, cloned, expressed, and biochemically characterized in both mycobacteria. Those genes were also investigated by homologous recombination methods and promoter activity assays. AroQ genes seem to be essential for the growth of M. tuberculosis and M. smegmatis, while an *aroQ mutant strain could be generated in M. smegmatis. PNP promotes the interconversion and recycling of purine bases, nucleosides, and nucleotides in the purine salvage pathway. Construction of deoD (which codes for PNP) deletion mutants of M. tuberculosis and M. smegmatis using an efficient two-step gene replacement technique was not possible. Thus, it is suggested that deoD is also essential for mycobacterial growth under the conditions tested. The identification of essential genes is of great importance, both because it indicates their biological significance, and because, with pathogens like M. tuberculosis, these may also indicate attractive molecular targets for the development of new antimycobacterial drugs.
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Ingvarsson, Henrik. "Structural studies of Caseinolytic protease 1 from Mycobacterium tuberculosis and Methionyl-tRNA synthetase from Mycobacterium smegmatis." Doctoral thesis, Uppsala universitet, Strukturell molekylärbiologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-121779.
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Tuberculosis is a severe disease that causes about 2 million deaths every year. It is a worldwide threat and it is estimated that one-third of the world’s population carries the infection. The severe side effects of the present drugs, and the more than 6 months long treatment, in addition to the development of resistant bacterial strains, are the incentives for the intensified search for new drugs. In this work two potential mycobacterial drug targets have been studied: Caseinolytic protease 1 (ClpP1) from Mycobacterium tuberculosis (Mt) and Methionyl-tRNA synthetase (MetRS) from Mycobacterium smegmatis (Ms). The X-ray stucture of ClpP1 was determined to 3.0 Å resolution. The study gives details on the tetradecameric arrangement of the enzyme. Two hepameric discs assemble to form a chamber containing the catalytic activity mediated by each of the monomers. The chamber can be reached by two pores. Comparison with the human homologue reveals important structural differences. The X-ray studies on Ms MetRS were done to 2.3 Å and 2.8 Å resolution. The study gives details on the flexibility of the enzyme and how this is related to activity. Important findings are identification of an intermediate structure in which the methionine to be adenylated is bound in the catalytic site in a tight complex. The catalytic site and the anticodon recognizing domains are separated and the structural results indicate communication between the domains. The possibility to allosterically inhibit the enzyme is discussed.
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VICENTE, Iêda Cristina da Silva. "Atividade bactericida de um novo derivado 1,2,4-oxadiazol-hidrazida frente ao Mycobacterium fortuitum e Mycobacterium smegmatis." Universidade Federal de Pernambuco, 2003. https://repositorio.ufpe.br/handle/123456789/1352.
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Previous issue date: 2003As infecções causadas pelas micobactérias atípicas são freqüentes em pacientes imunocomprometidos, particularmente aqueles contaminados com o HIV. Os quimioterápicos normalmente utilizados não são suficientes, devido a crescente deficiência no tratamento da tuberculose e micobacterioses. Por esta razão, novos experimentos são necessários para obtenção de novas drogas para o tratamento desses pacientes. O objetivo desse estudo foi determinar in vitro a atividade bactericida de uma nova hidrazida, [3-(4-hidroxi-fenil)-1,2,4-oxadiazol-5-il]- acil - hidrazida e de sua associação com o p-flúor-fenilalanina, um inibidor da biossíntese do micosídio-C. Os microrganismos testados M. fortuitum e M. smegmatis foram isolados de pacientes imunocomprometidos hospitalizados. A atividade bactericida in vitro do derivado hidrazínico e p-flúor-fenilalanina e sua associação foram estudadas pelo método da curva de morte versus tempo (Time Killing Curve). Após 120 horas de exposição foram observados um decréscimo no crescimento de 1,45 ± 0,39 log10 e 2,41 ± 0,17 log10 frente o M. fortuitum e M. smegmatis, respectivamente. Com a pflúor- fenilalanina não foi observada nenhuma diminuição do crescimento. Nas associações foram observadas as diminuições do crescimento de 1,08 ± 0,59 log10 frente ao M. fortuitum e de 2,08 ± 0,25 log10 para o M. smegmatis. Não foi verificado recrescimento nas culturas. O derivado hidrazínico demonstrou atividade contra o M. fortuitum e M. smegmatis, contudo sua atividade não foi acentuada na associação com o p-flúor-fenilalanina
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Almourfi, Feras. "Structural genomic studies of lipoproteins from Mycobacterium smegmatis for drug design." Thesis, University of Sheffield, 2014. http://etheses.whiterose.ac.uk/6666/.
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Tuberculosis (TB) is considered as an old infectious disease that leads to many fatalities in Man. Mycobacterium tuberculosis was discovered as the causative agent of tuberculosis by Robert Koch in 1882. Since then, scientists started the first move in order to develop such tools to prevent the disease. According to the WHO 2012 TB report, about one-third of the population is infected with M.tuberculosis; TB causes nearly 1.8 million deaths every year. Perhaps most worrying, new strains of M.tuberculosis resistant to most or even all-standard anti-TB drugs are spreading throughout the world, making treatment more costly and often impossible. Therefore, we urgently need to discover new drugs to overcome TB. The genomic sequence of M.tuberculosis has been completed in 1998 and has helped to shed light on new pathways as drug targets. As part of a drug discovery programme, a structural genomics study of lipoproteins has been launched using the Mycobacterium smegmatis as a model organism for M.tuberculosis. A lipid-anchored protein is a class of protein that is produced in the cytoplasm as a pre-prolipoprotein and attached to the cell membrane following posttranslational modification (lipidation). Such proteins represent about 3% of bacterial genomes. Furthermore, all bacteria apparently allocate particular proteins to the cell envelope by a process called post-translational lipid modification in order to produce membrane-anchored lipoproteins that are able to work in the aqueous environment at the membrane interface. Therefore, this project aims to identify new targets suitable for drug discovery and shed light on their role in the cell. Eight targets were identified and put into a pipeline of cloning, over-expression, purification and crystallization for structure determination. Five target proteins of different putative functions were successfully purified with one (Msmeg_0515) annotated as an ABC sugar transporter protein, leading to structure determination. The structure of Msmeg_0515 (AgaE) has been determined to a high resolution of 1.22 A. Structure comparison of AgaE with other sugar binding proteins revealed that AgaE shares a similar fold with the maltose/maltodextrin binding protein (MalE) from E.coli. Previous bioinformatics studies on the sugar transporters of M.smegmatis and M.tuberculosis suggested that AgaE is an α-galactoside sugar binding protein, however, structural analysis of the binding site of AgaE protein revealed that it's more similar to malto-oligosaccharide binding proteins. Also, binding assay by Circular dichroism (CD) has revealed a significant affinity of AgaE for maltose, glycerol 3-phosphate and acarbose but not α-galactoside sugars.
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Oliveira, Fábio Muniz de. "Mycobacterium smegmatis recombinante expressando a proteína CMX induz resposta imune contra Mycobacterium tuberculosis em camundongos BALB/c." Universidade Federal de Goiás, 2014. http://repositorio.bc.ufg.br/tede/handle/tede/3457.
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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
For hundreds years tuberculosis (TB), a contagious disease caused by Mycobacterium tuberculosis (Mtb), has been a global public health problem. Even after the development of the vaccine BCG, in 1921, tuberculosis control continues on slow pace. This comes to be as a result of the variable efficacy (from 0 to 80%) presented by the vaccine in the protection against TB in adults. Therefore, the development of a new vaccine against TB is necessary. In this study, it was evaluated a recombinant vaccine composed of Mycobacterium smegmatis expressing the CMX fusion protein (mc2- CMX), formed from three antigen epitopes of Mtb: Ag85C, MPT51 and HspX. M. smegmatis mc2 155 was transformed with pLA71-CMX by electroporation, and the presence of the CMX protein was confirmed by imuno blotting. BALB/c mice were distributed in four groups: saline, infection, BCG and mc2-CMX. The groups were immunized with their respective vaccines in two moments with an interval of fifteen days and the animal blood was collected fifteen days after the last immunization. Thirty days after the last immunization, the animals were challenged with Mtb H37Rv (intravenously) and thirty days after the challenge, the blood was collected to perform ELISA test. Seventy days after the challenge, the lungs from all mice were collected to obtain cells for flow-cytometry, histological analysis and also to determine the bacillary burden. The immunization with mc2-CMX induced higher levels of antibodies of IgG1 (1,910±0,70) and IgG2a (0,139±0,020) class anti-CMX when compared with BCG group (0,646±0,19 and 0,413±0,24; respectively, p<0,05). These results demonstrated the relevance of CMX antigen in the immunogenicity of the recombinant vaccine. Seventy days after the challenge, the amount of T CD4 cells in the lung producing Th1- type cytokines was assessed. It was observed a significant increase in the percentage of T CD4 cells positive for IFN-γ and TNF-α in the immunized mice with mc2-CMX vaccine, when compared with the group immunized with BCG. Mice challenged with Mtb presented significant higher percentage of IL-2 producer cells when compared with the non-immunized group. However, only the mice immunized with the vaccine mc2- CMX presented significant higher percentage when compared with the infection group. The immune response induced by the vaccine was effective in the control of Mtb infection, confirmed by the histological analysis and the bacillar burden determined. The groups vaccinated with mc2-CMX and BCG presented a significant reduction of the lung lesion induced by the Mtb infection, and also lung bacterial load, when compared with the infection group. Thus, the recombinant vaccine mc2-CMX presents potential characteristics to be used in the prevention of TB.
Há séculos a tuberculose (TB), doença infectocontagiosa causada por Mycobacterium tuberculosis (Mtb), vem sendo um problema de saúde pública mundial. Mesmo após o surgimento da vacina BCG em 1921, o controle da tuberculose continua a passos lentos. Isso se deve à eficácia variável de 0 a 80% apresentada pela vacina na proteção contra TB em indivíduos adultos. Deste modo, o desenvolvimento de uma nova vacina contra a TB é necessário. Neste estudo, avaliou-se uma vacina recombinante composta por Mycobacterium smegmatis expressando a proteína de fusão CMX (mc2-CMX), formada por três antígenos do Mtb: Ag85C, MPT51 e HspX. M. smegmatis mc2 155 foi transformado com pLA71-CMX por eletroporação, sendo a expressão da proteína CMX confirmada por imunoblot. Camundongos BALB/c foram distribuídos em quatro grupos: salina, infecção, BCG e mc2-CMX. Os grupos foram imunizados com suas respectivas vacinas em dois momentos com intervalos de 15 dias, e o sangue de todos os animais coletado quinze dias após a última imunização. Trinta dias após a imunização, os animais foram desafiados com Mtb H37Rv (via endovenosa) e trinta dias após o desafio, o sangue foi coletado para realização de ELISA. Setenta dias após desafio, o pulmão e o baço de todos os camundongos foi coletado para obtenção de células para realização de citometria, histopatológico e determinação da carga bacilar. A imunização com o mc2-CMX induziu níveis maiores de anticorpos da classe IgG1 (1,910±0,70) e IgG2a (0,139±0,020) anti-CMX quando comparado com o grupo BCG (0,646±0,19 e 0,413±0,24, respectivamente, p<0,05). Estes resultados demonstram a relevância do antígeno CMX na imunogenicidade da vacina recombinante. Após setenta dias do desafio, a quantidade de células T CD4 produtoras de citocinas do tipo Th1 foi analisada no pulmão. Foi observado um aumento significativo na porcentagem de células T CD4 positivas para IFN-γ e TNF-α nos camundongos imunizados com vacina mc2-CMX, quando comparado com o grupo BCG. Camundongos desafiados com Mtb apresentaram porcentagens maiores de células produtoras de IL-2, quando comparado com o grupo não desafiado. Todavia, somente os camundongos imunizados com a vacina mc2-CMX apresentaram porcentagens significativamente maiores em comparação ao grupo infecção. A resposta imune observada foi efetiva no controle da infecção por Mtb, sendo isto confirmado quando os pulmões dos camundongos foram analisados histologicamente e a carga bacilar determinada. Os grupos vacinados com as vacinas mc2-CMX e BCG apresentaram uma redução significativa da lesão pulmonar induzida pela infecção por Mtb, e também da carga bacilar no pulmão, quando comparados com o grupo infecção. Conclui-se que mc2-CMX tem um bom potencial para ser explorado como vacina contra a TB.
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Lamrabet, Otmane. "Modifications génétiques de Mycobacterium tuberculosis : interactions avec les organismes hôtes." Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM5027/document.
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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 bactériesMycobacteria 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 our laboratory we modified two species of the M. tuberculosis complex, M. tuberculosis H37Rv and Mycobacterium bovis BCG to observe the effect of these changes on their pathogenicity and survival
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Steyn, Natassja Lise. "Investigating the localisation of the ESX-3 secretion system in Mycobacterium smegmatis." Thesis, Stellenbosch : Stellenbosch University, 2012. http://hdl.handle.net/10019.1/71959.
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Thesis (MScMedSc)--Stellenbosch University, 2012.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 uitdrukking van ESX-3 in M. tuberculosis word gereguleer deur IdeR en Zur in reaksie op intrasellulêre yster en sink konsentrasies, onderskeidelik. ESX-3 word nie benodig vir die groei en oorlewing van die saprofitiese organisme M. smegmatis nie. Hierdie studie was gemik om die sub-sellulêre lokalisering van ESX-3 te identifiseer in die niepatogeniese en vinnig-groeiende organisme, M. smegmatis. Die “esx conserved component” (ecc) gene van ESX-3 is uitgedruk vanaf die episomale uitdrukkingsvektor pDMNI as gekombineerde proteïene met die groen fluoreserende proteïen (GFP). MSMEG_0615 (eccA3), MSMEG_0616 (eccB3), MSMEG_0623 (eccD3) en MSMEG_0626 (eccE3) is suksesvol gekloneer en die uitdrukking van die gekombineerde proteïene is bevestig deur Western oordrag vir MSMEG_0615-GFP, MSMEG_0616-GFP en MSMEG_0626-GFP in M. smegmatis. In die M. smegmatis ESX-3 uitklopmutant (met MSMEG_0615 tot MSMEG_0626 uitgeslaan) is uitdrukking bevestig vir MSMEG_0615-GFP en MSMEG0626-GFP. Fluoresensie mikroskopie het bepaal dat MSMEG_0615-GFP gelokaliseer is by ‘n enkele mikobakteriese pool in beide stamme. MSMEG_0616-GFP en MSMEG_0626-GFP was membraan-geassosieerd in M. smegmatis, terwyl en MSMEG_0626-GFP geassosieer het met die membraan in die M. smegmatis uitklopmutant. MSMEG_0615 het gelokaliseer by ‘n enkele pool in M. smegmatis en dit dui aan dat die saamgestelde ESX-3 sekresie sisteem apparaat slegs by ‘n enkele pool voorkom in M. smegmatis. Ons hipotiseer dat MSMEG_0615 dalk mag optree as ‘n werwer proteïen wat betrokke is by die samestelling van die ESX-3 sekresie sisteem by die mikrobakteriese pool.
Stellenbosch University
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MARINHO, Victor Hugo de Souza. "O papel do colesterol na biossíntese da parede celular de Mycobacterium smegmatis." Universidade Federal do Pará, 2015. http://repositorio.ufpa.br/jspui/handle/2011/7453.
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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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Joyce, Graham. "Organisation of the Mycobacterium smegmatis chromosome and its role in cell division." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/6831.
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Tuberculosis remains a global health problem, exacerbated by the increasing emergence of multi‐drug resistant strains. The identification of new drug targets and the discovery of new anti‐tuberculosis drugs is therefore a high priority. Although little is currently known about mycobacterial cell division, the process is essential for the survival and expansion of all bacterial species so may involve proteins that represent excellent drug targets. In this thesis, proven tools for the study of bacterial cell division such as live‐cell time‐lapse imaging and Fluorescent Repressor Operator System (FROS) were adapted for use in mycobacteria. Application of such techniques, fluorescent tagging of cell division proteins and deletion of parA in M. smegmatis helped to elucidate some interesting characteristics of mycobacterial cell division. In contrast to model organisms, live cell imaging and septal staining indicated that M. smegmatis can grow and divide asymmetrically and divides at a range of lengths suggesting a fundamentally different mechanism of division regulation. The chromosome was hypothesised to play a key role in cell division so was investigated further by labelling a specific chromosomal loci. The key finding was that M. smegmatis cells only contain 1 or 2 chromosomal copies and that regardless of cell length, the nucleoid occupies almost the entire intracellular space. To examine if the nucleoid organisation is important for cell division, a putative chromosome segregation gene parA was disrupted. The ΔparA mutant displayed a classic cell division phenotype characterised by the production of anuclear mini‐cells. The mechanism responsible for the ΔparA mutant phenotype was studied further by applying live cell imaging, FROS and expressing a ParA‐mCherry fusion protein. The data obtained from all work presented was collated and used to propose a novel model of bacterial cell division regulation applicable to mycobacteria where the nucleoid plays a central role and ParA is required to ensure correct nucleoid placement.
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O'Brien, Lyn. "An investigation of the killing of Mycobacterium tuberculosis by macrophages and the acid stress response of Mycobacterium smegmatis." Thesis, University of Leicester, 1995. http://hdl.handle.net/2381/35403.
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Attempts were made to activate human monocytes with immunomodulators and human macrophages with T-cell supernatants for in vitro antimycobacterial activity. Both these approaches failed. Alveolar macrophages from Mycobacterium bovis BCG-vaccinated guinea pigs have previously been shown to kill Mycobacterium tuberculosis in vitro. The guinea pig was therefore used to investigate macrophage antimycobacterial mechanisms. Tuberculocidal factors were found within lysosomes. Lysosomal fractions of macrophages from BCG-vaccinated guinea pigs significantly (P 0.001) killed M. tuberculosis. Tuberculocidal activity was not observed with macrophage lysosomal fractions from non-vaccinated guinea pigs. The specific activities of enzymes in macrophage homogenates were tested. None of the lysosomal enzymes had significantly different activities in macrophages from vaccinated and non-vaccinated guinea pigs. Strains of M. tuberculosis were sensitive to reactive nitrogen intermediates (RNI). However, guinea pig alveolar macrophages did not generate RNI on infection with mycobacteria. Macrophages from BCG-vaccinated guinea pigs killed M. tuberculosis in the presence of an inhibitor of RNI synthesis. Thus, no evidence was gained to suggest that RNI are responsible for the tuberculocidal activity of guinea pig macrophages. Aminoaldehydes have been shown to be toxic to M. tuberculosis. Ornithine decarboxylase (ODC) is the first enzyme in the pathway that synthesises aminoaldehydes. ODC activity was not elevated in macrophages twenty-four hours and six days after BCG vaccination. Increased ODC activity was not observed in macrophages infected with M. bovis BCG for twenty-four hours. ODC is therefore not responsible for any prolonged increase in aminoaldehyde production that may take place when macrophages are infected with mycobacteria. Mycobacterium tuberculosis was passaged once through the mouse. After passaging, the mycobacteria exhibited increased resistance to hydrogen peroxide but not RNI. This indicates that hydrogen peroxide is generated during the murine immune response to mycobacteria. Culturing Mycobacterium smegmatis at pH5.0 allowed the bacteria to survive subsequent incubation at pH3.5 better than bacteria grown solely at pH7.6. Mycobacterium smegmatis could therefore be adapted to lethal pH values by pre-exposing the bacteria to mildly acidic conditions.
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SHANAHAN, Erin Rose. "Molecular investigation of mycobacterium tuberculosis and the critical cell wall lipase culp6." Thesis, The University of Sydney, 2014. http://hdl.handle.net/2123/10066.
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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.
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Zhou, Wei [Verfasser], and Andreas [Akademischer Betreuer] Burkovski. "Signal transduction in nitrogen control of Mycobacterium smegmatis / Wei Zhou. Gutachter: Andreas Burkovski." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2013. http://d-nb.info/107547471X/34.
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Gebhard, Susanne, and n/a. "The Phn and Pst systems of Mycobacterium smegmatis : phosphate transport and gene regulation." University of Otago. Department of Microbiology & Immunology, 2006. http://adt.otago.ac.nz./public/adt-NZDU20070502.112113.
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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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Tadepalli, Adilakshmi. "Studies on the role of salicylic acid in iron metabolism of Mycobacterium smegmatis." Thesis, University of Hull, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310316.
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REVEL, VIRAVAU VALERIE. "Quinolones et mycobacteries : identification, caracterisation biochimique et role de l'adn gyrase de mycobacterium smegmatis dans la resistance aux quinolones." Paris 6, 1996. http://www.theses.fr/1996PA066679.
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L'interet des fluoroquinolones dans le traitement des infections a mycobacteries s'est considerablement accru au cours de ces dernieres annees. Malheureusement, l'utilisation des fluoroquinolones a entraine l'emergence de mutants resistants a ces antibiotiques. Notre objectif general etait de determiner le role de l'adn gyrase dans la resistance acquise aux fluoroquinolones chez mycobacterium smegmatis. Nous avons selectionne des mutants de m. Smegmatis resistants a l'ofloxacine. Deux mutations ont ete caracterisees dans une region conservee du gene gyra, appelee region determinant la resistance aux quinolones, l'une conduisant a la substitution ala-83val, et l'autre a la substitution asp-87gly. Nous avons caracterise les genes de structure, gyra et gyrb, de l'adn gyrase de m. Smegmatis. L'enzyme a ete purifiee par chromatographie d'affinite a partir de la souche sauvage mc#2155. Les mesures d'inhibition par les quinolones (ci#5#0) montrent qu'il faut environ 90 fois plus de quinolones classiques que de fluoroquinolones pour inhiber l'activite gyrase, ce qui explique en partie les differences de sensibilite de m. Smegmatis qui est naturellement resistante aux quinolones classiques et sensible aux fluoroquinolones. Enfin, les adn gyrases purifiees a partir des souches de m. Smegmatis resistantes aux fluoroquinolones ont ete utilisees pour determiner les ci#5#0 de differents composes de type quinolone. Les mutations conferant la resistance aux fluoroquinolones entrainent une augmentation moyenne des ci#5#0 et 125 fois pour le mutant ponctuel 87, et de 160 fois pour le double mutant 83-87. De plus, les ci#5#0 mesurees sur les adn gyrases resistantes montrent que les 8-chloro,6-fluoroquinolones ont une activite d'inhibition elevee par rapport aux fluoroquinolones classiques (environ 66 fois plus actives). Les caracteristiques structurales de ces composes sont donc d'un interet majeur pour la conception de nouvelles molecules actives vis-a-vis des mycobacteries resistantes aux fluoroquinolones.
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Safavi-Khasraghi, Mitra. "EXPRESSION AND CHARACTERIZATION OF MYCOBACTERIUM PARATUBERCULOSIS 19KDA WITH POSTTRANSLATIONAL MODIFICATION." Master's thesis, University of Central Florida, 2006. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/3080.
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Despite the fact that E. coli supports limited posttranslational modification, this bacterium has been universally used as the expression system of choice. Expression of modified proteins in E. coli may lead to expression of recombinant proteins that lack essential immunomodulatory or catalytic components essentials for infectious processes. Previously in our laboratory, pMptb#28 plasmid containing a 4.8 kb insert from M. paratuberculosis has been identified which expressed 16 kDa recombinant protein in E. coli and 19 kDa recombinant protein in Mycobacterium smegmatis. The objective of this study is to identify the ORF sequence, investigate possible posttranslational modification and characterize the protein forms in the two hosts. Earlier in the study, the genome sequence for M. paratuberculosis was not available and therefore sequencing both the 5' and 3' ends of the 4.8 kb insert did not help in the identification of the ORF. However, unidirectional Exonuclease deletion resulted in identification of subclones containing possible ORF sequence. Later on, the publication of the M. paratuberculosis genome sequence along with BLAST analysis of sequences from the subclones resulted in the identification of 486 bp ORF with significant identity to that from M. tuberculosis and M. leprae. Cloning of the 486 ORF coding sequence in E. coli resulted in the expression of 16 kDa protein similar to the calculated predicted size of translated peptide. Cloning of the 486 bp ORF coding sequence in M. smegmatis resulted in the expression of 19 kDa protein similar to that from M. paratuberculosis. The 16/19 kDa forms of the same protein were verified using rabbit anti-M. paratuberculosis antibodies adsorbed in E. coli and M. smegmatis lysates. The size of the 19 kDa proteins was not reduced following treatment with deglycosylation enzymes in absence of any enzyme inhibitors. The 19 kDa product was confirmed not be a glycoprotein when failed to react with ConA stain. The 16/19 kDa forms of the protein were evaluated against T-lymphocytes from Crohn's disease patients and normal controls. T- proliferation assay included controls such as PHA and PPD from M. paratuberculosis. There was not a significant difference between the two forms of the protein (16/19 kDa) against T-cell response from both populations. Overall, the study identified the ORF of the 19 kDa non-glycoprotein from M. paratuberculosis. Moreover, this is the first study which reports that the zoonotic M. paratuberculosis supports posttranslational modification similar to M. tuberculosis and M. leprae pathogens. Although the posttranslational modification component in this 19 kDa nonglycoprotein did not affect T- cell response, the finding is significant toward glycoproteins from M. paratuberculosis and their role in the pathogenesis of this bacterial infection in animals and humans.M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular Biology and Microbiology
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Raphela, Mabule Lucas. "Targeted depletion of RibF, a putative bifunctional FAD synthetase/ flavokinase in Mycobacterium smegmatis using CRISPR interference." Master's thesis, Faculty of Health Sciences, 2020. http://hdl.handle.net/11427/32943.
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Tuberculosis (TB) is the leading killer globally owing to an infectious disease. There is consequently an urgent need to develop novel TB drugs and shorter regimens to treat the causative agent, Mycobacterium tuberculosis, an imperative which demands the identification of new drug targets in essential mycobacterial pathways. To that end, the work presented in this dissertation aimed to functionally characterize ribF, an essential gene in the mycobacterial riboflavin (RF; vitamin B2) biosynthetic pathway. Given the role of RF as a core component of the essential flavin cofactors, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), it was hypothesized that silencing ribF would disrupt the biosynthesis of all flavoproteins, crippling numerous (essential) processes within the organism. Moreover, based on previous observations in Bacillus subtilis, it was predicted that the mycobacterial ribF homolog might play a role in regulating the rib operon (comprising a cluster of RF pathway genes) – either directly by binding to the FMN riboswitch, or indirectly through the production of FMN from RF, in turn enabling riboswitch-mediated repression of downstream genes. CRISPR interference (CRISPRi) technology was used to generate an anhydrotetracycline (ATc)-inducible ribF hypomorph of M. smegmatis, a widely exploited mycobacterial model. Consistent with other organisms, ribF was shown to be essential for in vitro growth of M. smegmatis: CRISPRi-mediated depletion of ribF was bacteriostatic, resulting in a 10-fold growth inhibition in liquid media and corresponding to no reduction (0 log-fold change) in colony forming units (CFU). Moreover, targeted metabolomic analyses revealed that ribF depletion was associated with accumulation of 6,7-Dimethylribityllumazine (DMRL), suggesting that the disruption of RibF function blocked conversion of RF to the essential cofactors, FMN and FAD, in turn inhibiting cell growth. Notably, the lethality of ribF depletion could not be complemented chemically by exogenous supplementation of growth media with RF, FMN or FAD. Downregulation of ribF also caused enhanced susceptibility to the known cell wall-targeting agent, vancomycin, but not to the putative RibF domain inhibitor, thonzonium bromide, suggesting an alternative mechanism of action or impaired bacillary permeation. In summary, these data support the inferred essentiality of ribF in mycobacteria, in turn supporting future work which aims to target this enzyme for new TB drug discovery.
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OLIVEIRA, Renato Antonio dos Santos. "Efeito pós-antibiótico de um novo derivado 1,2,4-oxadiazol-hidrazida e suas associações frente ao Mycobacterium fortuitum e Mycobacterium smegmatis." Universidade Federal de Pernambuco, 2004. https://repositorio.ufpe.br/handle/123456789/1503.
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Previous issue date: 2004A freqüente resistência das micobactérias atípicas às drogas antituberculose, a ausência de esquemas terapêuticos eficazes e os estudos concernentes à aplicação de novas moléculas sobre micobactérias motivaram a determinação do efeito pós-antibiótico (EPA) in vitro de um novo derivado 1,2,4-oxadiazol-hidrazida (HHBA) e de suas associações com o quimioterápico ofloxacino (OFLO), uma fluorquinolona, e o m-Flúor-Fenilalanina (m-FFen), um inibidor da síntese da parede celular. O EPA foi avaliado após o tempo de contato de 2 e 4 horas frente ao Mycobacterium fortuitum e ao Mycobacterium smegmatis. As drogas foram testadas em concentrações equivalentes a concentração inibitória mínima (CIM). Considerado um parâmetro farmacodinâmico de grande relevância em situações clínicas, o EPA exerce a sua maior importância na escolha do antimicrobiano, nos regimes de dosagem terapêutica e nos seus intervalos de administração. O EPA pode ser induzido pela exposição do microrganismo ao antimicrobiano por um período de tempo determinado, seguindo da rápida retirada deste antimicrobiano, e o acompanhamento do recrescimento deste microrganismo. Matematicamente, o EPA é calculado pela fórmula: EPA= T C, em que T representa o tempo necessário para que a cultura teste cresça o equivalente a 1log10 em relação à numeração efetuada logo após a retirada da droga e C representa o tempo necessário para que a cultura controle cresça igualmente de 1log10. O que concerne ao efeito pós-antibiótico sobre o M. fortuitum após 2 horas de contato a OFLO apresentou os maiores valores de EPA, 8,60 ± 1,50 horas. Todas as associações mostraram-se antagônicas com exceção da HHBA+m-FFen que induziu a um EPA indiferente de 1,20 ± 1,20 horas. Após 4 horas de contato, a OFLO continuou apresentando a melhor atividade cujo EPA foi equivalente a 11,50 ± 2,00 horas, sua associação com a m-FFen apresentou um EPA indiferente de 11,00 ± 2,70 horas. As demais associações apresentaram-se antagônicas. O efeito pós-antibiótico do HHBA frente ao M. smegmatis após um contato de 2 horas foi de 2,40 ± 2,80 horas e a sua associação com a OFLO mostrou ser a mais efetiva cujo valor do EPA foi de 3,30± 2,80 horas. Após 4 horas de contato o EPA do HHBA não foi alterado, permanecendo em 3,30 ± 2,60 horas. O aumento do tempo de contato induziu um aumento dos valores do EPA para as drogas estudadas e suas associações, exceto o m-FFen, as associações OFLO-HHBA e OFLO-HHBA-m-FFen frente ao M. smegmatis, e o HHBA e sua associação HHBA-m-FFen, frente ao M. fortuitum. A ofloxacino foi o antimicrobiano que induziu os melhores valores de EPA para o M. fortuitum, bem como o HHBA foi o antimicrobiano que induziu os melhores valores de EPA para o M. smegmatis. O aumento do tempo de contato de 2 para 4 horas potencializou os EPAs das drogas estudadas.Todas as associações testadas apresentaram um caráter antagônico, exceto a associação OFLO-m-FFen após 4 horas de contato, frente o M. smegmatis. E os tempos de contatos escolhidos, bem como a CMI utilizada para os ensaios, foram insuficientes para que as drogas e suas associações pudessem induzir EPAs sinérgicos. Como os mecanismos do EPA ainda não estão totalmente definidos é difícil lançar hipóteses acerca do qual ou quais mecanismos estão implicados na variação dos resultados dos EPAs de drogas testadas em associação
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Bakala, n'goma Jean-claude. "Etude In vitro de Phospholipases mycobactériennes impliquées dans la virulence." Thesis, Aix-Marseille 2, 2010. http://www.theses.fr/2010AIX22027/document.
Full textAbstract:
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 culturePhospholipases, 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
40
Vijay, Srinivasan. "Ultrastructural and Molecular Analyses of the Unique Features of Cell Division in Mycobacterium Tuberculosis and Mycobacterium Smegmatis." Thesis, 2013. http://etd.iisc.ernet.in/2005/3403.
Full textAbstract:
The Mycobacterium genus contains major human pathogens, like Mycobacterium tuberculosis and Mycobacterium leprae, which are the causative agents of Tuberculosis and Leprosy, respectively. They have evolved as successful human pathogens by adapting to the adverse conditions prevailing inside the host, which include host immune activation, nutrient depletion, hypoxia, and so on. During such adaptation for the survival and establishment of persistent infection inside the host, the pathogen, like M. tuberculosis, regulates its cell division. It is known that M. tuberculosis enters a state of non-replicating persistence (NRP) inside the host, to establish latent infection, which helps the survival of the pathogen under adverse host conditions such as hypoxia and nutrient depletion. The pathogen can reactivate itself, to come out of the NRP state, and establish active infection at a later stage, when conditions are suitable for its proliferation. The altered physiological state of the latent bacterium makes it tolerant to drugs, which are only effective against proliferating tubercle bacilli. In view of this unique behavioural physiology of tubercle bacilli, it is important to study the process of cell division and how it is regulated in the NRP and actively growing states. The work reported in the thesis is an attempt to understand these aspects of mycobacterial cell division.
iii
Chapter 1. Introduction: This chapter gives a detailed introduction to bacterial cell division and its regulation in various organisms, like Escherichia coli, Bacillus subtilis, Caulobacter crescentus, and others. In the background of this information, the major studies on mycobacterial cell division and its regulation are presented.
Chapter 2. Materials and Methods: This chapter describes in detail all the materials and methods used in the experiments, which are presented in the four data chapters, 3-6.
Chapter 3. Ultrastructural Study of the Formation of Septal Partition and Constriction in Mycobacteria and Delineation of its Unique Features: Mycobacteria have triple-layered complex cell wall, playing an important role in its survival under adverse conditions in the host. It is not known how these layers in the mother cell participate during cell division. Therefore, the ultrastructural changes in the different envelope layers of Mycobacterium tuberculosis, Mycobacterium smegmatis, and Mycobacterium xenopi, during the process of septation and septal constriction, were studied, using Transmission and Scanning Electron Microscopy. The unique aspects of mycobacterial septation and constriction were identified and were compared with those of E. coli and Bacillus subtilis septation. Further, based on all these observations, models were proposed for septation in M. tuberculosis and M. smegmatis.
Chapter 4. Identification of Asymmetric Septation and Division in Mycobacteria and Its Role in Generating Cell Size Heterogeneity: Bacterial populations are known to harbour phenotypic heterogeneity that helps survival under stress conditions, as this heterogeneity comprises subpopulations that have differential susceptibility to stress conditions. The
iv
heterogeneity has been known to lead to the requirement for prolonged drug treatment for the elimination of the tolerant subpopulation. Hence, it is important to study the different mechanisms, which operate to generate population heterogeneity. Therefore, in this chapter, studies were carried out to find out whether asymmetric septation and division occur in mycobacteria to generate cell size heterogeneity. Subpopulations of mycobacterial mid-log phase cells of M. tuberculosis, M. smegmatis, and M. xenopi were found to undergo asymmetric division to generate cell size heterogeneity. The asymmetric division and the ultrastructure and growth features of the products of the division were studied.
Chapter 5. Study of Mycobacterial Cell Division Using Growth-Synchronised Cells: In this chapter, different stages of cell septation and constriction were studied using growth-synchronised M. smegmatis cells. Phenethyl alcohol (PEA), which has been found to reversibly arrest mycobacterial cells, was used for growth synchronisation. The growth-synchronised mycobacterial cells, which were released from PEA block, were studied at different stages of septation and septal constriction, at the ultrastructural and molecular levels.
Chapter 6. Identification of the Stage of Cell Division Arrest in NRP Mycobacteria: The exact stage at which the NRP tubercle bacilli are arrested in cell division is currently unknown. In Wayne’s in vitro model for hypoxia-responsive tubercle bacilli, gradual depletion of oxygen leads to hypoxic stress, inducing the bacilli to enter non-replicating persistence (NRP) state. Using this model, the stage of cell division arrest in M. tuberculosis was characterised at the ultrastructural and molecular levels. Hypoxia-stressed M. smegmatis was used as an experimental system for contrast.
The thesis concludes with salient findings, a bibliography, and the list of publications.
41
Vijay, Srinivasan. "Ultrastructural and Molecular Analyses of the Unique Features of Cell Division in Mycobacterium Tuberculosis and Mycobacterium Smegmatis." Thesis, 2013. http://etd.iisc.ac.in/handle/2005/3403.
Full textAbstract:
The Mycobacterium genus contains major human pathogens, like Mycobacterium tuberculosis and Mycobacterium leprae, which are the causative agents of Tuberculosis and Leprosy, respectively. They have evolved as successful human pathogens by adapting to the adverse conditions prevailing inside the host, which include host immune activation, nutrient depletion, hypoxia, and so on. During such adaptation for the survival and establishment of persistent infection inside the host, the pathogen, like M. tuberculosis, regulates its cell division. It is known that M. tuberculosis enters a state of non-replicating persistence (NRP) inside the host, to establish latent infection, which helps the survival of the pathogen under adverse host conditions such as hypoxia and nutrient depletion. The pathogen can reactivate itself, to come out of the NRP state, and establish active infection at a later stage, when conditions are suitable for its proliferation. The altered physiological state of the latent bacterium makes it tolerant to drugs, which are only effective against proliferating tubercle bacilli. In view of this unique behavioural physiology of tubercle bacilli, it is important to study the process of cell division and how it is regulated in the NRP and actively growing states. The work reported in the thesis is an attempt to understand these aspects of mycobacterial cell division.
iii
Chapter 1. Introduction: This chapter gives a detailed introduction to bacterial cell division and its regulation in various organisms, like Escherichia coli, Bacillus subtilis, Caulobacter crescentus, and others. In the background of this information, the major studies on mycobacterial cell division and its regulation are presented.
Chapter 2. Materials and Methods: This chapter describes in detail all the materials and methods used in the experiments, which are presented in the four data chapters, 3-6.
Chapter 3. Ultrastructural Study of the Formation of Septal Partition and Constriction in Mycobacteria and Delineation of its Unique Features: Mycobacteria have triple-layered complex cell wall, playing an important role in its survival under adverse conditions in the host. It is not known how these layers in the mother cell participate during cell division. Therefore, the ultrastructural changes in the different envelope layers of Mycobacterium tuberculosis, Mycobacterium smegmatis, and Mycobacterium xenopi, during the process of septation and septal constriction, were studied, using Transmission and Scanning Electron Microscopy. The unique aspects of mycobacterial septation and constriction were identified and were compared with those of E. coli and Bacillus subtilis septation. Further, based on all these observations, models were proposed for septation in M. tuberculosis and M. smegmatis.
Chapter 4. Identification of Asymmetric Septation and Division in Mycobacteria and Its Role in Generating Cell Size Heterogeneity: Bacterial populations are known to harbour phenotypic heterogeneity that helps survival under stress conditions, as this heterogeneity comprises subpopulations that have differential susceptibility to stress conditions. The
iv
heterogeneity has been known to lead to the requirement for prolonged drug treatment for the elimination of the tolerant subpopulation. Hence, it is important to study the different mechanisms, which operate to generate population heterogeneity. Therefore, in this chapter, studies were carried out to find out whether asymmetric septation and division occur in mycobacteria to generate cell size heterogeneity. Subpopulations of mycobacterial mid-log phase cells of M. tuberculosis, M. smegmatis, and M. xenopi were found to undergo asymmetric division to generate cell size heterogeneity. The asymmetric division and the ultrastructure and growth features of the products of the division were studied.
Chapter 5. Study of Mycobacterial Cell Division Using Growth-Synchronised Cells: In this chapter, different stages of cell septation and constriction were studied using growth-synchronised M. smegmatis cells. Phenethyl alcohol (PEA), which has been found to reversibly arrest mycobacterial cells, was used for growth synchronisation. The growth-synchronised mycobacterial cells, which were released from PEA block, were studied at different stages of septation and septal constriction, at the ultrastructural and molecular levels.
Chapter 6. Identification of the Stage of Cell Division Arrest in NRP Mycobacteria: The exact stage at which the NRP tubercle bacilli are arrested in cell division is currently unknown. In Wayne’s in vitro model for hypoxia-responsive tubercle bacilli, gradual depletion of oxygen leads to hypoxic stress, inducing the bacilli to enter non-replicating persistence (NRP) state. Using this model, the stage of cell division arrest in M. tuberculosis was characterised at the ultrastructural and molecular levels. Hypoxia-stressed M. smegmatis was used as an experimental system for contrast.
The thesis concludes with salient findings, a bibliography, and the list of publications.
42
Páleníková, Petra. "Regulace exprese Ms1, sRNA z Mycobacterium smegmatis." Master's thesis, 2016. http://www.nusl.cz/ntk/nusl-351430.
Full textAbstract:
Bacteria are exposed to various environmental conditions during their growth. They have to cope with rapid changes in temperature, lack of nutrition, etc. To survive, bacteria alter their gene expression. One type of regulation of gene expression is regulation by small RNAs (sRNAs). In bacteria, a well-studied sRNA is 6S RNA that binds to the RNA polymerase holoenzyme. However, 6S RNA has not been identified in several bacterial species. Mycobacteria are a genus that probably does not have 6S RNA. Instead, Mycobacterium smegmatis possess another sRNA - Ms1. Ms1 structurally resembles 6S RNA and indeed it was first identified as a 6S RNA structural homologue. However, Ms1 binds to RNAP devoid of any sigma factor, and, therefore, is significantly distinct from 6S RNA. This work describes regulation of expression of Ms1. DNA fragments of different length from the region upstream of the Ms1 gene were prepared. These fragments were fused to the lacZ reporter gene and their activity was tested in different growth phases and under stress. This allowed identification and characterization of the core promoter sequence and regulatory sequences that might interact with transcription factor(s). Promoter activity increased with increased length of the promoter fragment and after transition into stationary...
43
Roy, Siddhartha. "Structural Studies On Mycobacterium Smegmatis Dps Molecules." Thesis, 2007. https://etd.iisc.ac.in/handle/2005/642.
Full textAbstract:
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 arrangement of the protein molecules. However, contrary to earlier suggestions, the spaces need not to be between layers of the protein molecules. The cubic form provides an arrangement in which grooves, which could hold DNA molecules, criss-cross the crystal.
M. smegmatis Dps is characterised by a 26 residue C-terminal tail which has been shown to be involved in DNA binding. The protein spontaneously degrades into a species in which 16 C-terminal residues are cleaved away. This species does not bind DNA, but forms dodecamers. A second species in which all the 26 residues constituting the tail were deleted not only does not bind to DNA, but also fails to assemble into dodecamers, indicating a role in assembly also for the C terminal tail. Therefore, the crystal structure of the species without the entire C-terminal tail was carried out. The molecule of the C-terminal mutant has an unusual open decameric structure, resulting from the removal of two adjacent subunits from the original dodecameric structure of the native form. It has been earlier shown that a Dps dodecamer could assemble with a dimer or one of two trimers (Trimer-A and Trimer-B) as intermediate and that Trimer-A is the intermediate species in the M. smegmatis protein. Estimation of surface area buried on trimerisation indicates that association within Trimer-B is weak. It further weakens when the C-terminal tail is removed, leading to the disruption of the dodecameric structure. Thus, the C-terminal tail has a dual role, one in DNA binding and the other in the assembly of the dodecamer. M. smegmatis Dps also has a short N-terminal tail of 9 residues. A species with this tail deleted, forms trimers in solution, but not dodecamers unlike wild type M. smegmatis Dps, under the same conditions. The crystal structure of this N-terminal mutant was also determined. Unlike in solution, the N-terminal mutant forms dodecamers in the crystal. In native Dps, the N-terminal stretch of one subunit and the C-terminal stretch of a neighbouring subunit lock each other into ordered positions. The deletion of one stretch results in the disorder of the other. This disorder appears to result in the formation of a trimeric species of the N-terminal deletion mutant contrary to the indication provided by the native structure. The ferroxidation site is intact in the mutants.
A second DNA binding protein from stationary phase cells of M. smegmatis (MsDps2) has been identified from the bacterial genome and its crystal structure determined. The core dodecameric structure of MsDps2 is the same as that of the Dps from the organism described earlier (MsDps1). However, MsDps2 possesses a long N-terminal tail instead of the C-terminal tail in MsDps1. This tail appears to be involved in DNA binding. It is also intimately involved in stabilizing the dodecamer. Partly on account of this factor, MsDps2 assembles straightway into the dodecamer while MsDps1 does so on incubation after going through an intermediate trimeric stage. The ferroxidation centre is similar in the two proteins while the pores leading to it exhibit some difference. The mode of sequestration of DNA in the crystalline array of molecules, as evidenced by the crystal structures, appears to be different in MsDps1 and MsDps2, highlighting the variability in the mode of Dps-DNA complexation. A sequence search led to the identification of 300 Dps molecules in bacteria with known genome sequences. 50 bacteria contain 2 or more types of Dps molecules each, while 195 contain only one type. Some bacteria, notably some pathogenic ones, do not contain Dps. A sequence signature for Dps could also be derived from the analysis
In addition to the work on Dps molecules, the author was also involved in studies on the crystal structures of the adipic acid complexes of L- and DL-arginine and supramolecular association in arginine-dicarboxylic acid complexes. This investigation, carried out primarily to obtain a good grounding in crystallography, is presented in an appendix.
44
Roy, Siddhartha. "Structural Studies On Mycobacterium Smegmatis Dps Molecules." Thesis, 2007. http://hdl.handle.net/2005/642.
Full textAbstract:
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 arrangement of the protein molecules. However, contrary to earlier suggestions, the spaces need not to be between layers of the protein molecules. The cubic form provides an arrangement in which grooves, which could hold DNA molecules, criss-cross the crystal.
M. smegmatis Dps is characterised by a 26 residue C-terminal tail which has been shown to be involved in DNA binding. The protein spontaneously degrades into a species in which 16 C-terminal residues are cleaved away. This species does not bind DNA, but forms dodecamers. A second species in which all the 26 residues constituting the tail were deleted not only does not bind to DNA, but also fails to assemble into dodecamers, indicating a role in assembly also for the C terminal tail. Therefore, the crystal structure of the species without the entire C-terminal tail was carried out. The molecule of the C-terminal mutant has an unusual open decameric structure, resulting from the removal of two adjacent subunits from the original dodecameric structure of the native form. It has been earlier shown that a Dps dodecamer could assemble with a dimer or one of two trimers (Trimer-A and Trimer-B) as intermediate and that Trimer-A is the intermediate species in the M. smegmatis protein. Estimation of surface area buried on trimerisation indicates that association within Trimer-B is weak. It further weakens when the C-terminal tail is removed, leading to the disruption of the dodecameric structure. Thus, the C-terminal tail has a dual role, one in DNA binding and the other in the assembly of the dodecamer. M. smegmatis Dps also has a short N-terminal tail of 9 residues. A species with this tail deleted, forms trimers in solution, but not dodecamers unlike wild type M. smegmatis Dps, under the same conditions. The crystal structure of this N-terminal mutant was also determined. Unlike in solution, the N-terminal mutant forms dodecamers in the crystal. In native Dps, the N-terminal stretch of one subunit and the C-terminal stretch of a neighbouring subunit lock each other into ordered positions. The deletion of one stretch results in the disorder of the other. This disorder appears to result in the formation of a trimeric species of the N-terminal deletion mutant contrary to the indication provided by the native structure. The ferroxidation site is intact in the mutants.
A second DNA binding protein from stationary phase cells of M. smegmatis (MsDps2) has been identified from the bacterial genome and its crystal structure determined. The core dodecameric structure of MsDps2 is the same as that of the Dps from the organism described earlier (MsDps1). However, MsDps2 possesses a long N-terminal tail instead of the C-terminal tail in MsDps1. This tail appears to be involved in DNA binding. It is also intimately involved in stabilizing the dodecamer. Partly on account of this factor, MsDps2 assembles straightway into the dodecamer while MsDps1 does so on incubation after going through an intermediate trimeric stage. The ferroxidation centre is similar in the two proteins while the pores leading to it exhibit some difference. The mode of sequestration of DNA in the crystalline array of molecules, as evidenced by the crystal structures, appears to be different in MsDps1 and MsDps2, highlighting the variability in the mode of Dps-DNA complexation. A sequence search led to the identification of 300 Dps molecules in bacteria with known genome sequences. 50 bacteria contain 2 or more types of Dps molecules each, while 195 contain only one type. Some bacteria, notably some pathogenic ones, do not contain Dps. A sequence signature for Dps could also be derived from the analysis
In addition to the work on Dps molecules, the author was also involved in studies on the crystal structures of the adipic acid complexes of L- and DL-arginine and supramolecular association in arginine-dicarboxylic acid complexes. This investigation, carried out primarily to obtain a good grounding in crystallography, is presented in an appendix.
45
Mukherjee, Raju. "Cell Surface Of Mycobacterium Smegmatis At The Stationary Phase : Regulation Of Gene Expression." Thesis, 2007. https://etd.iisc.ac.in/handle/2005/510.
Full textAbstract:
Tuberculosis remains one of the oldest diseases known to mankind but still persists as a very major risk. Discovery of several antimycobacterials was marked by a steady decline in the active cases of pulmonary tuberculosis. However, in the recent past there has been a surge in its clinical incidence. The reasons for this failure are the emergence of multi drug resistance and the ability of the organism to survive under extreme condition or for a very long period of time known as ‘persistence’. The latter one established itself as a major hindrance in the effective control of tuberculosis. The latent bacilli are confined for a very long period after the infection in caseous cavities, called granulomma, inside the host and gets reactivated any time when the host becomes immuno-compromised. Latency has been successfully simulated in vitro by various models which mimic the in vivo condition by depleting O2 (Wayne, 1977), nutrients (Nyka, 1974) or the carbon source (Ojha et al., 2000).
Stationary phase is exemplary of a stage in bacterial growth where the organism is exposed to various stresses like nutrient starvation, accumulation of toxic metabolites, low temperature, high osmolarity and acidity to name a few. Some evidences suggest that cells survive in nutrient deprived stationary phase. The present investigation was pursued with an objective to further our understanding on the mechanism of adaptation that the persistent mycobacterium may undertake to survive during the stationary phase of growth. The fast growing M.smegmatis, a nonpathogenic member in the non-tuberculous genera, however, with a genetic and metabolic similarity to M.tuberculosis has been used as a model for this study.
Chapter 1 introduces the challenges in tuberculosis therapy and discusses the reason for drug tolerance and persistence of M.tuberculosis and M.avium complexes that were uncovered recently. It focuses on the intricate lipid rich cell wall which forms the first barrier for drug delivery with an emphasis on the cell surface antigenic glycolipids, the glycopeptidolipids. A detail account of their structure, biosynthetic pathway, intracellular function and their implications on biofilm formation has been provided.
The evolution of the genetic approaches currently used in mycobacterial research is highlighted.
The transcription apparatus and the regulation of gene expression in mycobacteria at different environmental condition and stages of growth are also discussed. The need for a detail investigation of the stationary phase RNAP in mycobacteria is stressed.
Chapter 2 observes the changes in the cell surface of M.smegmatis at different ambience of growth. It focuses on the composition of glycopeptidolipid, one of the non-covalently attached free lipids and the mycolic-acids which are covalently attached to the inner layer of the cell wall. Composition of the mycobacterial cell wall with respect to the glycopeptidolipids and mycolic acids during biofilm mode of growth is also addressed. This chapter examines the conditional synthesis of a novel class of polar glycopeptidolipid in carbon starved cultures of M.smegmatis and determines their molecular structure.
Chapter 3 revisits the biosynthetic pathway of the glycopeptidolipids and justifies a need for a fresh perspective. It identifies a glycosyltransferase responsible for the transfer of an extra rhamnose to the existing rhamnose linked to the terminal alaninol in the new polar glycopeptidolipid. This chapter also identifies a conserved Polyketide synthase in the glycopeptidolipid biosynthetic cluster. Characterization of the domains present in its two distinct modules with a correlation to the structure of the fatty acylchain together with the formation of a hydroxy fatty acyl chain is delineated.
Chapter 4 deals with the construction of a suicide vector which when recombines to the mc2155 genome, incorporates a hexa-histidine tag at the C’ of the β΄ subunit of the RNAP. It details the strategy for the construction of the strain and established the genetic exchange event both genotypically and phenotypically. A single step procedure for purification of the holo-RNAP is also described in this chapter.
In chapter 5 the role of the mycobacterial principal likes sigma factor, SigB, at the stationary phase of growth is highlighted. An approach of expression proteomics involving differential display of the total protein was undertaken to investigate the genes that are under the SigB regular during the stationary phase of growth. This chapter also examines the stationary phase induced changes in the RNAP. Various proteins that interact with the assembly are identified and their role in conferring rifampicin resistance to the RNAP is described.
Appendix 1 details the preparation of the partially methylated deoxy monosaccharide using the esoteric reactions of organic synthesis. They were used extensively for glycosyl linkage analysis of the glycopeptidolipids by mass spectrometry, where they acted as standards.
46
Mukherjee, Raju. "Cell Surface Of Mycobacterium Smegmatis At The Stationary Phase : Regulation Of Gene Expression." Thesis, 2007. http://hdl.handle.net/2005/510.
Full textAbstract:
Tuberculosis remains one of the oldest diseases known to mankind but still persists as a very major risk. Discovery of several antimycobacterials was marked by a steady decline in the active cases of pulmonary tuberculosis. However, in the recent past there has been a surge in its clinical incidence. The reasons for this failure are the emergence of multi drug resistance and the ability of the organism to survive under extreme condition or for a very long period of time known as ‘persistence’. The latter one established itself as a major hindrance in the effective control of tuberculosis. The latent bacilli are confined for a very long period after the infection in caseous cavities, called granulomma, inside the host and gets reactivated any time when the host becomes immuno-compromised. Latency has been successfully simulated in vitro by various models which mimic the in vivo condition by depleting O2 (Wayne, 1977), nutrients (Nyka, 1974) or the carbon source (Ojha et al., 2000).
Stationary phase is exemplary of a stage in bacterial growth where the organism is exposed to various stresses like nutrient starvation, accumulation of toxic metabolites, low temperature, high osmolarity and acidity to name a few. Some evidences suggest that cells survive in nutrient deprived stationary phase. The present investigation was pursued with an objective to further our understanding on the mechanism of adaptation that the persistent mycobacterium may undertake to survive during the stationary phase of growth. The fast growing M.smegmatis, a nonpathogenic member in the non-tuberculous genera, however, with a genetic and metabolic similarity to M.tuberculosis has been used as a model for this study.
Chapter 1 introduces the challenges in tuberculosis therapy and discusses the reason for drug tolerance and persistence of M.tuberculosis and M.avium complexes that were uncovered recently. It focuses on the intricate lipid rich cell wall which forms the first barrier for drug delivery with an emphasis on the cell surface antigenic glycolipids, the glycopeptidolipids. A detail account of their structure, biosynthetic pathway, intracellular function and their implications on biofilm formation has been provided.
The evolution of the genetic approaches currently used in mycobacterial research is highlighted.
The transcription apparatus and the regulation of gene expression in mycobacteria at different environmental condition and stages of growth are also discussed. The need for a detail investigation of the stationary phase RNAP in mycobacteria is stressed.
Chapter 2 observes the changes in the cell surface of M.smegmatis at different ambience of growth. It focuses on the composition of glycopeptidolipid, one of the non-covalently attached free lipids and the mycolic-acids which are covalently attached to the inner layer of the cell wall. Composition of the mycobacterial cell wall with respect to the glycopeptidolipids and mycolic acids during biofilm mode of growth is also addressed. This chapter examines the conditional synthesis of a novel class of polar glycopeptidolipid in carbon starved cultures of M.smegmatis and determines their molecular structure.
Chapter 3 revisits the biosynthetic pathway of the glycopeptidolipids and justifies a need for a fresh perspective. It identifies a glycosyltransferase responsible for the transfer of an extra rhamnose to the existing rhamnose linked to the terminal alaninol in the new polar glycopeptidolipid. This chapter also identifies a conserved Polyketide synthase in the glycopeptidolipid biosynthetic cluster. Characterization of the domains present in its two distinct modules with a correlation to the structure of the fatty acylchain together with the formation of a hydroxy fatty acyl chain is delineated.
Chapter 4 deals with the construction of a suicide vector which when recombines to the mc2155 genome, incorporates a hexa-histidine tag at the C’ of the β΄ subunit of the RNAP. It details the strategy for the construction of the strain and established the genetic exchange event both genotypically and phenotypically. A single step procedure for purification of the holo-RNAP is also described in this chapter.
In chapter 5 the role of the mycobacterial principal likes sigma factor, SigB, at the stationary phase of growth is highlighted. An approach of expression proteomics involving differential display of the total protein was undertaken to investigate the genes that are under the SigB regular during the stationary phase of growth. This chapter also examines the stationary phase induced changes in the RNAP. Various proteins that interact with the assembly are identified and their role in conferring rifampicin resistance to the RNAP is described.
Appendix 1 details the preparation of the partially methylated deoxy monosaccharide using the esoteric reactions of organic synthesis. They were used extensively for glycosyl linkage analysis of the glycopeptidolipids by mass spectrometry, where they acted as standards.
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Senzani, Sibusiso. "Analysis of peptidoglycan degrading amidases in Mycobacterium smegmatis." Thesis, 2014.
Find full textAbstract:
Tuberculosis (TB) is a worldwide pandemic, which claims approximately 2 million lives annually. Though treatable through chemotherapy, TB still causes 8-10 million new infections annually. The problem is further complicated by the emergence of multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) the causative agent of TB. The unabated spread of this disease and associated high levels of mortality has prompted the search for new drugs with novel modes of action. The peptidoglycan (PG) component of the cell wall of Mtb is an incredibly complex structure and has been the focus of antimicrobial development in other organisms. In this study, we characterize PG remodelling N-acetylmuramyl-L-alanine amidases (cell wall amidases) in Mycobacterium smegmatis (Msm), a model organism for TB research by gene knockout/knockdown. Cell wall amidases cleave the bond between the stem peptide and the glycan backbone in PG and have been shown to play an essential role in cell growth, cell signalling and antibiotic resistance in other organisms. Our bioinformatics analysis revealed that M. smegmatis encodes three possible amidase homologues designated ami1, ami2 and ami3. Deletion mutagenesis in Msm resulted in successful deletion of ami1 whilst repeated attempts to delete ami2 did not yield a knockout mutant, suggesting that ami2 is essential for growth. Deletion of ami1 results in the formation of long filaments consisting of 3 to 8 cells attached to each other due to incomplete resolution of the septum. In these filaments, lateral growth at both the internal septation sites and extreme poles resulted in irregular cell width, strongly implicating Ami1 in bacterial cell division, the maintenance of bacterial cell shape and possibly balancing the growth at the cell pole. Since deletion of ami2 was not possible, a knockdown strain allowing for anhydrotetracycline (ATC)-regulated conditional gene expression of ami2 was generated. In this system, withdrawal of ATC results in repression of expression. Expression analysis of ami2 in this strain revealed that
whilst significant gene knockdown was achieved, ami2 expression was not completely abolished in the absence of the inducer ATC, suggesting the presence of basal level ami2 expression. The repression of ami2 expression results in retarded growth, diminished motility, unusual colony morphology consisting of miniature colonies lacking any form of serpentine cording and the formation of miniature cells consisting of globular bulges. These data implicate Ami2 in bacterial growth and maintenance of bacterial cell shape. Collectively, our data comprise the first demonstration of an important role for peptidoglycan degrading amidases in mycobacterial growth and cell division. Furthermore, the phenotypic defects in colony formation due to deletion or depletion of amidases suggest that these enzymes play an important role in cell-cell communication during colony formation. These data validate this class of enzymes as an untapped, legitimate source of novel targets for anti-tubercular drug discovery.
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Pradeep, Kumar *. "DNA Repair In Mycobacteria." Thesis, 2005. https://etd.iisc.ac.in/handle/2005/2199.
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Pradeep, Kumar *. "DNA Repair In Mycobacteria." Thesis, 2005. http://etd.iisc.ernet.in/handle/2005/2199.
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Chowdhury, Rakhi Pait. "Newer Insights On Structure, Function And Regulation Of Dps Protein From Mycobacterium smegmatis." Thesis, 2009. https://etd.iisc.ac.in/handle/2005/970.
Full textAbstract:
The first chapter will provide an introduction to the physiology, pathogenesis and biology of mycobacteria. Host-pathogen interactions, different modes of resistance of the bacteria, adaptations for survival under nutrient and oxygen depleted conditions has been discussed. This is followed by a general discussion on gene expression and regulation in the microbe. The physiology of bacteria under stresses from the view of the transcriptional regulation of specific genes has also been discussed. The scope and objective of the present study in M. smegmatis covered in the thesis has been considered at the end. The next chapter discusses the characterization of msdps promoter in vivo with the help of reporter gene assay technologies. With the advent of promoterless E. coli-mycobacterium shuttle vectors, activity assays can be easily performed to characterize unknown upstream putative promoter sequences of genes. Both the 1 kb upstream as well as a 200bp upstream region of msdps gene has been characterized by. Primer extension analysis and subsequent site directed mutagenesis studies reveal +1 transcription start site and the promoter consensus sequence for the msdps gene respectively. Next chapter comprises of the method of constructing heterologous in vitro transcription machinery in mycobacteria. It is followed by characterization of transcription initiation at two dps promoters of M. smegmatis. A novel pull-down assay has been designed which enabled us to identify the sigma factors in the reconstituted RNA polymerases to be associated with the respective dps promoters and to compare the regulation of the two genes at transcription level. Further characterization through single round in vitro transcription at mycobacterial promoters has been attempted. The following two chapters provide some newer insights into the structure-function relationship of the first Dps molecule, MsDps (MsDps1) with respect to its DNA binding activity. The DNA binding activity is associated with the higher oligomeric form only. With the help of time resolved anisotropy and Förster Resonance Energy Transfer (FRET) experiments, we have monitored the nature of Dps dodecamer-DNA complex and mapped the distance between the N and C169 position in the absence and the presence of DNA. A new computational programme, Maximum Entropy Method (MEM) has been applied successfully to analyze data obtained from phase-modulation (Phi-M) lifetime experiments in order to get distribution of lifetime. In the last chapter a new method is adopted to predict amino acids important for stabilizing the interface in a trimeric structure. Subsequently, single and double amino acid mutants of the native MsDps protein has been constructed through site directed mutagenesis and are scored for the ability of the mutants to oligomerize under conditions similar to that of the native protein. This helped us to propose a hypothetical model of the overall mechanism of the protein oligomerization process in solution.