P1 prophage is stably maintained in E. coli as a low-copy-number plasmid. Stable maintenance of P1 is dependent on the function of the plasmid encoded partition system, parABS. ParA is the partition ATPase, ParB is the partition-site binding protein, and parS is the partition site. The concerted action of these proteins results in dynamic movement of the plasmid over the bacterial nucleoid, which results in its stable maintenance. Plasmid movement has been proposed to be caused by interactions between parS bound ParB and nucleoid bound ParA. In this thesis, I have identified a complex of ParA, ParB, and DNA that is capable of promoting plasmid stability. ParA, ParB, DNA interactions required the ATP bound conformation of ParA. The ParA-ParB-DNA complex was dynamically regulated by nucleotide hydrolysis, which promoted complex disassembly. Complex formation resulted from the cooperative binding of ParA and ParB to DNA. ParA-ParB and ParB-DNA interactions were both necessary for complex formation. ParA-ParB-DNA complex size was regulated by ParB stimulation of ParA-ATP hydrolysis. Microscopy demonstrated that complexes resulted in the association of multiple DNA molecules due to protein binding. The properties of complex assembly, dynamics, and DNA grouping lead me to propose a model where associations between ParA bound to the bacterial nucleoid and the partition complex mediated plasmid movement and localization.

P1 prophage is stably maintained in E. coli as a low-copy-number plasmid. Stable maintenance of P1 is dependent on the function of the plasmid encoded partition system, parABS. ParA is the partition ATPase, ParB is the partition-site binding protein, and parS is the partition site. The concerted action of these proteins results in dynamic movement of the plasmid over the bacterial nucleoid, which results in its stable maintenance. Plasmid movement has been proposed to be caused by interactions between parS bound ParB and nucleoid bound ParA. In this thesis, I have identified a complex of ParA, ParB, and DNA that is capable of promoting plasmid stability. ParA, ParB, DNA interactions required the ATP bound conformation of ParA. The ParA-ParB-DNA complex was dynamically regulated by nucleotide hydrolysis, which promoted complex disassembly. Complex formation resulted from the cooperative binding of ParA and ParB to DNA. ParA-ParB and ParB-DNA interactions were both necessary for complex formation. ParA-ParB-DNA complex size was regulated by ParB stimulation of ParA-ATP hydrolysis. Microscopy demonstrated that complexes resulted in the association of multiple DNA molecules due to protein binding. The properties of complex assembly, dynamics, and DNA grouping lead me to propose a model where associations between ParA bound to the bacterial nucleoid and the partition complex mediated plasmid movement and localization.

<p>The relatively recent discoveries of bacterial small RNAs (sRNAs) and their important regulatory functions prompted us to conduct a genome wide survey for sRNAs in <em>Streptomyces coelicolor</em>. We used a combined bioinformatics and experimental approach to identify and characterize six sRNAs. sRNA expression profiles were determined throughout <em>S. coelicolor</em> development, including vegetative and reproductive growth, during growth on minimal and rich media. Additionally, we also tested sRNA expression in various <em>S. coelicolor</em> developmental mutants. Two sRNAs were expressed exclusively during growth on one medium type and all but one were expressed constitutively throughout growth apart from the late sporulation timepoint. One of the identified sRNAs, scr1906, appeared to be closely associated with development. scr1906 was only expressed in nutrient limiting conditions just prior to aerial development and sporulation. Expression of scr1906 was abolished in a mutant that was defective in sporulation (due to a mutation in the sporulation sigma factor gene, <em>whiG</em>); however, expression was detected in mutants of both known σ^WhiG target genes, <em>whiH</em> and <em>whiI</em>, which encode sporulation transcription factors. Intriguingly, <em>in silico</em> analysis predicted <em>whiH</em> to be a direct target for scr1906-mediated regulation based on potential nucleotide binding sites. The effects of deletion and overexpression of <em>scr1906</em> on WhiH levels were tested, but require further experimentation.</p> <p>In a separate line of investigation, we sought to characterize a novel actinobacterial-specific protein named sIHF. The <em>sIHF</em> mutant strain revealed that sIHF influenced DNA compaction and segregation during <em>S. coelicolor</em> sporulation and also affected antibiotic production. sIHF associated with the nucleoid, and <em>in vitro</em>, it bound to DNA non-specifically in a length dependent manner, although it was determined to have a preference for three distinct DNA motifs. Like most nucleoid-associated proteins, sIHF affected gene expression indicating the potential for an additional role as a transcription factor. Interestingly, sIHF impacted the activity of topoisomerase. Leveraging information that we have gained from the sIHF-DNA co-crystal complex, studies aimed at characterizing the sIHF regions that are important for DNA interaction and topoisomerase modulation are currently underway.</p> / Doctor of Philosophy (PhD)

Mycobacterium tuberculosis is one of the most successful human pathogens, estimated to have infected close to one-third of the global human population. In order to survive within its host, M. tuberculosis utilises multiple signalling strategies, one of them being synthesis and secretion of universal second messenger cAMP. This process is enabled by the presence of sixteen predicted adenylyl cyclases in the genome of M. tuberculosis H37Rv, ten of which have been characterised in vitro. The synthesized cAMP is recognised by ten putative cAMP-binding proteins in which the cyclic AMP-binding domain is associated with a variety of enzymatic domains. The cAMP signal can be extinguished by degradation by phosphodiesterase’s, secretion into the extracellular milieu or via sequestration of the nucleotide by upregulation of a high-affinity cAMP-binding protein. Of the sixteen adenylyl cyclases (ACs) encoded by M. tuberculosis H37Rv, a subset of multidomain adenylyl cyclases remain poorly characterised, primarily due to challenges associated with studying these in vitro. The adenylyl cyclase domain in these proteins is associated with an NB-ARC domain (nucleotide binding domain common to APAF-1, plant R proteins and CED-4), a TPR domain (tetratricopeptide repeat) and an LuxR-type HTH motif (helix-turn-helix). This architecture places these multidomain mycobacterial ACs within a larger group of STAND (Signal transduction ATPase’s with numerous domains) proteins, and hence they will be referred to as STAND ACs. The STAND proteins are a recently recognised class of multidomain ATPases which integrate a variety of signals prior to activation. Activation is accompanied by formation of large oligomeric signalling hubs which facilitate downstream signalling events. While most STAND proteins have a single effector domain followed by an NB-ARC domain and a scaffolding domain, the STAND ACs distinguish themselves by retaining two effector domains, the AC domain and the HTH domain, at the N- and C- termini respectively. The cyclase, NB-ARC, TPR and HTH domains have widely divergent taxonomic distributions making the presence of these four domains in a single polypeptide rare. In fact, proteins with cyclase-NB-ARC-TPR-HTH (C-A-T-H) domain organisation were found to be encoded almost exclusively by slow growing mycobacterial species, a clade that harbours most mycobacterial pathogens, such as M. tuberculosis and M. leprae. Notably, one of the STAND ACs, Rv0386, is the only mycobacterial AC shown till date to be required for virulence of M. tuberculosis in mice. Using phylogenetic, the evolutionary underpinnings of this domain architecture were examined. The STAND ACs appear to have most likely evolved via a domain gain event from a cyclase-ATPase-TPR progenitor encoded by a strain ancestral to M. marina. Subsequently, the genes duplicated and diverged, sometimes leading to frameshift mutations splitting the cyclase domain from the C-terminal domains. Consequently, M. tuberculosis encodes for three ‘full-length’ STAND ACs, namely, Rv0386, Rv1358 and Rv2488c and one split STAND AC. The split STAND AC is made up of Rv0891c, containing the AC domain, and Rv0890c, containing the NB-ARC, TPR and HTH domains. rv0891c and rv0890c were found to be expressed as an operatic transcript, though they were translationally uncoupled. Pertinently, M. Canetti, an early-branching species of the M. tuberculosis complex, contains an orthologue of Rv0891c and Rv0890c where all four domains are present in a single polypeptide. Sequence analysis of the four STAND ACs in M. tuberculosis allowed predictions of significant divergence in function. These proteins showed high sequence conservation in their HTH domains, with substantial sequence divergence in their TPR, NB-ARC and AC domains. Biochemical analysis on the AC domains revealed that Rv0891c and Rv2488c possessed poor or no AC activity, respectively. On the other hand, the cyclase domain of Rv0386 could catalyse cAMP synthesis. Moreover, for both Rv0891c and Rv0386, presence of the C-terminal domains potentiated adenylyl cyclase activity, suggestive of allosteric regulation within the STAND AC module. Studies on Rv0891c also revealed that the protein could inhibit the adenylyl cyclase activity of Rv0386 in trans. This result thus provided a novel mechanism by which proteins harbouring poorly active/inactive adenylyl cyclase domains could contribute to cAMP levels, by acting as inhibitors of other adenylyl cyclases. The STAND ACs were found to be inactive ATPases. Additionally, incubation with nucleotides did not stimulate oligomerisation of these proteins, unlike what has been shown for several other STAND proteins. However, mutations in the NB-ARC domain perturbed the basal oligomeric state of these proteins, indicating that the NB-ARC domain can influence self- association. A subset of NB-ARC domain mutants also showed increased adenylyl cyclase activity, reiterating the inter-domain cross-talk in the STAND ACs. Since the AC activity of these proteins was meagre, the properties of the HTH domain were examined, as an alternative effector domain. Genomic SELEX was performed using the TPR-HTH domains of Rv0890c, and revealed a set of sequences that bound to this protein, though they lacked common sequence features. Further analysis revealed that Rv0890c bound to DNA in a sequence-independent manner, through the HTH domain. This binding was cooperative with multiple protein units engaging in DNA-binding. Due to the cooperative nature of binding and the lack of sequence preference, Rv0890c appeared coat the DNA molecule. This was further proved by the ability of Rv0890c to protect DNA from DNaseI-mediated degradation, and the requirement for long DNA sequences to form stable DNA-protein complexes. Studies also revealed that Rv0890c interacted with RNA and ssDNA. In fact, the protein as purified from heterologously expressing E. coli cells was bound to RNA. RNA-binding by a LuxR-type HTH has not been reported previously, providing a new function for this class of HTHs. Interestingly, nucleic acid-binding by a fusion Rv0891c-Rv0890c protein, similar to the one encoded in M. canetti, was shown to stimulate adenylyl cyclase activity. This was likely due to a relief of inhibitory interactions between the TPR-HTH and the AC domains, on DNA-binding. Given the high sequence similarity between the HTH domains of the STAND ACs, they were expected to bind to DNA in an identical manner. Indeed, the HTH domains of Rv0386 and Rv1358 engaged with DNA with an identical affinity as Rv0890c. Sequence comparisons in the HTH domain enabled identification of conserved basic residues, of which one, R850 was essential for nucleic acid-binding. Surprisingly however, Rv0386 and Rv1358 did not exhibit RNA-binding, pointing towards functional divergence of Rv0890c from its paralogues. Since the HTH domains of the STAND ACs were highly conserved, it was possible that the ability to bind to RNA was instead dictated by the adjacent TPR modules. To examine this possibility, TPR domains were swapped between Rv0890c and Rv0386. Interestingly, both the chimeric proteins showed a reduced ability to bind to DNA, while showing a complete absence of RNA- binding. These results suggested that the TPR domains were critical in modulating nucleic acid-binding. Moreover, the effect of the TPR domain was context-dependent, since the presence of non-cognate TPR domains hampered nucleic acid-binding. However, the ability to bind to RNA was not solely governed by the TPR domain since the Rv0890cTPR-Rv0386HTH chimeric protein did not show RNA-binding, in spite of containing a permissive TPR domain. To further dissect the molecular requirements for RNA-binding, the conservation of basic residues between the HTH domains of Rv0890c versus Rv1358 and Rv0386 was examined. Interestingly the HTH domain Rv0890c contained two additional positively charged residues over Rv1358 and Rv0386. Mutations of these abolished RNA-binding by Rv0890c. Thus the evolution of two basic residues permit Rv0890c to diverge in its nucleic acid-binding properties, a possible example of defunctionalisation following gene duplication. In summary, this thesis attempts to understand the evolution and functions of the STAND ACs, a group of pathogenically relevant and uniquely mycobacterial multidomain proteins. Phylogenetic analysis revealed an expansion of this gene family in slow growing mycobacteria. Biochemical characterisation showed that following gene duplication, the resulting proteins diverge both in their ability to synthesize cAMP and in their association with nucleic acids. Studies on these proteins also revealed novel mechanisms of regulation of mycobacterial cAMP levels. Additionally, these proteins exhibited indiscriminate binding to DNA/nucleic acids indicating that they may be responsible for global functions in the cell which extend beyond cAMP synthesis.

The Dps (DNA-Binding Protein from Starved Cells) proteins are a class of stress-specific proteins with a major role in protecting DNA during the stationary phase of bacterial growth, through direct physical binding as well as ferroxidation. These proteins are characteristically dodecameric in nature. Mycobacterium smegmatis, which is the model organism used in this study has two Dps homologues- MsDps1 and MsDps2. MsDps1, that has previously been studied, is exceptional in having trimeric as well as dodecameric states in vitro. This work focuses on the functional domains of MsDps1, with respect to its oligomerisation and DNA binding property, the identification of a new Dps homologue MsDps2, the in vitro characterization of MsDps2 and elucidation of a possible function of the protein in the physiology of Mycobacterium smegmatis. The Thesis is organized as shown below: Chapter 1: The literature on the bacterial stationary phase physiology and the role of Dps has been reviewed in this chapter. It gives a brief introduction of the background of the present study including the stationary phase response of bacteria and the significance of studying bacteria under stress as apart from ideal conditions of growth, which has been the conventional approach until recently. The advantages of using Mycobacterium smegmatis as a model system, and its starvation-induced stationary phase are also discussed. An introduction to the Dps proteins as a family of proteins branched off from ferritins and nucleoid proteins is explained. A brief summary of the ferritin and nucleoid proteins is given. Similarities connecting Dps to both these protein families is described. The review of earlier work done in our laboratory on the mycobacterial MsDps1 protein is also presented. Chapter 2: involves the study of the solution properties of the protein including its ability to oligomerize in vitro. The MsDps1 protein exists in two forms, a trimer and a dodecamer. The trimer form is a unique feature of the M.smegmatis homologue. Dps proteins from other sources are characteristically dodecameric. Earlier studies have shown that the trimeric form of the protein can perform ferroxidation while the dodecamer can bind to DNA. The dodecamer can also perform ferroxidation and accumulate the oxidized iron in its negatively charged core. In this chapter, we show that the trimeric form is extremely stable, under various conditions of pHs. The protein, when over expressed in M.smegmatis, also shows the presence of the trimer, thus ruling out the effect of heterologous expression of the protein in E.coli. We further report here, the ideal conditions for dodecamerisation of the protein from trimer to dodecamer, which binds to DNA. The dodecamer once formed is also highly stable and does not revert back to the trimeric form. The structural stability of the dodecamer is expected, as it is the fully functional form of the protein that physically protects the DNA from stress. However, the high stability of the trimeric form and its precise conversion into a stable dodecamer is intriguing. It is interesting to study the functional significance in vivo of the oligomerisation process in MsDps1. In addition, we looked at the effect of over expression of the protein on the overall phenotype of Mycobacterium smegmatis, as evidenced by the colony morphology and find no visible alteration, when compared with the wild type. Chapter 3: deals with a more detailed structural analysis of the MsDps1 protein. The role of N and C termini of the protein in maintaining a stable oligomeric structure is studied by making an N-terminal deletion mutant of the protein which is found to be unable to form a dodecamer in solution. On the other hand, MsDps1 with a 16 amino acid C-terminal deletion, MsDpsΔC16, is able to form stable oligomeric structures, when the N-terminal is intact. A previous deletion reported from our laboratory with 26 amino acids deleted from the C-terminal tail, called MsDpsΔC26 showed inability to form stable oligomeric structures in vitro. Putting together all the above results, a model for the interaction of the N and C-terminal tails of the protein in maintaining a stable dodecamer is presented. A demarcation of the C-terminal tail of MsDps1 into regions determining the oligomeric stability and DNA binding was also inferred. The MsDpsΔC16 protein, does not bind to DNA although it forms a stable dodecamer. A further deletion of 10 amino acids, as seen in a previously made construct, MsDpsΔC26 disrupts both the DNA binding as well as the oligomeric stability of the protein. Chapter 4: describes the discovery of a new homolog of the Dps protein in M.smegmatis. It was named as MsDps2. Bio-informatics analysis carried out on the complete genome data of Mycobacterium smegmatis yielded a second homologue of Dps in addition to the one already present and characterized. Interestingly, out of the 300 homogues of Dps found in bacteria, only 195 are present as single copies in a bacterium. The rest exist as more than one homologue in the same bacterial genome. The basic characterization of this new Dps homologue and its confirmation as a Dps family member is the focus of this chapter. Chapter 5: deals with the possible functions of the new protein MsDps2. Electron micrography shows that the purified protein forms stable nucleoprotein-like complexes. Over expression of the MsDps2 proteins presents no difference in the colony morphology when compared with the wild-type. Western analysis shows that the MsDps2 protein is not expressed under normal conditions tested for growth. MsDps1, on the other hand shows expression under conditions of starvation and osmotic stress, as has been established previously in the laboratory. Hence, it can be inferred that the new protein MsDps2 does not perform the same function as MsDps1. However, the in vivo function of this protein remains an important question to be addressed. The appearance of in vitro nucleoid structures involving this protein under the electron microscope, suggests a possible role for this protein in the formation and stabilization of the mycobacterial nucleoid. Indeed extensive evidence for the same exists for the E.coli protein. Chapter 6: describes the results obtained from the sequence comparison of MsDps2 with other Dps proteins listed in the TIGR database. ClustalW sequence analysis, followed by the construction of a phylogenetic tree using the MEGA software, suggests that the mycobacterial Dps proteins fall into two separate groups, represented by the MsDps1 and MsDps2 homologues from Mycobacterium smegmatis. Chapter 7 Summary and Conclusions: A summary of the work presented in the thesis is given followed by the appendix sections. Appendix 1 includes list and maps of plasmids used. Appendix 2 details the theoretical DNA and protein sequences of the recombinant clones generated in the study and theoretical physical and chemical properties of the proteins studied, as calculated with the Expasy Protparam software. Appendix 3 includes raw data obtained from the bio-informatic analysis of MsDps2, obtained using ClustalW analysis.

In bacteria, nucleoid associated proteins (NAPs) represent a prominent group of global regulators that perform the tasks of genome compaction, establishing chromosomal architecture and regulation of various DNA transactions like replication, transcription, recombination and repair. HU, a basic histone like protein, is one of the most important NAPs in Eubacteria. Mycobacterium tuberculosis produces a homodimeric HU (MtbHU), which interacts with DNA non-specifically through minor groove binding. Exploration for essential genes in Mtb (H37Rv) through transposon insertion has identified HU coding gene [Rv2986c, hupB; Gene Id: 15610123; Swiss-Prot ID: P95109)] to be vital for the survival and growth of this pathogen. MtbHU contains two domains, the N-terminal domain which is considerably conserved among the HU proteins of the prokaryotic world, and a C–terminal domain consisting of Lys-Ala rich multiple repeat degenerate motifs. Sequence analysis carried out by the thesis candidate showed that MtbHU exhibits 86 to 100 percent identity within the N-term region among all the mycobacterium species and some of the members of actinobacteria, including important pathogens like M. tuberculosis, M. leprae, M. ulcerans, M. bovis, Nocardia; while C term repeat region varies relatively more. This strikingly high cross species identity establishes the MtbHU N-terminal domain (MtbHUN) as an important representative structural model for the above mentioned group of pathogens. The thesis candidate has solved the X-ray crystal structure of MtbHUN, crystallized in two different forms, P2 and P21. The crystal structures in combination with computational analyses elucidate the structural details of MtbHU interaction with DNA. Moreover, the similar mode of self assembly of MtbHUN observed in two different crystal forms reveals that the same DNA binding interface of the protein can also be utilized to form higher order oligomers, that HU is known to form at higher concentrations. Though the bifunctional interface involved in both DNA binding and self assembly is not akin to a typical enzyme active site, the structural analysis identified key interacting residues involved in macromolecular interactions, allowing us to develop a rationale for inhibitor design. Further, the candidate has performed virtual screening against a vast library of compounds, and design of small molecules to target MtbHU and disrupt its binding to DNA. Various biochemical, mutational and biological studies were performed in the laboratory of our collaborator Prof. V. Nagaraja, MCBL, IISc., to investigate these aspects. After a series of iterations including design, synthesis and validation, we have identified novel candidate molecules, which bind to MtbHU, disrupt chromosomal architecture and arrest M. tuberculosis growth. Thus, the study suggests that, these molecules can serve as leads for a new class of DNA-interaction inhibitors and HU as a druggable target, more so because HU is essential to Mtb, but absent in human. Our study proposes that, targeting the nucleoid associated protein HU in Mtb can strategize design of new anti-mycobacterial therapeutics. Perturbation of MtbHU-DNA binding through the identified compounds provides the first instance of medium to small molecular inhibitors of NAP, and augurs well for the development of chemical probe(s) to perturb HU functions, and can be used as a fundamental chemical tool for the system level studies of HU-interactome. Section I: “Crystal structure of Mycobacterium tuberculosis histone like protein HU and structure based design of molecules to inhibit MtbHU-DNA interaction: Leads for a new target.” of this thesis presents an elaborate elucidation of the above mentioned work. The candidate has additionally carried out structure based computational and theoretical work to elucidate the interaction of amino acid based metal complexes which efficiently bind to DNA via minor-groove, major-groove or base intercalation interaction and display DNA cleavage activity on photo-irradiation. This understanding is crucial for the design of molecules towards Photodynamic Therapy (PDT). PDT is an emerging method of non-invasive treatment of cancer in which drugs like Photofrin show localized toxicity on photoactivation at the tumor cells leaving the healthy cells unaffected. The work carried out in our group in close collaboration with Prof. A.R. Chakravarty of Inorganic and Physical Chemistry Department elaborates the structure based design of Amino acid complexes containing single Cu (II), such as [Cu(L-trp)(dpq)(H2O)]+ , [Cu (L-arg) 2](NO3)2 , Amino acid complexes containing oxobridged diiron Fe(III), such as [{Fe(L-his)(bpy)}2(μ-O)](ClO4)2 , [{Fe(L-his)(phen)}2(μ-O)](ClO4)2 , and Complexes containing Binuclear Cu(II) coordinated organic moiety, such as [{(dpq) CuII}2(μ-dtdp)2], which bind to DNA through minor groove/major groove/base intercalation interactions. Docking analysis was performed with the X-ray crystallographic structure of DNA as receptor and the metal complexes as ligands, to study the mode of binding to DNA and to understand the possible mode of DNA cleavage (single/double strand) when activated with laser. Section II: “Structure based computational and theoretical analysis of metal coordinated complexes containing amino acids and organic moieties designed for photo induced DNA cleavage” of this thesis presents a detailed presentation of the above mentioned work.