Mu is a temperate bacteriophage which infects Escherichia coli and several other Gram negative enteric bacteria. It is an extraordinary phage in several respects and has carved a special niche for itself both as a genetic tool and a paradigm in phage biology, almost rivaling phage lambda. It is also a predator that has adapted its hunting skills well in order to have an extraordinarily wide host range. While phage Mu finds a mention in almost every genetics textbook for several of its unique and well-studied characteristics, there are a few aspects of its biology that are far from understood. In this thesis, light has been shed on one such less understood feature of Mu biology, namely its anti-restriction function. The enigmatic mom gene of bacteriophage Mu is the center of this thesis work. Bacteriophages, through their sheer number and versatility of attack tactics, constitute an overwhelming threat to bacteria in the natural environment. While it is not always possible to completely prevent the entry of foreign DNA into the cell, it is in the interest of the bacterium to tame the xenogeneic DNA, whose expression may have adverse effects on bacterial fitness. Bacterial nucleoid associated proteins (NAPs) participate in chromosome structuring and global transcriptional regulation. Besides this canonical role, they furnish the job of regulating xenogeneic DNA as well. NAPs are known to regulate the expression of toxin-antitoxin modules, pathogenicity islands and other horizontally transferred DNA and have a profound role in regulating transposon dynamics and the lifestyle of many phages. Chapter 1 introduces the role of bacterial NAPs in silencing foreign DNA, especially after the DNA establishes itself in the host. This thesis examines the role of a bacterial NAP namely Fis in fine-tuning an immune evasion function of bacteriophage Mu. A general introduction to phage Mu and its host expansion strategies, with special focus on its DNA modification function is also presented. Owing to the various immune evasion strategies, phages often have an upper hand on their hosts in the ongoing evolutionary arms race. One such strategy is DNA modification which bacteriophages have evolved as a means to protect their genomes from restriction enzymes of the host. While most phages employ the commonplace methylation modification for their anti-restriction function, phage Mu employs an unusual acetamido modification, catalysed by its protein Mom. Mom modified DNA is refractory to several restriction enzymes from different bacterial species. However, the modification is toxic to the host and thus mom expression needs to be precisely regulated to prevent untimely expression. A crowded multifactorial regulatory circuit has evolved to ensure the expression of mom without jeopardizing the welfare of the bacterial host. Chapter 2 uncovers a new player in mom regulation. The study shows that the bacterial chromatin architectural protein Fis is a transcriptional repressor of mom promoter and that Fis mediates its repressive effect by denying access to RNA polymerase at mom promoter. Two distinct roles of Fis have been known previously in Mu biology. In addition to bringing about the overall downregulation of transposition events and transcription of early genes of phage Mu, Fis also stimulates tail fiber flipping by aiding the activity of a site-specific recombinase. The present study thus presents a novel facet of Fis function in Mu biology. While the regulation of mom has been a matter of intense investigation over the past few decades, most biochemical and structural aspects of the Mom protein per se have remained mysterious owing to the difficulties in cloning this toxic gene. Chapter 3 describes the expression, purification and biophysical characterization of Mom. A variety of techniques show Mom to be folded and dimeric in solution. SPR studies with Mom indicate its high affinity binding to DNA. Chapter 4 deals with the attempts to identify the elusive co-factor of Mom. To begin with, the in vivo activity of Mom was demonstrated by employing a simple plasmid cleavage assay based on the resistance of Mom modified DNA to certain restriction endonucleases. A variety of disparate in silico structure prediction tools such as I-TASSER, Robetta and PHYRE indicate Mom to be related to the GCN5-related N-acetyltransferase superfamily. Mutation of residues deemed important from this analysis indeed abolished or reduced Mom activity in vivo, validating the bioinformatics based prediction and shed light on the possible active site of Mom. However, acetyltransferases are not known to transfer acetamido groups. It was also necessary to establish beyond doubt, the chemical structure of the Mom modified nucleoside. High resolution mass spectrometry data showed the modification to be acetamido, corroborating the earlier sole report on this aspect. Based on the biochemical reactions that acetyl coenzyme A is known to participate in, it is difficult to explain the involvement of acetyl coenzyme A in acetamido addition. Notwithstanding the converging predictions of different bioinformatics tools, caution is recommended when inferring function from structurally similar family members. It is possible that a different chemistry might have converged on the same (acetyltransferase) fold, given that none of the known pathways utilizing acetyl coenzyme A can explain the Mom modification. Several likely candidates such as carboxy-SAM, glyoxylic acid and glycine were also tested for being donors of the two carbon entity transferred on adenine by Mom. Since these candidates tested negative in our genetic assays, a genome-wide genetic screen was subsequently devised to identify the host genes involved in mom modification. The assay exploited the phenotype of lethality associated with overexpression of Mom in E. coli in order to screen for mutations in the host genome that rescued the toxicity. However, the survivors which were obtained in this assay had emerged through mutations in the mom gene rather than abrogation of the co-factor synthesis pathway of the host. The results point at two possibilities: (i) utilization of essential gene(s) or (ii) existence of redundant pathways for the Mom modification reaction. Chapter 5 is an account of our attempts to trace the lineage of mom and its regulatory region, employing updated DNA and protein sequence databases. Despite the selective advantage conferred on the phage by the anti-restriction function of mom, in many Mu-like phages, mom is either absent or substituted with methyltransferases. However, in Mu-like genomes that do encode mom, in spite of a significant overall sequence divergence from Mu, the core elements of the mom regulatory circuit seem to have either co-evolved or have been selectively conserved. Although Mu appears to be unique in the possession of a regulatory circuit tailored for the purpose of mom regulation, recently discovered Mu-like genomes show that different types of regulatory features evolved several times in closely related genomes. It is very likely that a toxic gene like mom has earned its place in the phage genome by carrying along with itself a baggage of regulatory elements. Failure to sustain sufficient regulatory pressure may trigger the loss or replacement of the advantageous but potentially lethal mom function.

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.