3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Structure-function studies of a putative ribonuclease HI from Mycobacterium tuberculosis.(1999) Thomsen, Michelle LesleyBacterial Ribonuclease HI, which ensures that initiation of DNA replication occurs at the unique oriC; locus, is encoded by rnhA. The rnhA gene from Mycobacterium smegmatis encodes a protein that is closely related to other bacterial RNases HI (Dawes et al., 1995). Activity gel analysis rletected RNase HI activity associated with proteins in whole-cell extracts of Mycobacterium tuberculosis in the 14-25 kDa size range. A putative rnhA homologue was identified in M. tuberculosis and sequence analysis revealed that the rnhA open reading frame contains an apparent fusion of two genes (Cole et al., 1998). The 5' -region of the ORF corresponds to an rnhA homologue, whereas the 3'-region contains a gene, annotated herein as pgm, which encodes a protein belonging to the phosphoglycerate mutase (POM) family of proteins. The full-length ORF, as well as the individual mhA and pgm segments, were cloned into the pMAL-c2 expression vector and recombinant proteins were overexpresssed in E. coli as maliose binding fusion proteins. Recombinant proteins were purified and rabbit polyclonal antisera raised against each one were used to probe whole cell extracts of M. tuberculosis. Cross-reaction with polypeptides of unknown identity was observed. Limited proteolysis of the recombinant proteins suggest an instability of folding in E. coli. Functional investigation of the M. tuberculosis RNase HI included complementation of an E. coli RNase HI-defective mutant, and an M. smegmatis strain carrying a defective rnhA allele integrated at its rnhA locus, with M. tuberculosis rhns-pgm supplied in trans. No complementation in either hosts was observed. Upon completion of the genome sequence of H37Rv (Cole et al., 1998), it became apparent that the rnh/i-pgm ORF is the fourth gene in an operon which includes a gene known to be involved in cobalamin biosynthesis. Significant homlogy of the PGM to CobC phosphatase of Salmonella typhimurium implicates a role for rnh/: pgm in the cobalamin biosynthetic pathway of M. tuberculosis.Item Polymorphism and structural studies of isoniazid derivatives(2015-05-21) Hean, DuaneCrystal polymorphism is the capacity of a solid crystalline form to exist in more than one structural arrangement. In the pharmaceutical setting investigations into the polymorphic forms of potential drugs are of vital importance since different crystalline forms can affect bioavailability, mechanical, thermal, and chemical properties. One such example is isonicotinic acid-(1-phenylethylidene) hydrazide (IPH), a derivative of the popular drug isoniazid (used as first line treatment against Mycobacterium tuberculosis) was found to crystallise in six different polymorphic forms. Each crystal structure was determined using X-ray diffraction techniques and including the thermal phase relationships of the polymorphic compound were delineated. In addition to polymorph elucidation, isonicotinic acid-(1-phenylethylidene) hydrazide was modified with –OH and –NH2 at various aromatic positions, creating geometric pyridyl isomers. In-depth studies of these pyridyl isomers revealed a diverse range of supramolecular aggregates. Preliminary thermal screening suggests that only a small selection of these pyridyl isomers present potential polymorphic activity for further study.Item Molecular mechanisms of transport and metabolism of vitamin B12 in mycobacteria(2013-02-01) Moosa, AticaMycobacterium tuberculosis (MTB) encodes three enzymes that are dependent on vitamin B12–derived cofactors for activity, including a B12-dependent methionine synthase (MetH). Previously, work in the Molecular Mycobacteriology Research Unit (MMRU) demonstrated vitamin B12 auxotrophy in a mutant strain disrupted in the alternative, B12-independent methionine synthase, MetE. This observation established the ability of MTB to transport corrinoids despite the absence of an identifiable B12-specific transporter. In addition, it suggested that MTB does not synthesize vitamin B12 in vitro. Notably, bioinformatic analyses identified PPE2 as the only B12-related transport candidate in MTB, though as a putative B12-regulated cobalt transporter. PPE2 is unusual in possessing directly upstream of its predicted start codon one of only two B12-dependent riboswitches in the MTB genome, and it lies in a putative operon with B12 biosynthetic genes, cobU and cobQ1. In this study, the possibility that PPE2 functions in the transport of vitamin B12 or cobalt was investigated. Transcriptional and phenotypic data suggested that PPE2 was not involved in B12 transport. Instead, it was shown that cobalt can supplement the growth of an MTB metE mutant in liquid medium, strongly supporting the ability of MTB to synthesize B12 de novo. Moreover, the ability to utilise exogenous cobalt was dependent on functional PPE2, thereby establishing a role for a PPE-family member in cobalt assimilation in MTB. Vitamin B12 comprises a central corrin ring co-ordinated to 5,6-dimethylbenzimidazole (DMB) as α-axial ligand. Substituting DMB with adenine yields the alternate form, pseudo-B12. The ability of mycobacteria to utilize pseudo-B12 precursors (cobinamide and adenine) to support full function of B12-dependent metabolic pathways was evaluated. Although the pseudo-B12 precursors appeared to complement chemically the mycobacterial B12 auxotrophs, growth of the mutants on cobinamide alone complicated this interpretation. To address this limitation, DMB synthesis was targeted by disrupting the MTB bluB homologue, Rv0306. Neither site-directed mutagenesis of key Rv0306 residues, nor full-gene deletion was sufficient to eliminate growth on cobinamide. Instead, this observation highlights the need to establish biochemically the nature of the active B12 form synthesized and utilized by MTB under different conditions. In combination, the results presented here support the inferred flexibility of vitamin B12 biosynthesis in MTB, and reinforce the potential role of B12-dependent metabolism in mycobacterial pathogenesis.