In der vorliegenden Arbeit wurden neue Untersuchungsmöglichkeiten mit dem BacT/Alert 3D Mykobakterien-Testsystem erprobt. Erstens wurden Untersuchungen durchgeführt, um die Testkonzentrationen für Protionamid (PTH) und Linezolid (LIZ) für die standardmäßige Empfindlichkeitstestung von M. tuberculosis (Mtb) mit dem BacT/Alert 3D-System festzulegen. Dazu wurden die MHK-Werte für 32 Mtb-Stämme bestimmt: Referenzstamm Mtb H37Rv, sensible Patientenstämme, Patientenstämme mit verschiedenen Resistenzen (u. a. PTH-Resistenz) sowie eigens für die Arbeit isolierte LIZ-resistente Mutanten. Die PTH-MHK betrug für 20 von 21 sensiblen Mtb-Stämmen einschließlich des Referenzstammes Mtb H37Rv 0,125 - 1 mg/l (0,25 mg/l bei 11 von 21 Stämmen). Lediglich ein Stamm mit Resistenz gegenüber Isoniazid, Ethambutol und Streptomycin fiel mit einer etwas erhöhten PTH-MHK von 2 mg/l auf. Sechs PTH-resistente Stämme (z. T. mit anderen Resistenzen gegenüber Erstrang-Antituberkulotika) zeigten PTH-MHK von 4 - 16 mg/l. Die Gruppen der PTH-sensiblen und resistenten Stämme zeigten ein bimodales Verteilungsmuster, das mit einem Schwellenwert von 2 mg PTH/l gut zu differenzieren ist. Für die standardmäßige Durchführung der Empfindlichkeitstestung gegenüber PTH mit dem BacT/Alert 3D-System empfehlen wir deshalb eine PTH-Testkonzentration von 2 mg/l. Die LIZ-MHK betrug für 20 sensible Mtb-Stämme (inklusive Referenzstamm Mtb H37Rv) und sieben Stämme mit verschiedenen Resistenzen gegenüber Erstrang-Antituberkulotika 0,25 - 2 mg/l (0,5 mg/l bei 17 von 27 Stämmen). Für die vier isolierten LIZ-resistenten Mutanten betrug die LIZ-MHK 8 - 16 mg/l. Es zeigt sich auch bei der Verteilung der LIZ-MHK ein bimodales Verteilungsmuster; die Gruppen der sensiblen und resistenten Stämme sind gut zu differenzieren. Wir empfehlen für die standardmäßige Durchführung der Empfindlichkeitstestung gegenüber LIZ mit dem BacT/Alert 3D-System eine LIZ-Testkonzentration von 4 mg/l. Die festgestellten MHK-Werte von PTH und LIZ und die vorgeschlagenen Testkonzentrationen entsprechen Ergebnissen aus der Literatur, die mit ähnlichen Methoden erhoben wurden. Zweitens wurden mit dem BacT/Alert 3D-System Untersuchungen zur Kombinationstestung von Antituberkulotika bei Mtb und Stämmen des MAC-Komplexes durchgeführt, bisher liegen keine Publikationen für Untersuchungen von Wirkstoff-Kombinationen bei Mykobakterien mit diesem System vor. Es wurde geprüft, ob die MHK eines Antituberkulotikums durch die Zugabe einer subinhibitorischen Menge eines anderen Antituberkulotikums verändert wird. Bei Mtb wurden dazu folgende Kombinationen geprüft: Rifampicin (RMP) + LIZ, Moxifloxacin + LIZ, Isoniazid + PTH, RMP + PTH, PTH + LIZ. In keinem Fall konnten signifikante Effekte beobachtet werden. Ein tendenziell synergistischer Effekt der PTH-RMP-Kombination beim Stamm Mtb H37Rv (Reduktion der RMP-MHK um eine Stufe) wurde durch die Analyse der Wachstumskinetik des Stammes unterstützt. Bei zufällig ausgewählten Stämmen des MAC-Komplexes wurde die Kombination Ciprofloxacin (CIP) + Ethambutol (EMB) geprüft. Es zeigte sich bei sieben von zehn Stämmen eine Reduzierung der CIP-MHK um mindestens drei Stufen bei Zugabe einer subinhibitorischen Konzentration von EMB. Dieser synergistische Effekt wurde bereits in den 1990er Jahren mit einer ähnlichen Methode festgestellt, allerdings ohne die Stämme des MAC-Komplexes zu differenzieren (Arbeitsgruppe von S. Hoffner). Interessanterweise handelte es sich bei den von uns untersuchten Stämmen, bei denen dieser synergistische Effekt nachgewiesen wurde, um M. avium-Stämme. Diese Problematik sollte weiter verfolgt werden, da sich daraus Konsequenzen für die Therapieempfehlung ergeben könnten.

Emergence of drug resistance is a major problem in the treatment of many diseases including tuberculosis. To tackle the problem, it is essential to obtain a global perspective of the molecular mechanisms by which bacteria acquire drug resistance. Systems biology approaches therefore become necessary. This work aims to understand pathways to drug resistance and strategies for inhibition of the resistant strains by using a combination of experimental genomics and computational molecular systems approaches. Laboratory evolution of Mycobacterium smegmatis MC2 155 by treatment with isoniazid (INH), a front-line anti-tubercular drug, resulted in a drug-resistant strain (4XR), capable of growth even at about 10-times the minimum inhibitory concentration of the drug. Whole genome sequence of the 4XR was determined, which indicated only 31 variations in the whole genome, including 3 point mutations, 17 indels and 11 frame-shifts. Two mutations were in proteins required for the pharmacological action of the drug, albeit in regions distant from the drug binding site. The variations however were insufficient to explain the observed resistance to isoniazid. For a better understanding of the global changes associated with drug resistance, whole genome-wide gene expression data was obtained for the resistant strain and compared with that of the WT strain. 716 genes were found to be differentially regulated in 4XR, spanning different biochemical, signaling and regulatory pathways. From this, some explanations for the emergence of drug resistance were obtained, such as the up-regulation of the enzymes in the mycolic acid biosynthesis pathway and also of the drug efflux pumps. In addition, enrichment analysis indicated that up-regulated genes belong to functional categories of response to stress, carbohydrate metabolism, oxidation-reduction process, ion transport, signaling as well as lipid metabolism. The differential gene regulations seemed to be partially responsible for conferring the phenotype to the organism. Alterations in the metabolic pathways in 4XR were characterized using the phenotypic microarray technology, which experimentally scanned the respiratory ability of the resistant bacteria under 280 different nutrient conditions and 96 different inhibitors. Phenotypic gain, where the resistant strain grows significantly better than the wild type and phenotypic loss, where the growth of the resistant strain is compromised as compared to the sensitive strains were derived from the comparison of the phenotypic responses. Differences in survival ability and growth rates in different nutrient sources in the resistant phenotype as compared to the wild type were observed, suggesting rewiring in the metabolic network of the drug-resistant strain. In particular, the pathways of central carbon metabolism and amino acid biosynthesis exhibit significant differences. The strain-specific metabolic pathway differences may guide in devising strategies to tackle the drug-resistant strains selectively and in a rational manner. Scanning electron microscopy indicated the morphology of the drug-resistant strains to be significantly altered, as compared to the control drug-sensitive strain. It is well-known that isoniazid acts by inhibiting mycolic acid biosynthesis. The pathway turns out to be a target for many other anti-tubercular drugs also, since mycolic acids are major components of the cell wall. It is therefore important to understand what changes occur in the mycolic acid and the associated pathways in the drug-resistant variety so that strategies to tackle the latter can be chosen more judiciously. The lipidome of the cell wall was therefore quantitatively characterized by mass spectrometric analyses, which indeed confirmed that the 4XR strain has a significantly different composition profile. Among the six categories of lipids, the members of the glycerophospolipids category were abundant while the fatty acyls, polyketides and saccharolipids were lower in the 4XR strain as compared to the WT. The lipidomic data derived from the cell wall of INH-resistant strain shows that it results in the mycolic acid pathway function restoration, which would otherwise be lost upon drug exposure in the sensitive strain. Understanding the precise changes that occur in the lipidome in the drug-resistant strains is expected to be useful in developing new ways to tackle resistance. Next, to understand the implications of altered gene expression profiles, protein-protein interaction networks are constructed at a genome-scale that captures various structural and functional associations mediated by proteins in the mycobacterial cell. Using transcriptome data of 4XR, a response network is computed. Using an algorithm previously developed in the laboratory, the networks have been mined to identify highest differential activity paths and possible mechanisms that are deployed by the cells leading to drug resistance. Known resistance mechanisms such as efflux, cytochromes, SOS, are all seen to constitute the highest activities for achieving drug resistance in 4XR. Interestingly, such paths are seen to form a well-connected subnet, indicating such differential activities to be orchestrated. This clearly shows that multiple mechanisms are simultaneously active in the 4XR and may together generate drug resistance. Mechanisms of detoxification and antioxidant responses are seen to predominate in the 4XR subnet. Overall the analysis provides a shortlist of strategies for targeting the drug resistant strain. Next, the phenotypic microarray platform was used for screening for growth in Msm in the presence of various drugs. Data analysis and clustering resulted in identification of conditions that lead to phenotypic gain or loss in the 4XR as well as those that lead to differential susceptibility to various drugs. Drugs such as cephalosporins, tobramycin, aminotriazole, phenylarsine oxide, vancomycin and oxycarboxin were also found to inhibit growth in the resistant strain selectively. In other words, the 4XR is found to be collaterally sensitive to these drugs. The top-net formed by the highest differential activity paths, identified from the network described earlier has already indicated the involvement of proteins that generate antioxidant responses. Insights from the two methods, first from the targeted approach and second, from the phenotypic discovery approach were combined together to select only those compounds to which the 4XR strain was collaterally sensitive and targeted proteins responsible for antioxidant responses. These compounds are vancomycin, phenylarsine oxide, ebselen and clofazimine. These were further tested against the virulent M. tuberculosis H37Rv strain in a collaborator‘s laboratory. 3 of these compounds such as vancomycin, ebselen and phenylarsine oxide were found to be highly active in combinations with isoniazid against all tested Mtb strains, showed high levels of inhibition against H37Rv and 3 different single drug resistant, MDR and XDR strains. Moreover, they were observed to be highly potent when given in combinations. Clofazimine on the other hand, in combination with isoniazid showed activity but no significant synergy in the virulent drug-resistant strains of M. tuberculosis though synergistic to the sensitive strain. Thus, experiments with M. tuberculosis provide empirical proof that four different compounds, all capable of blocking antioxidant responses, are capable of inhibiting growth of single-, multiple- and extremely-drug-resistant clinical isolates of M. tuberculosis. Using transcriptome data from literature for M. tuberculosis exposed to six different drugs, similar drug specific response networks were constructed. These networks indicate differences in the cellular response to different drugs. Interestingly, the analysis suggests that different drug targets and hence different drugs could trigger drug resistance to various extents, leading to the possibility of prioritizing drug targets based on their resistance evolvability. An earlier study from the laboratory suggested the concept of target-co-target pairs, where-in the co-target could be a key protein in mediating drug resistance for that particular drug and hence for its target protein. Top ranked hubs in multiple drug specific networks such as PolA, FadD1, CydA, a monoxygenase and GltS, can possibly serve as co-targets. Simultaneous inhibition of the co-target along with the primary target could lower the chances of emergence of drug resistance. Such analyses of drug specific networks provide insights about possible routes of communication in the cell leading to drug resistance and strategies to inhibit such communication to retard emergence of drug resistance. Since mutations in the target proteins are known to form an important mechanism by which resistant strains emerge, an understanding of the nature of mutations in different drug targets and how they achieve resistance is crucial. Sequence as well as structural bases for the resistance from known drug-resistant mutants in different drug targets is deciphered and then positions amenable to such mutations are predicted in each target. Mutational indices of individual residues in each target structure are computed based on sequence conservation. Saturated mutagenesis is performed in silico and structural stability analysis of the target proteins has been carried out. Critical insights were obtained in terms of which amino acid positions are prone to acquiring mutations. This in turn suggests interactions that are not desirable, thus can be translated into guidelines for modifying the existing drugs as well as for designing new drugs. Finally, the work presented here describes application of the systems biology approaches to understand the underlying mechanisms of drug resistance, which has provided insights for drug discovery on multiple fronts though target identification, target prioritization and identification of co-targets. In particular, the work has led to a rational exploration of collateral drug sensitivity and cross-resistance of the drug-resistant strain to other compounds. Combinations of such compounds with isoniazid were first identified in the M. smegmatis model system and later tested to hold good for the virulent M. tuberculosis strain, in a collaborative study. The combinations were found to be active against three different clinical drug-resistant isolates of M. tuberculosis. Therefore, this study not only reveals the global view of resistance mechanisms but also identifies synergistic combinations of promising drug candidates based on the learnt mechanisms, demonstrating a possible route to exploring drug repurposing. The combinations are seen to work at a much reduced dosage as compared to the conventional tuberculosis drug regimens, indicating that the toxicity and any associated adverse effects may be greatly reduced, suggesting that the combinations may have a high chance to succeed in the next steps of the drug discovery pipeline.

M. tuberculosis (Mtb) senses and responds to changes in its environment primar-ily through two-component signalling systems (TCSs). Each TCS contains a trans-membrane histidine kinase (HK ) protein and a cytoplasmic response regulator (RR) protein. HK detects a stimulus and gets phosphorylated. It then binds and transfers the phosphoryl group to the RR of the same TCS. Activated RR then triggers gene ex-pression, including upregulation of the HK and RR involved, eliciting responses that are essential for the bacterium to adapt. Though di erent TCSs detect distinct stimuli, the binding regions of the HK s and RRs share signi cant similarity. This raises the possibil-ity of crosstalk, where HK s dissipate signals to RRs that do not belong to the same TCS. Studies have argued that such dissipation of signals impairs the fitness of the organism, as it decreases the output levels as well as triggers unwanted responses. In contrast, a recent experimental study has discovered that TCSs of Mtb share extensive crosstalk, violating the widely accepted specificity paradigm. In this study, we have attempted to unravel the evolutionary underpinnings of this extensive crosstalk observed in Mtb. We hypothesised that such crosstalk may be advantageous in programmed environments, where there are well-defined sequences of stimuli. In such situations, crosstalk can up-regulate HK s and RRs of non-cognate TCSs. This up-regulation primes the latter TCSs for upcoming signals, increasing their sensitivity. We constructed a mechanistic model of the functioning of TCSs and a fitness variable to qualitatively measure the response of a TCS to a signal, to test the hypothesis. We performed population genetics simulations of the evolution of phenotypes of different crosstalk patterns. We found that in a random environment, the phenotype without any crosstalk is selected over time, which is in agreement with prevalent arguments in favour of specificity of TCSs. But when the environment is programmed, the phenotype with a crosstalk pattern mirroring the pattern of stimuli dominates the population. Finally, we found evidence for the evolutionary preference to preserve crosstalk in gene sequences of HK s and RRs encoded in Mtb. We found that the binding domains of HK s and RRs, which were predicted to share crosstalk, are under greater pressure to be similar than those domains which do not crosstalk. Our study thus provides a plausible explanation of the unexpected presence of crosstalk in Mtb. Since these cross-interactions aid the pathogen to adapt in the host, inhibitors of such interactions are likely to have therapeutic potential.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, has long been the scourge of humanity, claiming millions of lives. It is the most devastating infectious disease of the world in terms of mortality as well as morbidity (WHO, 2009). The lack of a uniformly effective vaccine against TB, the development of resistance in the Mycobacterium tuberculosis against the present antitubercular drugs and its synergy with AIDS has made the situation very alarming. This therefore necessitates a search for new antitubercular drugs as well as the identification of new and unexplored drug targets (Broun et aI., 1992). Coenzyme A is an essential cofactor for all organisms and is synthesized in organisms from pantothenate by a universally conserved pathway (Spry et al., 2008; Sassetti and Rubin, 2003). The first enzyme of the pathway, pantothenate kinase catalyzes the most important step of the biosynthetic process, being the first committed step of CoA biosynthesis and the one at which all the regulation takes place (Gerdes et aI., 2002) This thesis describes the successful cloning of PanK from Mycobacterium tuberculosis, its expression in E. coli, single step affinity purification, and complete biochemical and biophysical characterization. In this work, pantothenol, a widely believed inhibitor of pantothenate kinase, has been shown to act as a substrate for the mycobacterial pantothenate kinase. Further it was shown that the product, 4'phosphopantothenol, thus formed, inhibited the next step of the CoA biosynthesis pathway in vitro. The study was extended to find outthe fate of pantothenol inside the cell and it was demonstrated that the CoA biosynthetic enzymes metabolized the latter into the pantothenol derivative of CoA which then gets incorporated into acyl carrier protein. Lastly, it was decisively shown that pantothenate kinase is not only regulated by feedback inhibition by CoA but, also regulated through feed forward stimulation by Fructose 1, 6 biphosphate (FBP), a glycolytic intermediate. The binding site of FBP was determined by docking and mutational studies of MtPanK. Chapter 1 presents a brief survey of the literature related to Coenzyme A biosynthesis pathway and describes the objective of the thesis. It also presents a history of TB and briefly reviews literature describing TB as well as the life cycle, biology, survival strategy, mode of infection and the metabolic pathways operational in the TB parasite, Mycobacterium tuberculosis. The chapter details the enzymes involved in CoA biosynthesis pathway from various organims. Chapter 2 In this chapter, cloning of the ORF (Rv1092c), annotated as pantothenate kinase in the Tuberculist database (http://genolist.pasteur.frfTubercuList), its expression in E. coli and purification using affinity chromatography has been described. Protein identity was confirmed by MALDI-TOF and by its ability to complement the pantothenate kinase temperature sensitive mutant, DV70. This chapter also illustrates the oligomeric status of MtPanK in solution and describes the biochemical characterization of MtPanK by means of two different methods, spectrophotometrically by a coupled assay and calorimetrically by using Isothermal Titration Calorimetry. Feedback inhibition of MtPanK by CoA is also discussed in this chapter. Chapter 3 describes the biophysical characterization of MtPanK. It discusses the enthalpy (~H) and free energy change (~G) accompanying the binding of a non-hydrolysable analogue of ATP; CoA; acetyl CoA and malonyl-CoA to MtPanK. The chapter details the energetics observed upon ATP binding to pantothenate-saturated MtPanK further elucidating the order of the reaction. This chapter also describes the various strategies which were designed and tested to remove CoA from the enzyme as the latter is always purified from the cell in conjunction with CoA. Validation of these strategies for complete CoA removal (by studying the n value from ITC studies) is further described. Chapter 4 discusses the interaction of the well-studied inhibitor of pantothenate kinases from other sources (e.g. the malarial parasite), pantothenol, with the mycobacterial enzyme. In order to investigate the interaction of this compound with MtPanK, its effect on the kinetic reaction carried out by the enzyme was studied by several methods. Surprisingly, a new band corresponding to 4'phosphopantothenol appeared when the reaction mix of MtPanK with pantothenol and ATP was separated on TLC. The identity of the new spot was confirmed by mass spectrometry analyses of the MtPanK reaction mixture.. These findings established the fact that pantothenol is a substrate of pantothenate kinase. To delve deeper into the mechanism of interaction of this compound with the enzymes of the coenzyme A biosynthesis pathway, the ability of pantothenol to serve as a substrate for the next step of the pathway, MtCoaBC was studied. Using various approaches it was established that pantothenol is actually a substrate for the MtPanK and the inhibition observed earlier (Saliba et aI., 2005) is actually due to the inability of CoaBC to utilize 4' -phosphopantothenol as substrate. Chapter 5 takes the story from Chapter 4 further detailing the effects of pantothenol on cultures of E. coli and M. smegmatis. I observed that pantothenol does not inhibit the culture of E. coli or M. smegmatis. So, further studies were carried out to know the fate of pantothenol once it is converted into 4'phosphopantothenoi. Since, the next enzyme of the pathway does not utilize 4'phosphopantothenol, I checked the further downstream enzyme of the pathway, CoaD, and found that it converts 4'-phosphopantothenol to thepantothenol derivative of dephospho-CoA. The next enzyme of the pathway, CoaE, took up this pantothenol derivative of dephospho-CoA as a substrate and converted it to the pantothenol derivative of CoA which was then transferred to apo-ACP by holo-ACP synthase. The holo-ACP thus synthesized enters into the dedicated pathway of fatty acid synthesis. Extensive investigations have been carried out on the regulation of pantothenate kinases, by the product of the pathway, Coenzyme A and its thioesters, xx establishing the latter as the feedback regulators of these enzymes. In order to determine if the cell employs mechanisms to sense available carbon sources and consequently modulate its coenzyme A levels by regulating activity of the enzymes involvedin CoA biosynthesis, glycolytic intermediates were tested against MtPanK for their possible role in the regulation of MtPanK activity. Chapter 6 details my identification of a novel regulator of MtPanK activity, fructose-I, 6-bisphosphate (FBP), a glycolytic intermediate, which enhances the MtPanK catalyzed phosphorylation of pantothenate by three fold. Further, the possible mechanisms through which FBP mediates MtPanK activation are also discussed. This chapter also describes the experiments carried out to identify the binding site of FBP on MtPariK.Interestingly, docking of FBP on MtPanK revealed that FBP binds close to the ATP binding site on the enzyme with one of its phosphates overlapping with the 3'~phosphate of CoA thereby validating its competitive binding relative to CoA on MtPanK. Based on these observations I propose that the binding of FBP to MtPanK lowers the activation energy of pantothenate phosphorylation by PanK. Chapter 7 presents a summary of the findings of this work. Coenzyme A biosynthesis pathway harbors immense potential in the development of drug against many communicable diseases, thanks to its essentiality for the pathogens and the differences between the pathogen and host CoA biosynthetic enzymes. The work done in this thesis extensively characterizes the first committed enzyme of the CoA biosynthetic pathway, pantothenate kinase, from Mycobacterium tuberculosis (MtPanK). The thesis also deals with the fate of a known inhibitor of PanK and proves it as a substrate for MtPanK. Finally this thesis describes a new link between glycolysis and CoA biosynthesis. Biotin, like coenzyme A, is another essential cofactor required by several enzymes in critical metabolic pathways. De novo synthesis of this critical metabolite has been reported only in plants and microorganisms. Therefore targeting the synthesis of biotin in the tubercular pathogen is another effective means of handicapping the tubercle pathogen. During the course of my studies, I also investigated the mycobacterial biotin biosynthesis pathway, studying the first enzyme of the pathway, 7-keto-8-aminopelargonic acid (KAPA) synthase (bioF) in extensive detail. Appendix 1 elucidates the kinetic properties of 7-keto-8aminopelargonic acid synthase (bioF) from Mycobacterium tuberculosis and proves beyond doubt that D-alanine which has previously been reported to act as a competitive inhibitor for the B. sphaericus enzyme (Ploux et al., 1999), is actually a substrate for the mycobacterial bioF.

Homologous recombination (HR) is a highly conserved cellular process involved in the maintenance of chromosomal integrity and generation of genetic diversity. Biochemical and genetic studies have suggested that HR is crucial for repair of damaged DNA arising from various endogenous or exogenous assaults on the genome of any organism. Further, HR is vital to repair fatal DNA damage during DNA replication. An instructive example of cross-talk between the processes of DNA recombination and replication can be construed in the processing of replication/recombination/repair intermediates. The impediment(s) to the progression of DNA replication fork is one of the underlying causes for increased genome instability and consequently this might compromise the survival of organism. Various processes manifest at stalled replication forks before they can be rendered competent for the replication-restart. One of the mechanisms of replication-restart involves replication fork reversal (RFR), which envisage unwinding of the blocked forks with simultaneous annealing of the parental and daughter strands o generate a Holliday junction intermediate adjacent to DNA double strand end. Genetic evidence shows that in E. coli dnaEts mutant, holD mutant and in helicase defective rep mutant, RFR is catalyzed by RuvAB complex. Classically, HJ intermediates are generated during the terminal stages of the HR pathway. In E. coli, branch migration and resolution of HJ intermediates is promoted by RuvA, RuvB and RuvC proteins, which participate at the late stages of HR. Structural, biochemical and mutational analysis suggest that E. coli RuvA binds Holliday junction DNA with high affinity and specificity. RuvB, a member of the AAA+ (ATPase associated with various cellular activities) family, is recruited to the RuvA-Holliday junction complex and functions as a motor protein. Together, RuvA and RuvB catalyze ATP dependent branch migration of HJ. The resolution of HJ is catalyzed by the RuvC endonuclease, which introduces coordinated cuts at two symmetrical sites across the junction. RuvAB complex, the Holliday junction branch migration apparatus, is ubiquitous in bacteria. Genetic, biochemical and structural studies have not only established the in vivo role of E. coli RuvAB, in context of HR pathway, but have also provided valuable insights into the mechanism of HJ processing by RuvAB complex. However, the paucity of extensive studies examining the biochemical properties of each member of the RuvABC protein complex restricts models in deciphering the functions of the individual components of this tripartite protein complex. Our current understanding of the biochemical function of E. coli RuvA is within the context of its interacting cellular partner, RuvB. Consequently, the inherent activities of RuvA in the context of DNA repair and HR are poorly understood. Moreover, it remains to be ascertained if RuvABC protein complex, its different sub-complexes, or the individual subunits can function differently in the processing of HJ intermediates generated during DNA repair and HR. The information from these studies would be helpful in understanding the mechanistic details of HR pathway in mycobacteria. Additionally, a number of important questions regarding the molecular basis of RuvAB catalyzed fork reversal remain unanswered. Therefore, exploration of biochemical details of the RuvAB mediated RFR would provide mechanistic insights into the dynamics of fork reversal process. Moreover, analysis of RuvAB catalyzed RFR might be helpful in validating the different assumptions of the RFR model that has been proposed on the basis of genetic analysis of certain E. coli replication mutants. Another interesting question that remains to be answered is, how under in vivo conditions, RuvABC protein complex or its individual subunits are regulated to function differently in the context of HR and DNA repair? Mycobacterium tuberculosis is an important intracellular pathogen which is likely to experience substantial DNA damage inside the host and thus may require an efficient DNA recombination and repair machinery for its survival. Our knowledge about the mechanistic aspects of genetic exchange in mycobacteria is rather limited. Therefore, understanding of the processes catalyzed by the components of HR pathway may help in molecular genetic analysis of mycobacteria. Sequence analysis of M. tuberculosis genome, followed by various comparative genomic studies, has revealed the presence of putative homologs of E. coli rec genes but it is not known whether these gene products are able to catalyze the reactions similar to their E. coli counterparts. In M. tuberculosis, the genes encoding for the enzymatic machinery required for branch migration and resolution of HJ intermediates are present. The ruvA, ruvB and ruvC genes form an operon, and are probably translationally coupled. Further, these ruv genes are DNA damage inducible. The transcript level of ruvC is regulated by both RecA dependent and independent mechanisms whereas ruvA and ruvB are induced only through RecA dependent SOS response. During M. tuberculosis infection of host cells, expression of ruvA and ruvB genes is upregulated. We therefore surmise that their gene product might be required for DNA replication, recombination or repair, and would be physiologically relevant under in vivo conditions. However, the details of reactions involved in the processing of HR intermediates and rescue of stalled replication forks in M. tuberculosis remains unknown. In the initial part of this study, we have investigated the function of M. tuberculosis RuvA protein using Holliday junctions containing either homologous or heterologous core. In the later part, we have explored the ability of M. tuberculosis RuvA and RuvB proteins to catalyze in vitro replication fork reversal. M. tuberculosis ruvA gene was isolated by PCR amplification and cloned in an expression vector to generate the pMTRA construct. Genetic complementation assays, using the pMTRA construct transformed into E. coli ΔruvA mutant, indicated that M. tuberculosis ruvA is functional in E. coli and suggested that it can substitute for E. coli RuvA in conferring resistance to MMS and survival following UV irradiation. Having established the functionality of M.tuberculosis ruvA, a method was developed for heterologous over-expression and purification of M. tuberculosis RuvA protein (MtRuvA). MtRuvA was purified to homogeneity and the identity of purified protein was verified using western blot analysis using the anti-MtRuvA antibodies. Purified MtRuvA was free of any contaminating endo- or exo-nuclease activity. Biochemical functions of MtRuvA were defined by performing detailed investigations of DNA-binding and Holliday junction processing activities. Substrate specificity of purified MtRuvA was examined,through DNA binding assays, by using oligonucleotide substrates mimicking differentintermediates involved in the pathway of recombinational DNA repair. Purified M. tuberculosis RuvA exhibited high affinity for HJ substrate but also formed stable complex with replication fork and flap substrate. DNase I footprinting of MtRuvA-homologous Holliday junction complex confirmed that MtRuvA bound at the junction center. The DNase I protection conferred by MtRuvA, on homologous HJ, was two-fold symmetric; the continuous footprint was 10 bp longon one pair of symmetrical arms and 7 bp on the opposite pair of arms. In parallel, DNase footprinting of MtRuvA-heterologous Holliday junction complex generated a footprint that encompassed 16 nucleotide residues on each strand of the Holliday junction. Different crystallographic studies have envisaged an important role for RuvA in base pair rearrangement atthe center of the junction. Also, in crystal structure of tetramer of EcRuvA-HJ complex twobases at the junction center were unpaired. To explore if RuvA binding leads to helical distortionof Holliday junction, MtRuvA-HJ complexes were subjected to chemical probing with KMnO4.In case of heterologous HJ, binding of MtRuvA resulted in appearance of sensitive T residues at the junction crossover. By contrast, binding of MtRuvA to homologous HJ rendered the T residues at the junction center and within the homologous core sensitive to oxidation by KMnO4.Taken together, these observations suggested that binding of MtRuvA distorts two base pairs at the junction crossover in heterologous HJ, whereas in case of homologous HJ base pairs distortion extends into the arms of the junction. These observations with KMnO4 probing were independently validated, in real time, by using sensitive to 2-aminopurine fluorescence spectroscopy measurements of MtRuvA-HJ complexes. To follow structural distortions upon interaction with MtRuvA, HJ variants carrying 2-AP substitution were generated for both homologous and heterologous HJ substrate. In each junction species, the 2-AP residue was uniquely present either at the junction center, adjacent to the center or away from the center. Incase of heterologous HJ, binding of MtRuvA resulted in increase of fluorescence emission of2-AP residues located at the junction crossover but not those of 2-AP residues that were present1-2 base pairs away from the junction center. Binding of MtRuvA to homologous HJ resulted in increase of fluorescence emission of 2-AP residues located at the junction crossover. Further, increase in fluorescence emission was also observed for 2-AP residues present within the homologous core or adjacent to the homologous core in a pair of symmetrically related arms. Thus, 2-AP fluorescence results suggested that binding of MtRuvA to homologous HJ causes base pair distortion within and adjacent to the homologous core whereas in case of heterologous HJ the base pair distortion is restricted to the junction center. Together, these results suggest thatMtRuvA causes two distinct types of base pair distortions between homologous and heterologous HJ substrates. To explore the relationship between binding of MtRuvA and alterations in global structure of the junction DNA, we employed the established technique of comparative gel electrophoresis. Analysis of data from comparative gel electrophoresis revealed that MtRuvA, upon binding to the Holliday junctions, converts the stacked-X structure of HJ to square-planar form and stabilizes the same for loading of RuvB rings and subsequent branch migration by RuvAB complex. Our results underline the possible existence of distinct pathways for RuvA function, which presumably depend on the structure and the nature of the DNA repair or HR intermediates. In summary, our results show that binding of MtRuvA to the HJ induced changes in the local conformation of junction, which might augment RuvB catalyzed branch migration. An unexpected finding is the observation that MtRuvA causes two distinct types of structural distortions, depending on whether the Holliday junction contains homologous or heterologous core. These observations support models wherein RuvA facilitates, in a manner independent of RuvB, base pair rearrangements at the crossover point of both homologous and heterologous Holliday junctions. Although the genetic basis of ruvA ruvB catalyzed RFR in E. coli has been understood in some detail but less is known about the genetic and molecular mechanism of fork reversal in mycobacteria or other organisms. Specifically, to examine if the E. coli paradigm can be generalized to other RuvAB orthologs, we explored the RFR activity of M. tuberculosis RuvAB using a series of oligonucleotides and plasmid-based substrates that mimic stalled replication fork intermediates. This approach might be useful in genetic analysis of factors involved in processing of stalled forks in M. tuberculosis wherein technical difficulties associated with the isolation and characterization of appropriate mutants have limited our understanding of DNA metabolism. Importantly, we have asked the questions as to how the structure at fork junction, extent of reversal and presence of sequence heterology might determine the outcome of RuvAB mediated RFR. The results from this study will be helpful in consolidating the proposed in vivo role for RuvAB complex in fork reversal. The open reading frame corresponding to M. tuberculosis ruvB gene was PCR amplified and cloned in an expression vector to generate the pMTRB construct. Genetic complementation assays were performed to assess the functionality of M. tuberculosis ruvB in E. coli ΔruvB mutant. The data from these assays suggested that M. tuberculosis ruvB is active in E. coli and it is able to make functional contacts with E. coli RuvA. Moreover, the efficient alleviation of MMS toxicity in E. coli ΔruvB mutant suggested that M. tuberculosis ruvB might have a role in relieving replication stress generated under specific in vivo conditions. For biochemical analysis, M. tuberculosis RuvB protein (MtRuvB) was over-expressed in a heterologous system and purified to homogeneity. The identity of purified MtRuvB was verified using western blot analysis using the anti-MtRuvB antibodies. Purified MtRuvB was free of any contaminating endo- or exo- nuclease activity. The DNA-binding properties of MtRuvB were analyzed, in conjunction with its cognate RuvA, by using different substrates that are most likely to occur as intermediates during the processes of DNA replication and/or recombination. MtRuvAB bound HJ, three-way junction and heterologous replication fork with high affinity but with relatively weaker affinity to flap and flayed duplex substrates. MtRuvB displayed very weak affinity for linear duplex and failed to bind linear single-stranded DNA. The high affinity of MtRuvB for HJ substrate, in presence of its cognate RuvA, is indicative of direct and functional interaction between RuvA and RuvB. To further test this idea, the catalytic activity of MtRuvB was assayed in the in vitro HJ branch migration assay. In this assay,MtRuvB, in association with its cognate RuvA, promoted efficient branch migration of homologous HJ over heterologous HJ. To decipher the role of MtRuvAB in processing of stalled replication fork we performed in vitro replication fork reversal (RFR) assay using both oligonucleotide and plasmid based model replication fork substrates. Initially, binding of MtRuvAB to different homologous fork (HomFork) substrates was analyzed using the electrophoretic mobility shift assays. MtRuvAB exhibited similar binding affinity towards different HomFork substrates bearing different spatial orientation of nascent leading and lagging strands. To gain insight into the role of MtRuvAB in processing of replication forks, in vitro RFR reactions were carried out using an array of synthetic homologous fork substrates. In all these reactions, MtRuvAB catalyzed efficient fork reversal leading to generation of both parental duplex and daughter duplex. In the kinetics of fork reversal reaction, for all the fork substrates,the accumulation of daughter duplex increased with time whereas the increase in parental or nascent strand DNA was negligible. Taken together, our results suggest that MtRuvAB can efficiently catalyze in vitro replication fork reversal reaction to generate a Holliday junction intermediate thus implicating that RuvAB mediated fork reversal involves concerted unwinding and annealing of nascent leading and lagging strands. Equally important, we demonstrate the reversal of forks carrying hemi-replicated DNA, thus indicating that MtRuvAB mediated fork reversal is independent of symmetry at the fork junction. For understanding the role of RuvAB mediated processing of stalled forks at chromosome level, the fork reversal assays were performed using plasmid derived model “RF” substrate. Fork reversal was monitored by restriction enzyme digestion mediated release of 5’ end labeled fragments of specific size from the fourth arm extruded at the branch point of fork junction. In these reactions MtRuvAB complex was proficient at generating the reversed arm de novo from the RF substrate. Further, MtRuvAB complex catalyzed extensive fork reversal as analyzed by release of linear duplex of2.9 kb from a JM substrate. Use of non hydrolysable analogs of ATP and analysis of restriction digestion mediated release of duplex fragments from the reversed arm suggested that MtRuvAB catalyzed RFR reaction is ATP hydrolysis dependent progressive and processive reaction. MtRuvAB complex catalyzed fork reversal on plasmid substrate that had been linearized thus indicating that MtRuvAB mediated RFR is uncoupled from DNA supercoils in the substrate. Notably, MtRuvAB promoted reversal of forks in a substrate containing short stretch of heterologous sequences, indicating that sequence heterology failed to impede fork reversal activity of MtRuvAB complex. These results are discussed in the context of recognition and processing of varied types of replication fork structures by RuvAB enzyme complex.

No description available.

Bacterial stationary phase is an interesting biological system to study, as the organism undergoes several metabolic changes during this period and new molecules are generated to support its survival. The stationary phase of mycobacteria has been extensively studied since the discovery of Mycobacterium tuberculosis, the causative agent of tuberculosis. The stationary phase of mycobacteria adds further complication as many antibacterial drugs become less effective. The M. tuberculosis infects the alveolar macrophages and dendritic cells or monocytes recruited from peripheral blood. Macrophages are supposed to provide an initial barrier against the bacterial infection, but fails. Mycobacteria have evolved several strategies to survive and set up an initial residence within these cells and grow actively inside the host. The host immune system tries to limit the bacterial growth and confines the organism to a latent state in which the organism can persist indefinitely, known as granuloma stage. During latency or granuloma stage mycobacteria can retain the ability to resume the growth in the future. Mycobacteria must adapt to a highly dynamic and challenging environment because the interior environment of granuloma is devoid of or in low level of oxygen, depleted nutrient, high carbon dioxide, and possess increased levels of aliphatic organic acids and hydrolytic enzymes. The survival of a bacterium in less nutrient supply or in depleted oxygen is important for its long-¬term persistence inside the host under harsh environmental conditions. Mycobacterium smegmatis is the closest non-¬pathogenic homologue of M. tuberculosis, and has been used widely as a model system to study gene regulation under such conditions. In these harsh environmental conditions bacteria need to sense the external environment to modulate their gene expression. More importantly, each individual cell should communicate with its neighbours, and the response takes place in a concerted manner, which is termed as quorum sensing. Thus, the quorum sensing is a cell-¬cell signaling process that allow the bacteria to monitor the presence of other bacteria in their surroundings by producing and responding to small signaling molecules, which are known as autoinducers. It is a density dependent phenomenon and regulates the expression of the genes in response to fluctuation in cell¬-population density. A minimum threshold level of autoinducers is necessary to detect the signal and respond to it. Quorum sensing enables bacteria to behave like multicellular organisms and controls group activities like biofilm formation, sporulation, bioluminescence, virulence, and pigment production, etc (Bassler, 1999; Camilli & Bassler, 2006; Fuqua et al., 1996; Miller & Bassler, 2001). In Gram-¬negative bacteria, small-¬molecules, which are known as autoinducers are produced. They are acyl homoserine lactones (AHLs), which are derived from S¬adenosyl methionine (SAM) and particular fatty acyl carrier protein by LuxI¬type AHL synthases (Fuqua et al., 1996). In Gram-¬positive bacteria small peptides autoinducers, 5¬12 amino acids long, play an active role in communication. These oligopeptides are post--translationally modified by the incorporation of lactone and thiolactone rings, lanthionines and isoprenyl groups. These oligopeptide autoinducers are detected by membrane-¬bound two-¬component signaling proteins, and signal transduction occurs by a phosphorylation cascade (Camilli & Bassler, 2006; More et al., 1996; Novick, 2003; Zhang et al., 2002). In bacteria, the cyclic adenosine monophosphate (cAMP), and guanosine pentaphosphate and/or tetraphosphate ((p)ppGpp) are well known second messengers, which play important role in relaying extracellular information, but recently cyclic diguanosine monophosphate (c-¬di¬-GMP) is being studied most comprehensively as a nucleotide-¬based second messenger. C-¬di¬-GMP was first discovered in Gluconacetobacter xylinus as a positive allosteric regulator of cellulose synthase (Ross et al., 1987; Tal et al., 1998; Weinhouse et al., 1997). The in vivo level of c-¬di-¬GMP in bacterial cell is maintained by the balance between diguanylate cyclase and phosphodiesterase activities. The GGDEF and EAL amino acids sequence are the signature motif for GGDEF and EAL domain protein within its active site, respectively. The GGDEF domain protein is involved in synthesis of c-¬di-¬GMP and the EAL domain protein is involved in the hydrolysis of c-¬di-¬GMP, and the majority of these proteins contain additional signal input domains (Paul et al., 2004; Ross et al., 1987; Ryjenkov et al., 2005; Tal et al., 1998). M. smegmatis has a single bi-¬functional protein having both the domains, GGDEF and EAL, for the diguanylate cyclase (DGC) and phosphodiesterase (PDE¬A) activities. In addition to GGDEF and EAL domain, one sensory domain, GAF, is also there at the N-terminal of MSMEG_2196 in M. smegmatis. In the present investigation, studies have been carried out to understand the regulation of c-¬di-¬GMP in M. smegmatis at protein and gene level. The entire study on mycobacterial MSMEG_2196 (msdgc¬1) can be broadly divided into five parts; the first part will cover the identification and biochemical characterization of MSDGC¬1 protein, responsible for the regulation of in vivo c-¬di-¬GMP concentration in M. smegmatis, and the presence of GGDEF¬EAL domain containing proteins in various mycobacterial species. The second part will cover the structure function relationship as a function of substrate, GTP and product, c-¬di-GMP, molecule using fluorescence spectroscopy as a tool, and the mutational and structural studies, which leads to the identification of a novel structural motif. The third part will cover the characterization of msdgc¬1 gene knockout and complementation studies in great detail. The fourth part will comprise in vivo and in vitro promoter characterization and regulation of the msdgc¬1 gene under nutritional starvation. The last chapter will cover the characterization of novel synthetic glycolipids, which are working as a growth and biofilm inhibitors in mycobacteria, and can be used as a new drug candidates. Chapter 1 outlines the signal transduction and quorum sensing mechanism, and small molecule signaling modules in brief. The importance of the study started with a brief introduction about the historical aspect of tuberculosis, the current scenario of the treatment of tuberculosis. The urgent need for new drug targets and drugs will be discussed. The important role of the novel second messenger, c-¬di¬-GMP has been explained in greater details in both Gram-¬positive and Gram-¬negative bacteria, and the information available on the different cellular targets has been documented. Chapter 2 describes the identification and biochemical characterization of M. smegmatis MSMEG_2196 protein. The domain architecture and individual domain role have been studied. The MSMEG_2196 proteins consist of three domains, GAF, GGDEF and EAL in tandem, and individual role of each domain has been studied. The diguanylate cyclases containing GGDEF and phosphodiesterases containing EAL domains have been identified as the enzymes involved in the regulation of in vivo cellular concentration of c-¬di-¬GMP. GAF domain has been identified as a metal binding domain in other bacteria and may be playing a role in the regulation of synthesis and hydrolysis activities of c-¬di¬-GMP. The identification, cloning expression and purification of MSMEG_2196 and MSMEG_2774 have been discussed. We have reported that mycobacterial MSDGC¬1 protein has dual activity, which means that it can synthesize and hydrolyse c¬-di-¬GMP; and also full-¬length protein is necessary for its either of the activities. The synthesis and hydrolysis products, c-¬di-¬GMP and pGpG, of MSDGC¬1 protein have been identified and characterized using radiolabelled alpha [α¬32P]GTP and Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI). The effects of temperature and pH on the activities of MSDGC¬1 have been studied. The circular dichroism studies show that the MSDGC¬1 protein is predominantly α¬helical in nature, and secondary structure does not alter upon GTP binding. The kinetic parameters for MSDGC¬1 protein have been calculated as a function of substrate, GTP. The protein, MSDGC¬1, exist as a monomer and a dimer in solution. The MSDGC¬1 protein has four cysteines, and we have shown here using mass spectrometric analysis that none of the cysteines is involved in the disulphide linkage. Chapter 3 deals with the structure-¬function relationship as a function of GTP and c¬-di-GMP molecules using fluorescence spectroscopy as a tool. In order to do so we have generated several cysteine mutants using site directed mutagenesis, and protein was labelled with thiol-¬specific fluorophores. The labelled protein was checked for its DGC and PDE¬A activities and specificity of labelling was confirmed using MALDI and radiometric analysis. The Fluorescence Resonance Energy Transfer (FRET) has been carried out to observe domain-¬domain interaction as a function of GTP and c¬-di-¬GMP. The bioinformatics, structural, and mutational analysis suggest that cysteine at 579 position is important for DGC and PDE¬A activities, and may be involved in the formation of a novel structural motif, GCXXXQGF, which is necessary for synthesis and degradation of c-¬di-¬GMP. Chapter 4 describes the construction of a deletion mutation of MSMEG_2196 gene in M. smegmatis. The strategy for the construction of the knockout strain has been shown and confirmation of the knockout event has been carried out using PCR and Southern hybridization. The effect of deletion of msdgc¬1 has been studied in great detail, and it was noticed that biofilm formation is not affected, but long-¬term survival is significantly compromised. It is hypothesized here that c-¬di¬-GMP is involved in the regulation of cell population density in mycobacteria. We have successfully detected the c-¬di¬-GMP in the total nucleotide extract using HLPC coupled with MALDI, and we have shown here that level of c-¬di-¬GMP increases many fold in the stationary phase of growth under nutritional starvation. Chapter 5 deals with the identification and characterization of the promoter element of msdgc¬1 in M. smegmatis. The study was undertaken to understand the mechanism of regulation at promoter level. We have observed here that msdgc¬1 promoter is starvation induced, and expression of msdgc¬1 increases many fold in the stationary phase under nutritional starvation. We have also tried to establish the link between the ppGpp and c-di¬-GMP signalling, and possible role of c-¬di-¬GMP in the regulation of cell population density have been discussed. Further, the +1 transcription start site has been identified using primer extension method. The putative ¬10 hexamer region for the RNA polymerase binding has been identified and confirmed using site-¬directed mutagenesis. It was found to be TCGATA, which is 14 bp upstream from the +1 transcription start site. The msdgc-1 promoter is specific for mycobacteria and does not function in E. coli. Moreover, we have identified the sigma factors, which regulate the msdgc¬1 promoter in growth phase dependent manner. Chapter 6 begins with the screening of synthetic glycolipids as a novel drug candidate. The different glycolipids have been tested for their effect on growth, biofilm formation, and sliding motility of M. smegmatis, and we have screened few of them, which were found to be effective in inhibiting the microbial growth, biofilm formation, and sliding motility. Chapter 7 summarizes the work presented in this thesis. Appendix: The protein sequences of MSDGC¬1 and MSDGC¬2, and the multiple sequence alignments of MSDGC¬1 protein have been documented. The FORTRAN program, which was used to calculate spectral overlap integral J, and the diagrams of the plasmids used in this study have been provided.

Homologous recombination (HR) is conserved across all three domains of life and is associated with a number of key biological processes. Over the years, numerous genetic, biochemical and structural studies have uncovered important mechanistic details and established a role for HR in DNA damage repair, control of DNA replication fidelity and suppression of various types of cancer. Much of our current understanding of the mechanistic aspects of HR is gained from the study of Escherichia coli paradigm. E. coli RecA is the founding member of a nearly ubiquitous family of multifunctional proteins and is substantially conserved among eubacterial species. During HR, RecA protein promotes homologous pairing followed by strand exchange reaction leading to heteroduplex formation. In addition to HR, RecA is a central component of SOS response, recombinational DNA repair and rescue of collapsed replications forks. Moreover, recent work has suggested that DNA recombination/repair mechanisms might contribute to genome evolution and consequently to the generation of multidrug-resistant strains of the pathogen. The disease caused by Mycobacterium tuberculosis, endemic in certain regions of the world, is a leading cause of disability and death. A thorough knowledge of the function and interaction of specific HR proteins/enzymes involved in the maintenance of genome integrity is essential in order to elucidate the impact of genome perturbation effects on M. tuberculosis. Toward this end, modulation of RecA protein activity, a central component of HR, represents a potential novel target for design of new drugs because of its involvement in various processes of DNA metabolism. Additionally, small molecule modulators of RecA activity may offer novel insights into the regulation and its role in cellular physiology and pathology. Traditionally, antibiotics have been used to treat infections caused by bacteria. Despite their importance, the development of new antibiotics against M. tuberculosis has considerably decreased over the past several years due to disappointing results in clinical trials. These failures may be due the fact that they suffer from low potency or low cell permeability. Therefore, one of the aims of studies described in this thesis was to test the effect of suramin, a known inhibitor of E. coli RecA, on various biochemical activities of mycobacterial RecA proteins and determine its mechanism of action. Furthermore, the most crucial step in the HR pathway and rescue of collapsed DNA replication forks is the resolution of Holliday junctions and other branched intermediates. Because Holliday junction resolvases are essential for the resolution of different types of DNA recombination/repair intermediates, therefore, we considered it worthwhile to study the genomic expression and biochemical properties of HJRs in M. tuberculosis. Suramin is a commonly used antitrypanosomal and antifiliarial drug, and a novel experimental agent for the treatment of several cancers. A forward chemical screen assay identified several small molecule inhibitors of E. coli RecA. In this screen, suramin (also called germanin), a polysulfonated naphthylurea, and suramin-like agents were found to inhibit EcRecA catalyzed ATPase and DNA strand exchange activity. However, the mechanism underlying such inhibitory action of suramin and whether it can exert antibacterial activity under in vivo conditions remains largely unknown. In an attempt to delineate the range of suramin action, we reasoned that it might be useful to test its effect on mycobacterium RecA proteins. We found that suramin is a potent inhibitor of all known biochemical activities of mycobacterial RecA proteins with IC50 values in the low μM range. The mechanism of action involves, in part, its ability to disassemble the nucleoprotein filaments of RecA-ssDNA. To validate the above results and to obtain quantitative data, a pull-down assay was developed to assess the effect of suramin on RecA–ssDNA filaments. The data indicated that suramin was able to dissociate >80% of RecA bound to ssDNA. Altogether, these results indicated the effectiveness of suramin in the disassembly of RecA nucleoprotein filament. Next, we sought to test whether suramin binds to RecA by using a CD spectropolarimeter. Significant spectral changes were observed upon addition of increasing concentrations of suramin, indicating alterations in the secondary structure of RecA protein. Additional evidence revealed that suramin impaired RecA catalyzed proteolytic cleavage of LexA repressor and blocked ciprofloxacin-inducible recA gene expression and SOS response. More importantly, suramin potentiated the cidal action of ciprofloxacin and reduced the growth of Mycobacterium smegmatis recA+ strain but not its isogenic recA∆ mutant, consistent with the idea that it acts directly on RecA protein. This approach, which appears as an appealing concept, opens up new possibilities to chemically disrupt the pathways controlled by RecA and treat drug-sensitive as well as drug-resistant strains of M. tuberculosis for better infection control and the development of new therapies. The annotated genome sequence of M. tuberculosis revealed the presence of putative homologues of E. coli DNA recombination/repair genes. However, it is unknown whether these putative genes have the ability to encode catalytically active proteins or participate in biochemical reactions intrinsic to the process of HR or DNA repair. Studies in the second half of the thesis originated from an in silico analysis for genes that encode functional equivalents of E. coli RuvC HJ resolvase(s) in M. tuberculosis. The central intermediate formed during mitotic and meiotic recombination is a four-way DNA junction, also known as the Holliday junction (HJ), and its efficient resolution is essential for proper segregation of chromosomes. The resolution of HJ is mediated by a group of structure specific endonucleases known as the Holliday junction resolvases (HJR) which have been identified in a wide variety of organisms based on their shared biochemical characteristics. Bioinformatics analyses of the evolutionary relationships among HJ resolvases suggests that HJR function has arisen independently from four distinct structural folds, namely RNase H, endonuclease VII-colicin E, endonuclease and RusA. Furthermore, similar analyses of HJRs identified another family within the RNaseH fold, along with previously characterized RuvC family of junction resolvases. This new family of putative HJRs is typified by E. coli Yqgf protein. The yqgf gene is highly conserved among bacterial genomes. Nuclear magnetic resonance structural studies have disclosed notable structural similarities between E. coli RuvC and YqgF proteins. Utilizing homology-based molecular modelling, YqgF is predicted to function as a nuclease in various aspects of nucleic acid metabolism. Sequence analysis of M. tuberculosis genome has revealed the presence of two putative HJ resolvases, ruvC (Rv2594c) and ruvX (Rv2554c, yqgF homolog). Previous studies have demonstrated that M. tuberculosis ruvC is induced following DNA damage and ruvX is expressed during active growth phase of M. tuberculosis. More importantly, the absence of ruvC increased the potency of moxifloxacin in M. smegmatis. Although, these results imply that the ruv genes play crucial roles in DNA recombination and repair in M. tuberculosis, the biochemical properties of their gene products have not been characterized. In this study, we have isolated M. tuberculosis ruvC and yqgF genes and purified their encoded proteins, M. tuberculosis RuvC (MtRuvC) and M. tuberculosis RuvX (MtRuvX), respectively, to near homogeneity. Protein-DNA interaction assays conducted with purified MtRuvC and MtRuvX revealed that both can bind HJ, albeit with different affinities. However, in contrast to MtRuvC, MtRuvX showed robust HJ resolvase activity. The endonuclease activity of MtRuvX was completely dependent on Mg2+and Mn2+ partially substituted for Mg2+. Additional experiments showed that RuvX exhibits >2-fold higher binding affinity for HJ over other recombination/ replication intermediates. As demonstrated for other HJRs, MtRuvX failed to cleave static HJ and linear duplex DNA. The cleavage sites were mapped within the homologous core of a branch-migratable HJ. To identify catalytic residues in RuvX, we conducted mutational analysis of an acidic amino acid residue guided by the bioinformatics data. The product of MtRuvXD28N retained full HJ-binding activity, but showed extremely reduced HJ-specific endonuclease activity. Further biochemical characterization revealed that MtRuvX exists as a homodimer in solution. Notably, we found that disulfide-bond mediated intermolecular homodimerization is crucial for the ability of MtRuvX to cleave Holliday junctions, implicating that stable junction binding is necessary to promote branch migration and to create cleavable sites. Analysis of qPCR data suggested that the pattern of yqgF gene expression was similar to those of ruvC and recA genes following DNA damage. Together, these data indicate that ruvX expression is induced by DNA-damaging agents and that RuvX might be functionally involved in recombinational DNA repair in M. tuberculosis. These findings are all consistent with the idea that RuvX might be the bona fide HJ resolvase in M. tuberculosis analogous to that of E. coli RuvC. More importantly, we provide the first detailed characterization of RuvX and present important insights into the mechanism of HJ resolution, which could be directly linked to the regulation of different DNA metabolic processes, including HR, DNA replication and DNA repair. Overall, this study opens a new avenue in the understanding of HR in this human pathogen, together with elucidation of the function of some of the uncharacterized genes may represent a novel set of recombination enzymes.

Bacteria lack nucleus and any other membrane-bound organelles. Hence all the cellular components, including proteins, DNA, RNA and other components are located within the cytoplasm. The region of the cell which encompasses the bacterial genomic DNA is termed ‘Nucleoid’. The nucleoid is composed largely of DNA and small amounts of proteins and RNA. The genomic DNA is organized in ways that are compatible with all the major DNA-related processes like replication, transcription and chromosome segregation. Proteins that play important role(s) in the structuring of DNA and having the potential to influence gene expression have been explored in all kingdoms of life. The organization of bacterial chromosome is influenced by several important factors. These factors include molecular crowding, negative supercoiling of DNA and NAPs (nucleoid-associated proteins) and transcription. Nucleoid-associated proteins are abundant and relatively low-molecular mass proteins which can bind DNA and function as architectural constituents in the nucleoid. Additionally, NAPs are involved in all the major cellular processes like replication, repair and gene transcription. At least a dozen distinct NAPs are known to be present in E. coli. HU, IHF (integration host factor), H-NS (histone-like nucleoid-structuring), Fis (Factor for inversion stimulation), Dps (DNA protection from starvation) are some of the abundant NAPs in E. coli. Most of these proteins bind DNA and show either DNA bending, bridging or wrapping which are directly relevant to their physiological role(s). As most of these proteins are involved in the regulation of transcription of many genes, they act as factors unifying gene regulation with nucleoid architecture and environment. Pathogenic bacteria have the ability to grow and colonize different environments and thus need to adapt to constantly changing conditions within the host. H-NS and IHF, being able to link environmental cues to the regulation of gene expression, play an important role in the bacterial pathogenesis. H-NS is one of the well studied NAPs in enterobacteria, and is known as a global gene silencer. It is also an important DNA structuring protein, involved in chromosome packaging. H-NS protein is a small (~15 kDa) protein, which is present at approximately 20000 copies/ cell. The most striking feature of H-NS is that although it binds DNA in a relatively sequence-independent fashion but is known to preferentially recognize and bind intrinsically curved DNA. It also constrains DNA supercoils in vitro, thereby affects DNA topology. H-NS also influences replication, recombination and genomic stability. In addition, it functions as a global regulator by regulating the expression of various genes which are linked to environmental adaptation. Various studies have shown the association of H-NS to AT-rich regions of the genome. About 5% of E. coli genes are regulated by H-NS, bulk of which are (~80%) negatively regulated. H-NS is involved in the silencing of horizontally-acquired genes, many of which are involved in pathogenesis, in a process known as xenogeneic silencing. H-NS is known to regulate the expression of various virulence factors like cytotoxins, fimbriae and siderophores in several pathogenic bacteria. Several studies have revealed that hns mutants show increased frequency of illegitimate recombination and reduction in intra-chromosomal recombination, indicating the involvement of H-NS in DNA repair/recombination. H-NS is known to act in several transposition systems, which it does so due to its ability to interact with other proteins involved and due to its DNA structure-specific binding activity. The prototypical IHF (Integration Host Factor) was originally discovered in E. coli as an essential co-factor for the site-specific recombination of phage λ. E. coli IHF belongs to DNABII structural family, along with HU and other proteins and consists of two subunits, IHFα and IHFβ. Thesubunits are ~10 kDa each and are essential for full IHF activity. Apart from its role in bacteriophage integration/excision, IHF also has roles in various processes such as DNA replication, transcription and also in several site-specific recombination systems. In most of these processes, IHF acts as an architectural component by facilitating the formation of nucleoprotein complexes by bending DNA at specific sites. IHF acts as a transcriptional regulator, influencing the global gene expression in E. coli and S. Typhimurium. Gene regulation by IHF requires its DNA architectural role, facilitating interactions between RNA polymerase and regulatory protein. The high intracellular concentration of IHF indicates that it might associate with DNA in a non-specific manner and contribute to chromatin organization. The binding of E. coli IHF causes the DNA to adopt U-turn and brings the non-adjacent sequences into close juxtaposition. IHF is also involved in gene regulation in several pathogenic organisms and is shown to regulate expression of many virulence factors. Despite extensive literature on NAPs, very little is known about NAPs and nucleoid architecture in M. tuberculosis. In the light of significant physiological roles played by NAPs in adaptation to environmental changes and in growth and virulence of bacteria, elucidation of their roles in M. tuberculosis is of paramount importance for a better understanding of its pathogen city. M. tuberculosis Rv3852 (hns) gene is predicted to encode a 134 amino acid protein with a molecular mass of 13.8 kDa. The amino acid sequence alignment revealed that M. tuberculosis H-NS and E. coli H-NS showed very low degree of sequence identity (6%). To explore the biochemical properties of M. tuberculosis H-NS, the sequence corresponding to Rv3852 was amplified via PCR, cloned and plasmid expressing M. tuberculosis hns was constructed. M. tuberculosis H-NS was over expressed and purified to homogeneity. E. coli H-NS was also over expressed and purified. Comparison of experimentally determined secondary structure showed considerable differences between M. tuberculosis and E. coli H-NS proteins. Chemical cross linking suggested that M. tuberculosis H-NS protein exists in both monomeric and dimeric forms in solution, consistent with the diametric nature of E. coli H-NS protein. Our studies have revealed that M. tuberculosis H-NS binds in a more structure-specific manner to DNA replication and repair intermediates, but displays lower affinity for double stranded DNA with relatively higher GC content. It bound to the Holliday junction (HJ), the central recombination intermediate, with high affinity. Furthermore, similar to M. tuberculosis H-NS, E. coli H-NS was able to bind to replication and recombination intermediates, but at a lower affinity than M. tuberculosis H-NS. To gain insights into homologous recombination in the context of nucleoid, we investigated the ability of M. tuberculosis RecA to catalyze DNA strand exchange between single-strand DNA and linear duplex DNA in the presence of increasing amounts of H-NS. We found that M. tuberculosis H-NS inhibited strand exchange mediated by its cognate RecA in a concentration dependent manner. Similar effect was seen in the case of E. coli H-NS, where it was able to suppress DNA strand exchange promoted by E. coli RecA, but at relatively higher concentrations, suggesting that H-NS proteins act as ‘roadblocks’ to strand exchange promoted by their cognate RecA proteins. H-NS and members of H-NS-family of NAPs are known to form rigid nucleoprotein filament structures on binding to DNA, which results in gene-silencing and is also implicated in chromosomal organization. Studies have also shown that H-NS mutants defective in gene silencing also lack the ability to form rigid nucleoprotein filament structure and that nucleoprotein filament structure is responsive to environmental factors. Our studies employing ligase-mediated DNA circularization assays reveal that both E. coli and M. tuberculosis H-NS proteins abrogate the circularization of linear DNA substrate by rigidifying the DNA backbone. These results suggest that M. tuberculosis H-NS could form nucleoprotein filament-like structures upon binding to DNA and these structures might be involved in transcriptional repression, chromosomal organization and protection of genomic DNA. In summary, these findings provide insights into the role of M. tuberculosis H-NS in homologous and/or homeologous recombination as well as transcriptional regulation and nucleoid organization. The second part of the thesis concerns the characterization of M. tuberculosis integration host factor (mIHF). The annotation of whole-genome sequence of M. tuberculosis H37Rv showed the presence of Mtihf gene (Rv1388) which codes for a putative 20-kDa integration host factor (mIHF). Amino acid sequence alignment revealed very low degree of sequence identity between mIHF and E. coli IHFαβ subunits. Unlike E. coli IHF, mIHF is essential for the viability of M. tuberculosis. The three-dimensional molecular modeling of mIHF based upon co crystal structure of Streptomycin coelicolor IHF (sIHF) duplex DNA, showed the presence of conserved Arg170, Arg171, Arg173, which were predicted to be involved in DNA binding and a conserved Pro150, in the tight turn. The coding sequence corresponding to the M. tuberculosis H37Rv ihf gene (Rv1388) was amplified, cloned and plasmid over expressing M. tuberculosis ihf (pMtihf) was constructed. Using pMtihf as a template and using specific primers, mutant ihf encoding plasmids were constructed in which, the arginine at position 170, 171, or 173 was replaced with alanine or aspartate and proline at position 150 was substituted with alanine. To explore the role of mIHF in cell viability, we investigated the ability of M. tuberculosis ihf to complement E. coli ΔihfA or ΔihfB strains against genotoxic stress. Despite low sequence identity between Mtihf and E. coli ihfA and ihfB, wild type Mtihf was able to rescue the UV and MMS sensitive phenotypes of E. coli ΔihfAand ΔihfBstrains, whereas Mtihf alleles bearing mutations in the DNA-binding residues failed to confer resistance against DNA-damaging agents. To further characterize the functions of mIHF, wild type and mutant versions of mIHF proteins were over expressed and purified to near homogeneity. Circular dichroism spectroscopy of wild type mIHF and mIHF mutant proteins revealed that they have similar secondary structures. By employing size-exclusion chromatography and blue-native PAGE, we determined that mIHF exists as a dimmer in solution. To understand the mechanistic basis of mIHF functions, we carried out electrophoretic mobility shift assays. In these assays, we found that wild-type mIHF showed high affinity and stable binding to DNA containing attB and attP sites and also to curved DNA, but not those mIHF mutants bearing mutations in DNA-binding residues. Because wild type mIHF was able to rescue the UV and MMS sensitive phenotypes of E. coli ΔihfA and ΔihfB strains, we ascertained the effect of overexpression of mIHF proteins on the bacterial nucleoid. Our results revealed that wild type mIHF was also able to cause significant nucleoid compaction upon its overexpression, but mutant mIHF proteins were unable to cause compaction of E. coli nucleoid structure. M. smegmatis IHF is known to stimulate L5 phage integrase mediated site-specific recombination, we investigated the ability of mIHF to promote site-specific recombination. In vitro recombination assays showed that M. tuberculosis IHF effectively stimulated the L5 integrase mediated site-specific recombination. Since DNA-bending activity of E. coli IHF is necessary for its functions in various processes like initiation of replication, site-specific recombination, transcriptional regulation and chromosomal organization, we asked whether mIHF possesses DNA bending activity. We employed ligase mediated DNA circularization assays, which revealed that like E. coli IHF, mIHF was able to bend DNA resulting in the covalent closure of DNA to yield circular DNA molecules. Interestingly mIHF also resulted in the formation of slower migrating linear DNA multimers, albeit to a lesser extent, which suggest that both E. coli IHF and mIHF show DNA-bending, but the mechanism is distinct. Further studies using atomic force microscopy showed that depending upon the placement of preferred binding site (curved-DNA sequence) mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. Together, these findings provide insights into functions of mIHF in the organization of bacterial nucleoid and formation of higher-order nucleoprotein structures. Importantly, our studies revealed that the DNA-binding residues, the DNA bending mechanism and mechanism of action of mIHF during site-specific recombination were different from E. coli IHF protein. Together with extensive biochemical and in vitro data of bacterial growth, the findings presented in this thesis provide novel insights into the biological roles of H-NS and mIHF in M. tuberculosis.

Made available in DSpace on 2014-07-29T15:30:40Z (GMT). No. of bitstreams: 1 Dissertacao Alyne Melo Pereira.pdf: 2782543 bytes, checksum: 9841bfc0f181c8d2c6af086d835eadc3 (MD5) Previous issue date: 2012-03-07 / Tuberculosis (TB) is a chronic infectious bacterial disease, very contagious, that, despite almost 130 years of biomedical research since the discovery of the tubercle bacillus, it continues to be a major threat to global health and is one of the leading causes of death by a bacterial organism, particularly in the developing world. The etiologic agent, Mycobacterium tuberculosis, is a bacterium that is easily transmitted by air. The disease control depends on several factors, being the correct diagnosis; efficient treatment; and active disease patient management to avoid transmission, essential ones. In order to improve the success in diagnosis, the knowledge of genotypic profiles by molecular techniques has provided positive results. Additionally, the correlation between the geographical locations of TB cases is a useful tool to aid epidemiological control strategies, especially when it is performed together with the knowledge of genetic variability of the studied TB strains. In this study we used the 15 loci MIRU-VNTR technique to identify polymorphisms among Mycobacterium tuberculosis isolates. A total of 119 M. tuberculosis samples, isolated between 2006 and 2007 from patients attending two reference hospitals of Goiânia city, were genotyped and the results compared with the gold standard RFLP-IS6110 technique. The 15 loci MIRU-VNTR analysis of 119 TB isolates provided 110 distinct genotypes, 105 of which contained only one isolate while 14 isolates were grouped in five genetic groups (clusters). This technique showed a good discriminatory power (0.9986). The 15 loci MIRU-VNTR technique was more discriminatory than RFLP-IS6110 (0.9942). We also performed the georeferencing of 241 TB cases, corresponding to all clinical forms reported in Goiânia during the year 2007 by the City´s Secretary of Health Department. The cases were randomly distributed throughout the city. The distribution of cases showed that, visually, there was no relationship between disease and socio-economic situation of the population. Among the georeferenced cases, we were able to genotype 50 isolates by 15 loci MIRU-VNTR. A great genetic variability of genotypes among the 50 isolates was observed, and the few ones that were clustered did not show epidemiological links. Based on the observed data, no transmission source the TB was identified in the city of Goiânia, and consequently we can infer that TB in the City of Goiânia could be resulted from a previously acquired infection due to the high degree of heterogeneous genetic profiles observed. / A tuberculose (TB) é uma doença bacteriana crônica infecto-contagiosa que, apesar de quase 130 anos de pesquisas desde a descoberta do bacilo, continua a ser um importante agravo à saúde global e uma das principais causas de morte, particularmente nos países em desenvolvimento. A doença tem como agente etiológico o Mycobacterium tuberculosis, uma bactéria de fácil transmissão uma vez que os bacilos se propagam pelo ar. O controle da doença depende de vários fatores, dentre os quais, o correto diagnóstico, o tratamento completo e o manejo adequado dos pacientes com a doença ativa para evitar a transmissão são fundamentais. Neste cenário, o conhecimento dos perfis genotípicos dos microorganismos circulantes em uma região, através de técnicas moleculares apropriadas, tem contribuído com bons resultados. Além disso, a correlação entre o espaço geográfico de casos da doença é uma ferramenta útil para a definição de estratégias epidemiológicas, sobretudo quando se conhece a variabilidade genética das cepas presentes em uma determinada população associada a sua localização geográfica. Neste estudo, foi empregada a análise de MIRU-VNTR para identificar o polimorfismo de 15 loci em amostras de Mycobacterium tuberculosis. Foram analisadas 119 amostras, coletadas entre 2006 e 2007, de pacientes com TB pulmonar em dois hospitais de referência no município de Goiânia. Os resultados obtidos pela técnica de 15 loci MIRU-VNTR foram posteriormente, comparados com resultados gerados pela técnica padrão ouro RFLP-IS6110. Pela análise dos padrões moleculares dos 119 isolados, foram encontrados 110 genótipos distintos. Destes, 105 continham um único isolado enquanto que 14 amostras se agruparam em cinco grupos (cluster) genéticos. Esta técnica apresentou um bom poder discriminatório (0,9986). Nossos resultados mostraram que a técnica de tipagem por 15 loci MIRU-VNTR se mostrou mais discriminatória que a técnica de RFLP-IS6110 (0,9942). Adicionalmente, foram georreferenciadas 241 amostras de pacientes diagnosticados com qualquer forma clínica de TB em 2007 de acordo com a Secretaria Municipal de Saúde e destas, 50 amostras foram genotipadas por 15 loci MIRU-VNTR. No georreferenciamento de 241 casos da doença, observou-se uma distribuição aleatória destes pelo município sem aglomerados significativos. A distribuição dos casos mostrou que, visualmente, não houve relação entre a doença e a situação sócio-ecônomica da população. O estudo genotípico de 50 isolados georreferenciados demonstrou que existe grande variabilidade genética de cepas circulantes na cidade e essas, quando agrupadas, não apresentam associação geo-espacial que possibilite estabelecer uma ligação epidemiológica entre os casos. Podemos concluir que não existem focos recentes de transmissão da doença, podendo esta ser proveniente de reativação endógena de infecção latente adquirida anteriormente, já que a maioria dos isolados apresentaram perfis únicos.