Department of Biological Sciences
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Item Dual Role for Zn2+ in Maintaining Structural Integrity and Inducing DNA Sequence Specificity in a Promiscuous Endonuclease(Elsevier, 2007-11) Ghosh, SoumitraWe describe two uncommon roles for Zn2+ in enzyme KpnI restriction endonuclease (REase). Among all of the REases studied, KpnI REase is unique in its DNA binding and cleavage characteristics. The enzyme is a poor discriminator of DNA sequences, cleaving DNA in a promiscuous manner in the presence of Mg2+. Unlike most Type II REases, the active site of the enzyme comprises an HNH motif, which can accommodate Mg2+, Mn2+, or Ca2+. Among these metal ions, Mg2+ and Mn2+ induce promiscuous cleavage by the enzyme, whereas Ca2+-bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase.Item Moonlighting function of glutamate racemase from Mycobacterium tuberculosis: racemization and DNA gyrase inhibition are two independent activities of the enzyme(American Society for Microbiology, 2008-09) Ghosh, SoumitraGlutamate racemase (MurI) provides d-glutamate, a key building block in the peptidoglycan of the bacterial cell wall. Besides having a crucial role in cell wall biosynthesis, MurI proteins from some bacteria have been shown to act as an inhibitor of DNA gyrase. Mycobacterium tuberculosis and Mycobacterium smegmatis MurI exhibit these dual characteristics. Here, we show that the two activities of M. tuberculosis MurI are unlinked and independent of each other. The racemization function of MurI is not essential for its gyrase-inhibitory property. MurI–DNA gyrase interaction influences gyrase activity but has no effect on the racemization activity of MurI. Overexpression of MurI in vivo provides resistance to the action of ciprofloxacin, suggesting the importance of the interaction in gyrase modulation. We propose that the moonlighting activity of MurI has evolved more recently than its racemase function, to play a transient yet important role in gyrase modulation.Item Regulation of Lipid Biosynthesis, Sliding Motility, and Biofilm Formation by a Membrane-Anchored Nucleoid-Associated Protein of Mycobacterium tuberculosis(American Society for Microbiology, 2013-03) Ghosh, SoumitraBacteria use a number of small basic proteins for organization and compaction of their genomes. By their interaction with DNA, these nucleoid-associated proteins (NAPs) also influence gene expression. Rv3852, a NAP of Mycobacterium tuberculosis, is conserved among the pathogenic and slow-growing species of mycobacteria. Here, we show that the protein predominantly localizes in the cell membrane and that the carboxy-terminal region with the propensity to form a transmembrane helix is necessary for its membrane localization. The protein is involved in genome organization, and its ectopic expression in Mycobacterium smegmatis resulted in altered nucleoid morphology, defects in biofilm formation, sliding motility, and change in apolar lipid profile. We demonstrate its crucial role in regulating the expression of KasA, KasB, and GroEL1 proteins, which are in turn involved in controlling the surface phenotypes in mycobacteria.Item HupB, a Nucleoid-Associated Protein of Mycobacterium tuberculosis, Is Modified by Serine/Threonine Protein Kinases In Vivo(American Society for Microbiology, 2014-06) Ghosh, SoumitraHU, a widely conserved bacterial histone-like protein, regulates many genes, including those involved in stress response and virulence. Whereas ample data are available on HU-DNA communication, the knowledge on how HU perceives a signal and transmit it to DNA remains limited. In this study, we identify HupB, the HU homolog of the human pathogen Mycobacterium tuberculosis, as a component of serine/threonine protein kinase (STPK) signaling. HupB is extracted in its native state from the exponentially growing cells of M. tuberculosis H37Ra and is shown to be phosphorylated on both serine and threonine residues. The STPKs capable of modifying HupB are determined in vitro and the residues modified by the STPKs are identified for both in vivo and the in vitro proteins through mass spectrometry. Of the identified phosphosites, Thr65 and Thr74 in the DNA-embracing β-strand of the N-terminal domain of HupB (N-HupB) are shown to be crucial for its interaction with DNA. In addition, Arg55 is also identified as an important residue for N-HupB–DNA interaction. N-HupB is shown to have a diminished interaction with DNA after phosphorylation. Furthermore, hupB is shown to be maximally expressed during the stationary phase in M. tuberculosis H37Ra, while HupB kinases were found to be constitutively expressed (PknE and PknF) or most abundant during the exponential phase (PknB). In conclusion, HupB, a DNA-binding protein, with an ability to modulate chromatin structure is proposed to work in a growth-phase-dependent manner through its phosphorylation carried out by the mycobacterial STPKs.Item Direct regulation of topoisomerase activity by a nucleoid-associated protein(OUP, 2014-09) Ghosh, SoumitraThe topological homeostasis of bacterial chromosomes is maintained by the balance between compaction and the topological organization of genomes. Two classes of proteins play major roles in chromosome organization: the nucleoid-associated proteins (NAPs) and topoisomerases. The NAPs bind DNA to compact the chromosome, whereas topoisomerases catalytically remove or introduce supercoils into the genome. We demonstrate that HU, a major NAP of Mycobacterium tuberculosis specifically stimulates the DNA relaxation ability of mycobacterial topoisomerase I (TopoI) at lower concentrations but interferes at higher concentrations. A direct physical interaction between M. tuberculosis HU (MtHU) and TopoI is necessary for enhancing enzyme activity both in vitro and in vivo. The interaction is between the amino terminal domain of MtHU and the carboxyl terminal domain of TopoI. Binding of MtHU did not affect the two catalytic trans-esterification steps but enhanced the DNA strand passage, requisite for the completion of DNA relaxation, a new mechanism for the regulation of topoisomerase activity. An interaction-deficient mutant of MtHU was compromised in enhancing the strand passage activity. The species-specific physical and functional cooperation between MtHU and TopoI may be the key to achieve the DNA relaxation levels needed to maintain the optimal superhelical density of mycobacterial genomes.Item Targeting Mycobacterium tuberculosis nucleoid-associated protein HU with structure-based inhibitors(Springer Nature, 2014-06) Ghosh, SoumitraThe nucleoid-associated protein HU plays an important role in maintenance of chromosomal architecture and in global regulation of DNA transactions in bacteria. Although HU is essential for growth in Mycobacterium tuberculosis (Mtb), there have been no reported attempts to perturb HU function with small molecules. Here we report the crystal structure of the N-terminal domain of HU from Mtb. We identify a core region within the HU–DNA interface that can be targeted using stilbene derivatives. These small molecules specifically inhibit HU–DNA binding, disrupt nucleoid architecture and reduce Mtb growth. The stilbene inhibitors induce gene expression changes in Mtb that resemble those induced by HU deficiency. Our results indicate that HU is a potential target for the development of therapies against tuberculosis.Item Transcriptional regulation of topology modulators and transcription regulators of Mycobacterium tuberculosis(Elsevier, 2016-07) Ghosh, SoumitraMycobacterium tuberculosis (Mtb) is a formidable pathogen which has the ability to survive the hostile environment of the host by evading the host defense system. The re-configuration of its transcriptional and metabolic process allows the pathogen to confront the adverse environment within the host macrophages. The factors that assist the transcription and modulate the DNA topology would have to play a key role in the regulation of global gene expression of the organism. How transcription of these essential housekeeping genes alters in response to growth conditions and environmental stress has not been addressed together in a set of experimental conditions in Mtb. Now, we have mapped the transcription start sites (TSS) and promoters of several genes that play a central role in the regulation of DNA topology and transcription in Mtb. Using in vivo reporter assays, we validated the activity of the identified promoter elements in different growth conditions. The variation in transcript abundance of these essential genes was also analyzed in growth phase-dependent manner. These data provide the first glimpse into the specific adaptive changes in the expression of genes involved in transcription and DNA topology modulation in Mtb.Item Lysine acetylation of the Mycobacterium tuberculosis HU protein modulates its DNA binding and genome organization(Wiley, 2016-01) Ghosh, SoumitraNucleoid-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.Item A Sir2 family protein Rv1151c deacetylates HU to alter its DNA binding mode in Mycobacterium tuberculosis(Elsevier, 2017-11) Ghosh, SoumitraTill 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.Item Convergent roles of ATF3 and CSL in chromatin control of cancer-associated fibroblast activation(JEM, 2017) Ghosh, SoumitraCancer-associated fibroblasts (CAFs) are important for tumor initiation and promotion. CSL, a transcriptional repressor and Notch mediator, suppresses CAF activation. Like CSL, ATF3, a stress-responsive transcriptional repressor, is down-modulated in skin cancer stromal cells, and Atf3 knockout mice develop aggressive chemically induced skin tumors with enhanced CAF activation. Even at low basal levels, ATF3 converges with CSL in global chromatin control, binding to few genomic sites at a large distance from target genes. Consistent with this mode of regulation, deletion of one such site 2 Mb upstream of IL6 induces expression of the gene. Observed changes are of translational significance, as bromodomain and extra-terminal (BET) inhibitors, unlinking activated chromatin from basic transcription, counteract the effects of ATF3 or CSL loss on global gene expression and suppress CAF tumor-promoting properties in an in vivo model of squamous cancer–stromal cell expansion. Thus, ATF3 converges with CSL in negative control of CAF activation with epigenetic changes amenable to cancer- and stroma-focused intervention.