Oxidative stress is a universal phenomenon experienced by both aerobic and anaerobic organisms. Reactive oxygen species (ROS) are generated during the stress, which can damage most cellular components including proteins, lipids and DNA. Naturally, organisms have evolved defence mechanisms to prevent oxidative damage. In prokaryotic systems, Dps (DNA binding protein from stationary phase cells) forms an important component of the mechanisms. Dps is known to be produced maximally during the stationary phase of bacterial growth. They exhibit ferroxidase activity as well. Dps homologs have been identified in a variety of distantly related bacteria, thus implying that this protein has a crucial function. The crystal structures of these proteins from a few bacteria are available. The work reported here is concerned with structural studies on Dps molecules from Mycobacterium smegmatis. Well-established X-ray crystallographic techniques were used to study the structures reported here. Hanging drop vapour diffusion and microbatch methods were used for crystallization. X-ray intensity data were collected on MAR Research imaging plates mounted on Rigaku X-ray generators. The data were processed using the HKL program suite. All the structures were solved by the molecular replacement method using the programs AMoRe and PHASER. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using FRODO and COOT. PROCHECK, ALIGN, INSIGHT, NACCESS, HBPLUS, CONTACT and ESCET were used for validation and analysis of the refined structures. Figures were prepared using MOLSCRIPT, BOBSCRIPT, RASTER3D and PYMOL. The structure of the first Dps identified in M. smegmatis has been determined in three crystal forms and has been compared with those of similar proteins from other sources. The dodecameric molecule can be described as a distorted icosahedron. The interfaces among subunits are such that the dodecameric molecule appears to have been made up of stable trimers. The situation is similar in the proteins from Escherichia coli and Agrobacterium tumefaciens, which are closer to the M. smegmatis protein in sequence and structure than those from other sources, which appear to form a dimer first. Trimerisation is aided in the three proteins by the additional N-terminal stretches they possess. The M. smegmatis protein has an additional C-terminal stretch compared to other related proteins. The stretch, known to be involved in DNA binding, is situated on the surface of the molecule. A comparison of the available structures permits a delineation of the rigid and flexible regions in the molecule. The subunit interfaces around the molecular dyads, where the ferroxidation centres are located, are relatively rigid. Regions in the vicinity of the acidic holes centred around molecular threefold axes, are relatively flexible. So are the DNA binding regions. The crystal structures of the protein from M. smegmatis confirm that DNA molecules can occupy spaces within the crystal without disturbing the arrangement of the protein molecules. However, contrary to earlier suggestions, the spaces need not to be between layers of the protein molecules. The cubic form provides an arrangement in which grooves, which could hold DNA molecules, criss-cross the crystal. M. smegmatis Dps is characterised by a 26 residue C-terminal tail which has been shown to be involved in DNA binding. The protein spontaneously degrades into a species in which 16 C-terminal residues are cleaved away. This species does not bind DNA, but forms dodecamers. A second species in which all the 26 residues constituting the tail were deleted not only does not bind to DNA, but also fails to assemble into dodecamers, indicating a role in assembly also for the C terminal tail. Therefore, the crystal structure of the species without the entire C-terminal tail was carried out. The molecule of the C-terminal mutant has an unusual open decameric structure, resulting from the removal of two adjacent subunits from the original dodecameric structure of the native form. It has been earlier shown that a Dps dodecamer could assemble with a dimer or one of two trimers (Trimer-A and Trimer-B) as intermediate and that Trimer-A is the intermediate species in the M. smegmatis protein. Estimation of surface area buried on trimerisation indicates that association within Trimer-B is weak. It further weakens when the C-terminal tail is removed, leading to the disruption of the dodecameric structure. Thus, the C-terminal tail has a dual role, one in DNA binding and the other in the assembly of the dodecamer. M. smegmatis Dps also has a short N-terminal tail of 9 residues. A species with this tail deleted, forms trimers in solution, but not dodecamers unlike wild type M. smegmatis Dps, under the same conditions. The crystal structure of this N-terminal mutant was also determined. Unlike in solution, the N-terminal mutant forms dodecamers in the crystal. In native Dps, the N-terminal stretch of one subunit and the C-terminal stretch of a neighbouring subunit lock each other into ordered positions. The deletion of one stretch results in the disorder of the other. This disorder appears to result in the formation of a trimeric species of the N-terminal deletion mutant contrary to the indication provided by the native structure. The ferroxidation site is intact in the mutants. A second DNA binding protein from stationary phase cells of M. smegmatis (MsDps2) has been identified from the bacterial genome and its crystal structure determined. The core dodecameric structure of MsDps2 is the same as that of the Dps from the organism described earlier (MsDps1). However, MsDps2 possesses a long N-terminal tail instead of the C-terminal tail in MsDps1. This tail appears to be involved in DNA binding. It is also intimately involved in stabilizing the dodecamer. Partly on account of this factor, MsDps2 assembles straightway into the dodecamer while MsDps1 does so on incubation after going through an intermediate trimeric stage. The ferroxidation centre is similar in the two proteins while the pores leading to it exhibit some difference. The mode of sequestration of DNA in the crystalline array of molecules, as evidenced by the crystal structures, appears to be different in MsDps1 and MsDps2, highlighting the variability in the mode of Dps-DNA complexation. A sequence search led to the identification of 300 Dps molecules in bacteria with known genome sequences. 50 bacteria contain 2 or more types of Dps molecules each, while 195 contain only one type. Some bacteria, notably some pathogenic ones, do not contain Dps. A sequence signature for Dps could also be derived from the analysis In addition to the work on Dps molecules, the author was also involved in studies on the crystal structures of the adipic acid complexes of L- and DL-arginine and supramolecular association in arginine-dicarboxylic acid complexes. This investigation, carried out primarily to obtain a good grounding in crystallography, is presented in an appendix.

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.

The first chapter will provide an introduction to the physiology, pathogenesis and biology of mycobacteria. Host-pathogen interactions, different modes of resistance of the bacteria, adaptations for survival under nutrient and oxygen depleted conditions has been discussed. This is followed by a general discussion on gene expression and regulation in the microbe. The physiology of bacteria under stresses from the view of the transcriptional regulation of specific genes has also been discussed. The scope and objective of the present study in M. smegmatis covered in the thesis has been considered at the end. The next chapter discusses the characterization of msdps promoter in vivo with the help of reporter gene assay technologies. With the advent of promoterless E. coli-mycobacterium shuttle vectors, activity assays can be easily performed to characterize unknown upstream putative promoter sequences of genes. Both the 1 kb upstream as well as a 200bp upstream region of msdps gene has been characterized by. Primer extension analysis and subsequent site directed mutagenesis studies reveal +1 transcription start site and the promoter consensus sequence for the msdps gene respectively. Next chapter comprises of the method of constructing heterologous in vitro transcription machinery in mycobacteria. It is followed by characterization of transcription initiation at two dps promoters of M. smegmatis. A novel pull-down assay has been designed which enabled us to identify the sigma factors in the reconstituted RNA polymerases to be associated with the respective dps promoters and to compare the regulation of the two genes at transcription level. Further characterization through single round in vitro transcription at mycobacterial promoters has been attempted. The following two chapters provide some newer insights into the structure-function relationship of the first Dps molecule, MsDps (MsDps1) with respect to its DNA binding activity. The DNA binding activity is associated with the higher oligomeric form only. With the help of time resolved anisotropy and Förster Resonance Energy Transfer (FRET) experiments, we have monitored the nature of Dps dodecamer-DNA complex and mapped the distance between the N and C169 position in the absence and the presence of DNA. A new computational programme, Maximum Entropy Method (MEM) has been applied successfully to analyze data obtained from phase-modulation (Phi-M) lifetime experiments in order to get distribution of lifetime. In the last chapter a new method is adopted to predict amino acids important for stabilizing the interface in a trimeric structure. Subsequently, single and double amino acid mutants of the native MsDps protein has been constructed through site directed mutagenesis and are scored for the ability of the mutants to oligomerize under conditions similar to that of the native protein. This helped us to propose a hypothetical model of the overall mechanism of the protein oligomerization process in solution.

The DNA binding proteins under starvation (Dps) from M. smegmatis are cage-like structures which internalize iron and bind DNA. They provide resistance to the cells from free radical damage, and physically protect the DNA from the harmful effects of reactive oxygen species by DNA compaction. The work compiled in this thesis has been an effort to study oligomerization and dynamics of iron metabolism by these nano-protein compartments. Chapter 1 gives a general introduction on stress, especially oxidative stress, and the ways bacteria fight back the host resistance systems. This has been elaborated from the point of view of the Dps proteins which is the focus of our work. Also, the competition for iron among the host and pathogens, and the modes of iron trafficking of the pathogens from host organisms has been summarized. Finally, the structural aspects of ferritin family proteins to which Dps belongs, has been discussed. Chapter 2 elaborates on the oligomerization pathways of the first M. smegmatis Dps MsDps1, which exists in vitro as two oligomeric forms. The GFP-tagging has been used to locate the Dps1 proteins by live cell imaging and the over-expression of these proteins during nutrient limiting conditions has been studied. The crystal structure of a point mutant F47E in the background of MsDps1, which shows no dodecamerization in vitro, has been solved. The possible ways of dodecamerization of MsDps1 has been concluded by analyzing the intermediates via glutaraldehyde cross-linking and native electrospray mass spectrometry. Chapter 3 documents the gating machinery of iron in MsDps2 protein, the second M. smegmatis Dps protein. Through graph theoretical approaches, a tight histidine-aspartate cluster was identified at the ferritin-like trimeric pore which harbors the channel for the entry and exit of iron. Sitespecific variants of MsDps2 were generated to disrupt this ionic knot, and the mutants were further assayed for ferroxidation, iron uptake and iron release properties. Our studies in MsDps2 show the importance of counter-acting positive and negatively charged residues for efficient assimilation and dispersion of iron. Chapter 4 describes crystallization studies of MsDps2 pore variants, done in an attempt to connect the changes in functional properties described in chapter 3, with structural alterations of the point mutants. We show here that the gating mechanism happens by alterations in side chain configuration at the pore and does not alter the over-all stability of the proteins. Chapter 5 is the final section where we have employed site specific mutations and cocrystallization studies to elucidate the behaviour of MsDps2 proteins upon the addition of iron. By studying the effect of substitutions at conserved sites near ferroxidation center, we attempt to arrive at a pathway which iron atoms take to reach the ferroxidation site. Also, by crystallization of proteins loaded with varying amounts of iron we tried to map the changes in the protein structure in the presence of its ligand. Chapter 6 concludes briefly the work that has been documented in this thesis. Appendix I relates the role of N-terminal tail for DNA binding in MsDp2. Appendix II gives the technical details of a modified protein preparation and oligomerization process for his-tagged MsDps1 protein. Appendix III gives the maps of the plasmids used in this study.