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dc.contributor.advisorCook, Gregory
dc.contributor.advisorBerney, Michael
dc.contributor.advisorMcLellan, Alexander
dc.contributor.authorEkanayaka, Nandula Vidumini
dc.date.available2013-02-24T22:10:00Z
dc.date.copyright2013
dc.identifier.citationEkanayaka, N. V. (2013). Growth and Energetics of Cholesterol Utilisation by Mycobacterium smegmatis mc2155 (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/3743en
dc.identifier.urihttp://hdl.handle.net/10523/3743
dc.description.abstractMembers of the genus Mycobacterium are able to degrade cholesterol and for pathogenic members like Mycobacterium tuberculosis this degradation has been implicated in persistence in the lungs of the chronically infected host. The physiological role of cholesterol and the enzymes involved in its breakdown by mycobacteria requires further investigation. The aims of this thesis were to determine how Mycobacterium smegmatis degrades cholesterol and identify the genetic components involved in this degradation. To address this goal, I studied the transcriptional response of M. smegmatis mc2155 to cholesterol (compared to glycerol) at very low growth rate (i.e. ~ 69 h doubling time) using continuous culture and microarray analysis. M. smegmatis was able to utilise cholesterol as a sole carbon and energy source at low growth rate. During growth in continuous culture, 75 µM cholesterol remained in the medium suggesting that M. smegmatis did not use an active transport system to accumulate cholesterol from this medium. This was confirmed by transport studies using [4-14C]-cholesterol. Microarray analysis revealed 243 genes upregulated and 269 genes downregulated when grown on cholesterol compared to glycerol (p-value ≤ 0.1). The majority of these genes encoded hypothetical proteins. Genes implicated in a cholesterol degradation pathway were significantly upregulated (p-value ≤ 0.1). Clusters of genes in the KstR and KstR2 lipid degradation regulons were upregulated along with four lipid-transfer proteins some of which have previously been shown to be essential for intracellular survival of M. tuberculosis. Comparing the cholesterol and hypoxia transcriptomes revealed a 35 % overlap, but also a large cholesterol-specific set of genes were present and are collectively designated as the cholesterol specific transcriptome in this study. The expression of a putative lysine exporter (lysE; MSMEG_0467) was upregulated 28-fold along with the downregulation (50-fold) of an enzyme involved in lysine transamination (L-lysine-ε-aminotransferase; MSMEG_1764) indicating accumulation and export of lysine during growth on cholesterol. We propose that lysine export may represent a novel “overflow” metabolism during growth on cholesterol as a ∆lysE mutant of M. smegmatis showed impaired growth on cholesterol and other fatty acids, but not on glycerol or glucose (Berney, Ekanayaka and Cook, unpublished data). During growth on cholesterol, there was a concurrent induction of the glyoxylate shunt, the methylcitrate cycle and the methylmalonate pathway, which results in an increased flux towards succinate and this was reflected by the significant upregulation of one of the two annotated succinate dehydrogenase gene clusters (Sdh2) of M. smegmatis. Whereas the second annotated succinate dehydrogenase operon (Sdh1) was significantly downregulated. Sdh1 was encoded in an operon of five genes (MSMEG_0420 to 0416) and Sdh2 was encoded in an operon of four genes (MSMEG_1672 to 1669) as confirmed by RT-PCR. Using 5’RACE analysis the transcriptional start sites and putative –10 and –35 promoter regions of Sdh1 and Sdh2 were identified. Both –10 regions showed similarity to the Pribnow box consensus sequence (TATAAT) whereas the –35 regions displayed poor matches to known consensus sequences. To characterise the promoter regions and identify key promoter elements of Sdh1 and Sdh2, a series of sdh-lacZ fusions were constructed. Important promoter regions of 10 bp and 43 bp for Sdh1 and Sdh2, respectively, located about 300 bp upstream of the transcriptional start sites were identified to be required for maximal expression of both operons. Five consecutive base pairs (CACCG) of similar sequence were present in both these identified DNA regions. Sdh1 and Sdh2 did not exhibit classic catabolite repression in the presence of glucose in the growth medium. Successful deletion of the entire Sdh1 operon was achieved with no phenotypic differences from the wild-type observed. Moreover, the levels of succinate dehydrogenase activity in cell-free crude cell lysates of the Sdh1 mutant were comparable to wild-type levels. No deletion mutants of Sdh2 could be achieved suggesting it may be essential for growth. Future work will focus on determining the role of Sdh2 in mycobacterial growth on cholesterol and energetics.
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectMycobacteria
dc.subjectcholesterol
dc.subjectsuccinate dehydrogenase
dc.subjectcontinuous culture
dc.subjectmicroarray
dc.titleGrowth and Energetics of Cholesterol Utilisation by Mycobacterium smegmatis mc2155
dc.typeThesis
dc.date.updated2013-02-23T08:45:59Z
dc.language.rfc3066en
thesis.degree.disciplineMicrobiology and Immunology
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.interloanyes
otago.openaccessAbstract Only
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