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Living organisms employ different kinds of mechanisms, to regulate the functions of genes or their products, which may help in maintaining homeostasis inside the cell or may help in fighting hostile environment in the case of pathogenic organisms. These mechanisms act at the transcriptional, post-transcriptional, translational, and post-translational levels. In order to understand the physiology of an organism, it is essential to obtain an in-depth knowledge of such mechanisms, in which several proteins participate in interlinked pathways. In this regard, the present study focuses on two such proteins: (i). the newly identified Fic (Filamentation induced by cAMP) protein; and (ii). NDK (Nucleoside Diphosphate Kinase), which had been studied for decades. Fic protein and NDK share several common features: (i). both use nucleoside triphosphate (NTPs) or nucleoside diphosphate (NDPs) or their derivatives as one of their substrates; (ii). they have been found to be involved in diverse cellular pathways, involving different types of substrates that form the second substrate of these proteins; (iii). both are ubiquitously present in all the living organisms - from bacteria to humans to plants. However, there is very little information on these proteins from mycobacterial systems, which include some major human pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, which are the causative agents of Tuberculosis and Leprosy, respectively. In view of these reasons, in the present study, the structural and/or functional features of the Fic and NDK proteins from Mycobacterium tuberculosis, were analysed, as it might be of medical significance for effectively combating the pathogen. The Chapter 1 of the thesis contains the Introduction to the research work and Chapter 2 is on the overall Materials and Methods. The remaining chapters pertain to the data obtained on the structural and/or functional features of the Fic and NDK proteins from Mycobacterium tuberculosis. Chapter 3. Cloning, Expression and Purification of Mycobacterium tuberculosis Fic The role of FIC (Filamentation induced by cAMP) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylylation), in microorganisms, higher eukaryotes, and plants is emerging. In order to understand the biological role of FIC domain containing proteins in mycobacteria, the gene for the FIC domain containing protein of the human pathogen, Mycobacterium tuberculosis, MtuFic, was cloned, overexpressed, purified to homogeneity, and biochemically characterised. Neither the His-tagged nor the GST-tagged MtuFic protein, overexpressed in Escherichia coli, nor expression of Mtufic in Mycobacterium smegmatis, yielded the protein in the soluble fraction. However, the maltose binding protein (MBP) tagged MtuFic (MBP-MtuFic) could be obtained partly in the soluble fraction. Denatured-refolded protein was used for the antibody generation in mice and rabbit. The cellular localisation and secretion of MtuFic were characterised using the antibody. Chapter 4. Biochemical Characterisation of Mycobacterium tuberculosis Fic Sequence alignment with several FIC motif containing proteins, complemented with homology modeling on the FIC motif containing protein, VbhT of Bartonella schoenbuchensis as the template, showed conservation and interaction of residues constituting the FIC domain. MtuFic, possesses the critical His144 residue, in the characteristic FIC Motif, HPFREGNGRSTR (HPFxxGNGRxxR), spanning 144th to 155th residue. Site-specific mutagenesis of the His144, or Glu148, or Asn150 of the FIC motif, or of Arg87 residue that constitutes the FIC domain, or complete deletion of the FIC motif, abolished the NTP to NMP conversion activity. The activity of MtuFic was consistent with the biochemical activities hitherto reported for a variety of bacterial FIC domain containing proteins. Studies were also carried out on NMPylylation in the presence of eukaryotic proteins and eukaryotic and mycobacterial cell lysates. Although formation of NMPs from NTPs mediated by MBP-MtuFic could be detected, we could not identify any protein as the target substrate either in the human macrophage (THP1) cells or in the M. tuberculosis cells. VopSΔ30 (kind gift from Dr. Kim Orth), along with human G proteins as targets, were used as the positive controls. Various possibilities for the inability to detect a protein target substrate are discussed. Chapter 5. Transcriptional Analysis of Mycobacterium tuberculosis fic Gene (Mtufic) In parallel, in order to understand the transcriptional regulation of Mtufic, primer extension analysis was carried out. The Transcription Start Site (TSS; +1 site) of Mtufic were mapped under different growth/stress conditions, which tubercle bacilli encounter in human host. Mtufic got expressed mainly through two transcripts, T1 and T2, arising from two different transcription start sites (TSS). Putative promoter regions were cloned in a promoter probe vector, which expresses a GFP protein of very high intensity, in order to qualitatively detect the activity of the promoters. The half-life of the gfp mRNA was determined to be 4 min and therefore justifiably quantitated the Mtufic promoter activity by determining the gfp mRNA levels. The levels of Mtufic mRNA were two-fold higher under nutrient-depleted stationary phase of growth, as compared to the levels at mid-log phase. The activity of P1 and P2, as quantitated real-time using the short half-life gfpm2+ mRNA levels in Mycobacterium smegmatis transformants, showed that the activity of P2 was upregulated two-fold under nutrient-depleted stationary phase of growth, while that of P1 remained unaltered while of P1 and P2 were low under hypoxia. Co-transcription of Mtufic, with the immediate upstream gene, Rv3642c, of unknown function, was observed. Taken together, the data strongly indicated that the expression of Mtufic gets altered under nutrient-depleted and hypoxic conditions, which are the stress conditions experienced by tubercle bacilli in granuloma in tuberculosis patients. Chapter 6. Functional Characterisation of Mycobacterial FtsZ-NDK Interaction During the past few decades, our laboratory has been carrying out extensive molecular and functional studies on the cytokinetic protein, FtsZ, of different mycobacterial species, and of a variety of other mycobacterial proteins that are believed to be interacting with the cell division machinery. In this regard, in parallel to the work on MtuFic, we carried out work on the identification and characterisation of the proteins that interact with mycobacterial FtsZ. In this context, we found for the first time that the nucleoside diphosphate kinase (NDK), which can generate NTPs from ATP/GTP and NDPs, interacts with FtsZ and that the interaction was conserved across several mycobacterial species. Therefore, the FtsZ-NDK interaction was extensively characterised in vitro, using the recombinant, purified FtsZ and NDK proteins from different mycobacterial species. This novel finding on the interaction of NDK with FtsZ adds another role to NDK, namely in bacterial cell division.

Two mechanisms, intrinsic and factor-dependent, have evolved for accomplishing the termination of transcription in eubacteria. In this thesis, the first chapter is an introduction to the topic that presents what is known about the mechanisms of termination. The properties of the primary and secondary ‘players’- intrinsic terminators, Rho protein, rho-dependent terminators, RNA polymerse and Nus factors - are presented and the known mechanisms by which termination functions are discussed. In Chapter 2, a detailed analysis of intrinsic terminators – their differential distribution, similarity and divergence - has been penned. The database, compiled using the program GeSTer (Genome Scanner for Terminators), comprises ~2000 sequences and is one of the largest of its kind. Furthermore, analyzing the data from over 700 bacteria reveals how different species have fine-tuned intrinsic terminators to suit their cellular needs. Non-canonical intrinsic terminators emerge to be a significant fraction of the observed structures. The conserved structural features of identified intrinsic terminators are discussed and the relationship between the two modes of termination is assessed. Chapter 3 deals with the importance of transcription termination in regulating horizontally acquired DNA. The results show that genomic islands are scarce in intrinsic terminators and thus constitute most likely sites for Rho-dependent termination. Plausible reasons for why such a scenario has evolved are discussed and a generally applicable model is presented. Chapters 4 and 5 focus on Rho protein from Mycobacterium tuberculosis. In silico identification of M. tuberculosisgenes that rely on MtbRho-dependent termination is followed by experimental validation. The data show that Rho-dependent termination is the predominant mechanism in this species.MtbRho is a majorly expressed protein that governs termination of protein-coding and non-protein coding genes. Further, MtbRho can productively interact with RNA that has considerable secondary structure. Such interactions cause conformational changes in the enzyme. Given that MtbRho has to function with a GC-rich transcriptome, the altered properties could have evolved for optimal function. Taken together, the thesis extends our current understanding of both modes of termination. The importance of non-canonical intrinsic terminators in mycobacteria and other organisms is discussed. The unusual function of Rho and its predominant role in mycobacteria is elucidated. Finally, the inter-relationship between the two modes of termination is also discussed.

Protein synthesis is a process by which organisms manufacture their proteins that perform various cellular activities either alone or in combination with other similar or different molecules. In eubacteria, protein synthesis proceeds at a rate of around 15 amino acids per second. The ribosomes, charged tRNAs and mRNAs can be considered as the core components of protein synthesis system which, in addition, involves a panel of non-ribosomal proteins that regulate the speed, specificity and accuracy of the process. Peptidyl-tRNA hydrolase (Pth) and ribosome recycling factor (RRF) are two such non-ribosomal proteins involved in protein synthesis. These two proteins are essential for eubacterial survival and the work reported in this thesis involves structural characterization of these two proteins from the bacterial pathogen, Mycobacterium tuberculosis. The protein structures were solved using established techniques of protein crystallography. Hanging drop vapour diffusion method and crystallization under oil using microbatch plates were the methods employed for protein crystallization. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator in all the cases. The data were processed using DENZO and MOSFLM. The structures were solved by molecular replacement method using the program PHASER. Structure refinements were carried out using programs CNS and REFMAC. Model building was carried out using COOT. PROCHECK, ALIGN, CHIMERA, and PYMOL were used for structure validation and analysis of the refined structures. Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA that has dropped off from ribosome before reaching the stop codon, in order to avoid the toxicity resulting from peptidyl-tRNA accumulation and to free the tRNA to make it available for further rounds in protein synthesis. To begin with, the structure of the enzyme from M. tuberculosis (MtPth) was determined in three crystal forms. This structure and the structure of the same enzyme from Escherichia coli (EcPth) in its crystal differ substantially on account of the binding of the C-terminus of the E.coli enzyme to the peptide binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body of the molecule and a polypeptide stretch involving a loop and a helix. This stretch is in open conformation when the enzyme is in the free state as in the crystals of MtPth. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the EcPth crystal mimics the peptide-bound conformation of the enzyme. The peptide stretch involving a loop and a helix, referred to earlier, now closes on the bound peptide. Concurrently, a gate connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of the two residues. Thus, the crystal structure of MtPth when compared with that of EcPth, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. A discrepancy between the X-ray results and NMR results, which subsequently became available, led to X-ray studies on new crystal forms of the enzyme. The results of these studies and those of the enzyme from different sources that became available, confirmed the connection deduced previously between the closure of the lid at the peptide-binding site and the opening of the gate that separates the peptide-binding site and tRNA binding site. The plasticity of the molecule indicated by X-ray structures is in general agreement with that deduced from the available solution NMR results. The correlation between the lid and the gate movement is not, however, observed in the NMR structure of MtPth. The discrepancy between the X-ray and NMR structures of MtPth in relation to the functionally important plasticity of the molecule, referred to earlier, also led to molecular dynamics simulations. The X-ray and the NMR studies along with the simulations indicated an inverse correlation between crowding and molecular volume. A detailed comparison of proteins for which X-ray and the NMR structures are available appears to confirm this correlation. In consonance with the reported results of the investigation in cellular components and aqueous solutions, the comparison indicates that the crowding results in compaction of the molecule as well as change in its shape, which could specifically involve regions of the molecule important for function. Crowding could thus influence the action of proteins through modulation of the functionally important plasticity of the molecule. After termination of protein synthesis at the stop codon, the ribosome remains as a post-termination complex (PoTC), consisting of the 30S and the 50S subunits, mRNA and a deacylated tRNA. This complex has to be disassembled so that the ribosome is available for the next round of translation initiation. Ribosome recycling factor (RRF) binds to ribosome and in concert with elongation factor G (EF.G), performs the recycling of ribosome that results in disassembly of PoTC. The structure of this L-shaped protein with two domains connected by a hinge, from Mycobacterium tuberculosis (MtRRF) was solved previously in our laboratory. The relative movement of domains lies at the heart of RRF function. Three salt bridges were hypothesized to reduce the flexibility of MtRRF when compared to the protein from E.coli (EcRRF), which has only one such salt bridge. Out of these three bridges, two are between domain 1 and domain 2, whereas the third is between the hinge region and the C-terminus of the molecule. These salt bridges were disrupted with appropriate mutations and the structure and activity of the mutants and their ability to complement EcRRF were explored. An inactive C-terminal deletion mutant of MtRRF was also studied. Major, but different, structural changes were observed in the C-terminal deletion mutant and the mutant involving the hinge region. Unlike the wild type protein and the other mutants, the hinge mutant complements EcRRF. This appears to result from the increased mobility of the domains in the mutant, as evidenced by the results of librational analysis. In addition to the work on PTH and RRF, the author was involved during the period of studentship in carrying out X-ray studies of crystalline complexes involving amino acids and carboxylic acids, which is described in the Appendix of the thesis. The complexes studied are that of tartaric acid with arginine and lysine.

The treatment of tuberculosis (TB) is currently a challenge due to multi- and extensively drug resistant strains of Mycobacterium tuberculosis. Mycobacterium bovis and M. tuberculosis cause clinically indistinguishable tuberculosis in humans. Both M. bovis and M. tuberculosis have been isolated from humans and animals. Plant species contain antimicrobial compounds that may lead to new anti-TB drugs. To conduct in vitro antimycobacterial assays, it is important to include current clinical isolates as new strains of bacteria might be circulating under the ongoing climate change environment. The overall goal and objectives of this study were to isolate and characterize mycobacteria species from South Africa, to test some selected plant species of the Anacardiaceae family for antimycobacterial activity using some of the newly isolated and reference strains of mycobacteria followed by cytotoxicity evaluation of the most active plant species, and finally the isolation and characterization of at least one compound from the most active and least toxic plant. This study led to the discovery of a new isolate of Mycobacterium Avium Complex species from black wildebeest. Other non-tuberculous mycobacteria and M. bovis isolates were identified from other animal species. Five out of 15 plant species screened showed good activity against Mycobacterium species. Five antimycobacterial compounds were isolated from Searsia undulata, the most active plant species. Two out of the five compounds were identified, and one compound appears to be novel, but both compounds have been isolated for the first time from Searsia undulata. An incidental finding was the potential anticancer property of extracts of Searsia undulata. Recommended future activities include isolation and identification of more active compounds from Searsia undulata which were visible in bioautography analysis, as well as synergy evaluation of antimycobacterial activities of the different compounds with current anti-tubercular drugs. / Environmental Sciences / Ph. D. (Environmental Science)