Nucleoid-associated proteins of mycobacteria come with a distinctive flavor.

In every bacterium, nucleoid-associated proteins (NAPs) play crucial roles in chromosome organization, replication, repair, gene expression, and other DNA transactions. Their central role in controlling the chromatin dynamics and transcription has been well-appreciated in several well-studied organisms. Here, we review the diversity, distribution, structure, and function of NAPs from the genus Mycobacterium. We highlight the progress made in our understanding of the effects of these proteins on various processes and in responding to environmental stimuli and stress of mycobacteria in their free-living as well as during distinctive intracellular lifestyles. We project them as potential drug targets and discuss future studies to bridge the information gap with NAPs from well-studied systems.

Identification of a 1-acyl-glycerol-3-phosphate acyltransferase from Mycobacterium tuberculosis, a key enzyme involved in triacylglycerol biosynthesis.

Latent tuberculosis, caused by dormant Mycobacterium tuberculosis (Mtb), poses a threat to global health through the incubation of undiagnosed infections within the community. Dormant Mtb, which is phenotypically tolerant to antibiotics, accumulates triacylglycerol (TAG) utilizing fatty acids obtained from macrophage lipid droplets. TAG is vital to mycobacteria, serving as a cell envelope component and energy reservoir during latency. TAG synthesis occurs by sequential acylation of glycerol-3-phosphate, wherein the second acylation step is catalyzed by acylglycerol-3-phosphate acyltransferase (AGPAT), resulting in the production of phosphatidic acid (PA), a precursor for the synthesis of TAG and various phospholipids. Here, we have characterized a putative acyltransferase of Mtb encoded by Rv3816c. We found that Rv3816c has all four characteristic motifs of AGPAT, exists as a membrane-bound enzyme, and functions as 1-acylglycerol-3-phosphate acyltransferase. The enzyme could transfer the acyl group to acylglycerol-3-phosphate (LPA) from monounsaturated fatty acyl-coenzyme A of chain length 16 or 18 to produce PA. Complementation of Escherichia coli PlsC mutant in vivo by Rv3816c confirmed that it functions as AGPAT. Its active site mutants, H43A and D48A, were incapable of transferring the acyl group to LPA in vitro and were not able to rescue the growth defect of E. coli PlsC mutant in vivo. Identifying Rv3816c as AGPAT and comparing its properties with other AGPAT homologs is not only a step toward understanding the TAG biosynthesis in mycobacteria but has the potential to explore it as a drug target.

Distinct subunit architecture and assembly pattern of DNA gyrase from mycobacteria.

DNA gyrase, the sole negative supercoiling type II topoisomerase, is composed of two subunits, GyrA and GyrB, encoded by the gyrA and gyrB genes, respectively, that form a quaternary complex of A2 B2 . In this study, we have investigated the assembly of mycobacterial DNA gyrase from its individual subunits, a step prerequisite for its activity. Using analytical size-exclusion chromatography, we show that GyrA from Mycobacterium tuberculosis and Mycobacterium smegmatis forms tetramers (A4 ) in solution unlike in Escherichia coli and other bacteria where GyrA exists as a dimer. GyrB, however, persists as a monomer, resembling the pattern found in E. coli. GyrB in both mycobacterial species interacts with GyrA and triggers the dissociation of the GyrA tetramer to facilitate the formation of catalytically active A2 B2 . Despite oligomerisation, the GyrA tetramer retained its DNA binding ability, and DNA binding had no effect on GyrA's oligomeric state in both species. Moreover, the presence of DNA facilitated the assembly of holoenzyme in the case of M. smegmatis by stabilising the GyrA2 B2 tetramer but with little effect in M. tuberculosis. Thus, in addition to the distinct organisation and regulation of the gyr locus in mycobacteria, the enzyme assembly also follows a different pattern.

Identification and characterization of a new HNH restriction endonuclease with unusual properties.

Restriction-modification (R-M) systems form a large superfamily constituting bacterial innate immunity mechanism. The restriction endonucleases (REases) are very diverse in subunit structure, DNA recognition, co-factor requirement, and mechanism of action. Among the different catalytic motifs, HNH active sites containing REases are the second largest group distinguished by the presence of the ßßα-metal finger fold. KpnI is the first member of the HNH-family REases whose homologs are present in many bacteria of Enterobacteriaceae having varied degrees of sequence similarity between them. Considering that the homologs with a high similarity may have retained KpnI-like properties, while those with a low similarity could be different, we have characterized a distant KpnI homolog present in a pathogenic Klebsiella pneumoniae NTUH K2044. A comparison of the properties of KpnI and KpnK revealed that despite their similarity and the HNH motif, these two enzymes have different properties viz oligomerization, cleavage pattern, metal ion requirement, recognition sequence, and sequence specificity. Unlike KpnI, KpnK is a monomer in solution, nicks double-stranded DNA, recognizes degenerate sequence, and catalyses the degradation of DNA into smaller products after the initial cleavage at preferred sites. Due to several distinctive properties, it can be classified as a variant of the Type IIS enzyme having nicking endonuclease activity. KEY POINTS: • KpnK is a distant homolog of KpnI and belongs to the ßßα-metal finger superfamily. • Both KpnI and KpnK have widespread occurrence in K. pneumoniae strains. • KpnK is a Type IIS restriction endonuclease with a single-strand nicking property.

Intrinsic and Rho-dependent termination cooperate for efficient transcription termination at 3' untranslated regions.

The intrinsic, and the Rho-dependent mechanisms of transcription termination are conserved in bacteria. Generally, the two mechanisms have been illustrated as two independent pathways occurring in the 3' ends of different genes with contrasting requirements to halt RNA synthesis. However, a majority of intrinsic terminators terminate transcription inefficiently leading to transcriptional read-through. The unwanted transcription in the downstream region beyond the terminator would have undesired consequences. To prevent such transcriptional read-through, bacteria must have evolved ways to terminate transcription more efficiently at or near the termination sites. We describe the participation of both the mechanisms, where intrinsic terminator and Rho factor contribute to prevent transcriptional read-through. Contribution from both the termination processes is demonstrated at the downstream regions of the genes both in vitro and in vivo in mycobacteria. Distinct patterns of cooperation between the two modes of termination were observed at the 3' untranslated regions of the genes to ensure efficient termination. We demonstrate similar mode of operation between the two termination processes in Escherichia coli suggesting a likely prevalence of this cooperation across bacteria. The reporter system developed to assess the Rho - intrinsic termination collaboration in vivo for mycobacteria and E. coli can readily be applied to other bacteria.

Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu.

The broad host range bacteriophage Mu employs a novel 'methylcarbamoyl' modification to protect its DNA from diverse restriction systems of its hosts. The DNA modification is catalyzed by a phage-encoded protein Mom, whose mechanism of action is a mystery. Here, we characterized the co-factor and metal-binding properties of Mom and provide a molecular mechanism to explain 'methylcarbamoyl'ation of DNA by Mom. Computational analyses revealed a conserved GNAT (GCN5-related N-acetyltransferase) fold in Mom. We demonstrate that Mom binds to acetyl CoA and identify the active site. We discovered that Mom is an iron-binding protein, with loss of Fe2+/3+-binding associated with loss of DNA modification activity. The importance of Fe2+/3+ is highlighted by the colocalization of Fe2+/3+ with acetyl CoA within the Mom active site. Puzzlingly, acid-base mechanisms employed by >309,000 GNAT members identified so far, fail to support methylcarbamoylation of adenine using acetyl CoA. In contrast, free-radical chemistry catalyzed by transition metals like Fe2+/3+ can explain the seemingly challenging reaction, accomplished by collaboration between acetyl CoA and Fe2+/3+. Thus, binding to Fe2+/3+, a small but unprecedented step in the evolution of Mom, allows a giant chemical leap from ordinary acetylation to a novel methylcarbamoylation function, while conserving the overall protein architecture.

DNA flowerstructure co-localizes with human pathogens in infected macrophages.

Herein, we characterize the cellular uptake of a DNA structure generated by rolling circle DNA amplification. The structure, termed nanoflower, was fluorescently labeled by incorporation of ATTO488-dUTP allowing the intracellular localization to be followed. The nanoflower had a hydrodynamic diameter of approximately 300 nanometer and was non-toxic for all mammalian cell lines tested. It was internalized specifically by mammalian macrophages by phagocytosis within a few hours resulting in specific compartmentalization in phagolysosomes. Maximum uptake was observed after eight hours and the nanoflower remained stable in the phagolysosomes with a half-life of 12 h. Interestingly, the nanoflower co-localized with both Mycobacterium tuberculosis and Leishmania infantum within infected macrophages although these pathogens escape lysosomal degradation by affecting the phagocytotic pathway in very different manners. These results suggest an intriguing and overlooked potential application of DNA structures in targeted treatment of infectious diseases such as tuberculosis and leishmaniasis that are caused by pathogens that escape the human immune system by modifying macrophage biology.

Direct control of type IIA topoisomerase activity by a chromosomally encoded regulatory protein.

Precise control of supercoiling homeostasis is critical to DNA-dependent processes such as gene expression, replication, and damage response. Topoisomerases are central regulators of DNA supercoiling commonly thought to act independently in the recognition and modulation of chromosome superstructure; however, recent evidence has indicated that cells tightly regulate topoisomerase activity to support chromosome dynamics, transcriptional response, and replicative events. How topoisomerase control is executed and linked to the internal status of a cell is poorly understood. To investigate these connections, we determined the structure of Escherichia coli gyrase, a type IIA topoisomerase bound to YacG, a recently identified chromosomally encoded inhibitor protein. Phylogenetic analyses indicate that YacG is frequently associated with coenzyme A (CoA) production enzymes, linking the protein to metabolism and stress. The structure, along with supporting solution studies, shows that YacG represses gyrase by sterically occluding the principal DNA-binding site of the enzyme. Unexpectedly, YacG acts by both engaging two spatially segregated regions associated with small-molecule inhibitor interactions (fluoroquinolone antibiotics and the newly reported antagonist GSK299423) and remodeling the gyrase holoenzyme into an inactive, ATP-trapped configuration. This study establishes a new mechanism for the protein-based control of topoisomerases, an approach that may be used to alter supercoiling levels for responding to changes in cellular state.

Rv0802c is an acyltransferase that succinylates and acetylates Mycobacterium tuberculosis nucleoid-associated protein HU.

Among the nucleoid-associated proteins (NAPs), HU is the most conserved in eubacteria, engaged in overall chromosome organization and regulation of gene expression. Unlike other bacteria, HU from Mycobacterium tuberculosis (MtHU), has a long carboxyl terminal domain enriched in basic amino acids, resembling eukaryotic histone N-terminal tails. As with histones, MtHU undergoes post-translational modifications and we have previously identified interacting kinases, methyltransferases, an acetyltransferase and a deacetylase. Here we show that Rv0802c interacts and succinylates MtHU. Although categorized as a succinyltransferase, we show that this GNAT superfamily member can catalyse both succinylation and acetylation of MtHU with comparable kinetic parameters. Like acetylation of MtHU, succinylation of MtHU caused reduced interaction of the NAP with DNA, determined by electrophoretic mobility shift assay and surface plasmon resonance. However, in vivo expression of Rv0802c did not significantly alter the nucleoid architecture. Although such succinylation of NAPs is rare, these modifications of the archetypal NAP may provide avenues to the organism to compensate for the underrepresentation of NAPs in its genome to control the dynamics of nucleoid architecture and cellular functions.

Genome-wide mapping of Topoisomerase I activity sites reveal its role in chromosome segregation.

DNA Topoisomerase I (TopoI) in eubacteria is the principle DNA relaxase, belonging to Type 1A group. The enzyme from Mycobacterium smegmatis is essential for cell survival and distinct from other eubacteria in having several unusual characteristics. To understand genome-wide TopoI engagements in vivo, functional sites were mapped by employing a poisonous variant of the enzyme and a newly discovered inhibitor, both of which arrest the enzyme activity after the first transestrification reaction, thereby leading to the accumulation of protein-DNA covalent complexes. The cleavage sites are subsets of TopoI binding sites, implying that TopoI recruitment does not necessarily lead to DNA cleavage in vivo. The cleavage protection conferred by nucleoid associated proteins in vitro suggest a similar possibility in vivo. Co-localization of binding and cleavage sites of the enzyme on transcription units, implying that both TopoI recruitment and function are associated with active transcription. Attenuation of the cleavage upon Rifampicin treatment confirms the close connection between transcription and TopoI action. Notably, TopoI is inactive upstream of the Transcription start site (TSS) and activated following transcription initiation. The binding of TopoI at the Ter region, and the DNA cleavage at the Ter indicates TopoI involvement in chromosome segregation, substantiated by its catenation and decatenation activities.

Physical and functional interaction between nucleoid-associated proteins HU and Lsr2 of Mycobacterium tuberculosis: altered DNA binding and gene regulation.

Nucleoid-associated proteins (NAPs) in bacteria contribute to key activities such as DNA compaction, chromosome organization and regulation of gene expression. HU and Lsr2 are two principal NAPs in Mycobacterium tuberculosis (Mtb). HU is essential for Mtb survival and is one of the most abundant NAPs. It differs from other eubacterial HU proteins in having a long, flexible lysine- and arginine-rich carboxy-terminal domain. Lsr2 of Mtb is the functional analogue of the bacterial NAP commonly called H-NS. Lsr2 binds to and regulates expression of A/T-rich portions of the otherwise G/C-rich mycobacterial chromosome. Here, we demonstrate that HU and Lsr2 interact to form a complex. The interaction occurs primarily through the flexible carboxy-terminal domain of HU and the acidic amino-terminal domain of Lsr2. The resulting complex, upon binding to DNA, forms thick nucleoprotein rods, in contrast to the DNA bridging seen with Lsr2 and the DNA compaction seen with HU. Furthermore, transcription assays indicate that the HU-Lsr2 complex is a regulator of gene expression. This physical and functional interaction between two NAPs, which has not been reported previously, is likely to be important for DNA organization and gene expression in Mtb and perhaps other bacterial species.

Revisiting intrinsic transcription termination in mycobacteria: U-tract downstream of secondary structure is dispensable for termination.

In Escherichia coli, G/C-rich hairpin structure followed by a U-tract in the 3' region of the nascent RNA are crucial determinants for intrinsic or factor independent transcription termination. In mycobacteria, there is a scarcity of such intrinsic terminators. However, secondary structures having G/C-rich stem devoid of any U's or with suboptimal U-tracts were identified earlier as terminators and found to be functional both in vitro and in vivo. Two different observations - that a mycobacterial RNA polymerase (RNAP) does not function at intrinsic terminators devoid of U-tracts and the identification of an altogether new motif for termination in mycobacteria necessitated re-examining a number of putative terminators for their function as terminators. When these in silico identified non-canonical terminators were subjected to experimental validation, they were found to dissociate RNA from the elongating RNAP. Termination is observed when the U-tracts were reduced, or totally absent both in vitro and in vivo. Our results, thus indicate that the presence of U-tract following the G/C-rich stem in an intrinsic terminator may not be an essential determinant for transcription termination in mycobacteria.

Transcription facilitated genome-wide recruitment of topoisomerase I and DNA gyrase.

Movement of the transcription machinery along a template alters DNA topology resulting in the accumulation of supercoils in DNA. The positive supercoils generated ahead of transcribing RNA polymerase (RNAP) and the negative supercoils accumulating behind impose severe topological constraints impeding transcription process. Previous studies have implied the role of topoisomerases in the removal of torsional stress and the maintenance of template topology but the in vivo interaction of functionally distinct topoisomerases with heterogeneous chromosomal territories is not deciphered. Moreover, how the transcription-induced supercoils influence the genome-wide recruitment of DNA topoisomerases remains to be explored in bacteria. Using ChIP-Seq, we show the genome-wide occupancy profile of both topoisomerase I and DNA gyrase in conjunction with RNAP in Mycobacterium tuberculosis taking advantage of minimal topoisomerase representation in the organism. The study unveils the first in vivo genome-wide interaction of both the topoisomerases with the genomic regions and establishes that transcription-induced supercoils govern their recruitment at genomic sites. Distribution profiles revealed co-localization of RNAP and the two topoisomerases on the active transcriptional units (TUs). At a given locus, topoisomerase I and DNA gyrase were localized behind and ahead of RNAP, respectively, correlating with the twin-supercoiled domains generated. The recruitment of topoisomerases was higher at the genomic loci with higher transcriptional activity and/or at regions under high torsional stress compared to silent genomic loci. Importantly, the occupancy of DNA gyrase, sole type II topoisomerase in Mtb, near the Ter domain of the Mtb chromosome validates its function as a decatenase.

Two negative regulators of biofilm development exhibit functional divergence in conferring virulence potential to Candida albicans.

Candida albicans, a human pathogen, carries an expanded family of Zn(II)2Cys6 transcription factors. A CTG clade-specific protein Zcf32 and its closely related protein Upc2, a well-conserved transcription factor across the various fungal species, belong to this family of proteins. Unlike Upc2, Zcf32 is poorly studied in C. albicans. Here, we examined roles played by these two related transcription factors in biofilm development and virulence of C. albicans. Our data show that the null mutants of each of Zcf32 or Upc2 form better biofilms than the wild-type suggesting that both of them negatively regulate the biofilm development. While acting as negative regulators of biofilm formation, these two transcription factors target a different set of biofilm genes. A mouse model of candidiasis reveals that zcf32/zcf32 was hypervirulent, while upc2/upc2 shows compromised virulence compared to the wild-type. Notably, the absence of Zcf32 enhances detrimental inflammation brought about by TNFα, IFNß and IFNγ. upc2/upc2 failed to generate a similar feedback, instead demonstrated an elevated anti-inflammatory (IL4 and IL10) host response. Taking together, we show how a recently evolved transcription factor Zcf32 retained functional resemblance with a more ubiquitous member Upc2 but also functionally diverged from the latter in the regulation of biofilm development and virulence of the pathogen.

Restriction endonuclease triggered bacterial apoptosis as a mechanism for long time survival.

Programmed cell death (PCD) under certain conditions is one of the features of bacterial altruism. Given the bacterial diversity and varied life style, different PCD mechanisms must be operational that remain largely unexplored. We describe restriction endonuclease (REase) mediated cell death by an apoptotic pathway, beneficial for isogenic bacterial communities. Cell death is pronounced in stationary phase and when the enzyme exhibits promiscuous DNA cleavage activity. We have elucidated the molecular mechanism of REase mediated cell killing and demonstrate that released nutrients from dying cells support the growth of the remaining cells in the population. These findings illustrate a new intracellular moonlighting role for REases which are otherwise established host defence arsenals. REase induced PCD appears to be a cellular design to replenish nutrients for cells undergoing starvation stress and the phenomenon could be wide spread in bacteria, given the abundance of restriction-modification (R-M) systems in the microbial population.

Regulation of the gyr operon of Mycobacterium tuberculosis by overlapping promoters, DNA topology, and reiterative transcription.

DNA gyrase introduces negative supercoils into DNA to maintain topological homeostasis. The genes encoding gyrase, gyrB and gyrA, form a dicistronic operon in Mycobacterium tuberculosis (Mtb) and other actinobacteria. Earlier work indicated that DNA relaxation stimulates the expression of the gyr genes, a phenomenon termed relaxation-stimulated transcription (RST). The present study addresses the underlying mechanism of gyr operon regulation. The operon is regulated by overlapping and divergently oriented promoters located upstream of gyrB. The principal promoter, PgyrB1, drives transcription of the operon, while a weak "reverse" promoter, PgyrR, transcribes in opposite direction. We demonstrate that PgyrR plays a role in fine tuning gyr gene expression by reiterative transcription (RT), a regulatory mechanism hitherto not found in Mtb. In vitro transcription assays showed that RT at PgyrR depended on the negatively supercoiled state of the DNA template. The principal promoter, PgyrB1, was also sensitive to DNA supercoiling, but it was stimulated by DNA relaxation. Moreover, RNA polymerase binding to the promoter was efficient at PgyrB1 when template DNA was relaxed, whereas binding to PgyrR was preferred when DNA was supercoiled. Thus, a collaboration between RST and RT governs the regulation of the gyr operon; the differing sensitivity of the two overlapping promoters to superhelix density explains how gyrase expression responds to changes in supercoiling to determine the efficiency of transcription initiation.

Total Synthesis of Ripostatin B and Structure-Activity Relationship Studies on Ripostatin Analogs.

Described is the total synthesis of the myxobacterial natural product ripostatin B and of a small number of analogs. Ripostatin B is a polyketide-derived 14-membered macrolide that acts as an inhibitor of bacterial RNA-polymerase, but is mechanistically distinct from rifamycin-derived RNA-polymerase inhibitors that are in use for tuberculosis treatment. The macrolactone ring of ripostatin B features two stereocenters and a synthetically challenging doubly skipped triene motif, with one of the double bonds being in conjugation with the ester carbonyl. Appended to the macrolactone core are an extended hydroxy-bearing phenylalkyl side chain at C13 and a carboxymethyl group at C3. The triene motif was established with high efficiency by ring-closing olefin metathesis, which proceeded in almost 80% yield. The side chain-bearing stereocenter α to the ester oxygen was formed in a Paterson aldol reaction between a methyl ketone and a ß-chiral ß-hydroxy aldehyde with excellent syn selectivity (dr >10:1). The total synthesis provided a blueprint for the synthesis of analogs with modifications in the C3 and C13 side chains. The C3-modified analogs showed good antibacterial activity against efflux-deficient Escherichia coli but, as ripostatin B, were inactive against Mycobacterium tuberculosis, in spite of significant in vitro inhibition of M. tuberculosis RNA-polymerase.

Autoregulation of topoisomerase I expression by supercoiling sensitive transcription.

The opposing catalytic activities of topoisomerase I (TopoI/relaxase) and DNA gyrase (supercoiling enzyme) ensure homeostatic maintenance of bacterial chromosome supercoiling. Earlier studies in Escherichia coli suggested that the alteration in DNA supercoiling affects the DNA gyrase and TopoI expression. Although, the role of DNA elements around the promoters were proposed in regulation of gyrase, the molecular mechanism of supercoiling mediated control of TopoI expression is not yet understood. Here, we describe the regulation of TopoI expression from Mycobacterium tuberculosis and Mycobacterium smegmatis by a mechanism termed Supercoiling Sensitive Transcription (SST). In both the organisms, topoI promoter(s) exhibited reduced activity in response to chromosome relaxation suggesting that SST is intrinsic to topoI promoter(s). We elucidate the role of promoter architecture and high transcriptional activity of upstream genes in topoI regulation. Analysis of the promoter(s) revealed the presence of sub-optimal spacing between the -35 and -10 elements, rendering them supercoiling sensitive. Accordingly, upon chromosome relaxation, RNA polymerase occupancy was decreased on the topoI promoter region implicating the role of DNA topology in SST of topoI. We propose that negative supercoiling induced DNA twisting/writhing align the -35 and -10 elements to facilitate the optimal transcription of topoI.

Lysine acetylation of the Mycobacterium tuberculosis HU protein modulates its DNA binding and genome organization.

Nucleoid-associated protein HU, a conserved protein across eubacteria is necessary for maintaining the nucleoid organization and global regulation of gene expression. Mycobacterium tuberculosis HU (MtHU) is distinct from the other orthologues having 114 amino acid long carboxyl terminal extensions with a high degree of sequence similarity to eukaryotic histones. In this study, we demonstrate that the DNA binding property of MtHU is regulated by posttranslational modifications akin to eukaryotic histones. MtHU purified from M. tuberculosis cells is found to be acetylated on multiple lysine residues unlike the E. coli expressed recombinant protein. Using coimmunoprecipitation assay, we identified Eis as one of the acetyl transferases that interacts with MtHU and modifies it. Although Eis is known to acetylate aminoglycosides, the kinetics of acetylation showed that its protein acetylation activity on MtHU is robust. In vitro Eis modified MtHU at various lysine residues, primarily those located at the carboxyl terminal domain. Acetylation of MtHU caused reduced DNA interaction and alteration in DNA compaction ability of the NAP. Over-expression of the Eis leads to hyperacetylation of HU and decompaction of genome. These results provide first insights into the modulation of the nucleoid structure by lysine acetylation in bacteria.

A Sir2 family protein Rv1151c deacetylates HU to alter its DNA binding mode in Mycobacterium tuberculosis.

Till recently, knowledge about epigenetic regulation in bacterial world confined largely to DNA methylation. Lysine acetylation/deacetylation of histones is a major contributor for chromatin dynamics in eukaryotes. However, little is known about such epigenetic changes brought about by post-translational modifications in bacteria. Here, we describe an example of such mechanism occurring in a histone like protein, HU from Mycobacterium tuberculosis (Mtb). Previously, we demonstrated the interaction and acetylation of Mtb HU (MtHU) by one of the acetyl transferases, Eis. In this work, we demonstrate the deacetylation of acetylated HU (MtHUAc) by Rv1151c, the only Sir2 like protein discovered in Mtb. The DNA binding properties of MtHU are significantly altered upon acetylation but reversed consequent to deacetylation by the deacetylase. Deacetylated HU (MtHUdAc) bound to relaxed DNA leading to the formation of looped and dense molecules as compared to open structures formed by its acetylated form. Interaction of MtHUdAc with linear DNA modifies its organization leading to formation of highly bridged compact structures while binding of MtHUAc leads to the formation of stiff and straight rods. That a nucleoid associated protein can undergo acetylation/deacetylation to alter its DNA binding and architectural role opens up a new dimension of investigation of epigenetic regulation in mycobacteria.