No description available.

No description available.

No description available.

Helicobacter pylori is one of the most genetically diverse bacterial species that successfully colonizes at least 50% of the world population. It has been associated with humans for thousands of years and most probably evolved from ancestral gastric Helicobacter species in early mammals. One of the important characteristics of this pathogen is the degree of allelic diversity and genetic variability which helps it to adapt and colonize. Phase variation is one of the mechanisms used by H. pylori to generate variation. The presence of homopolymeric nucleotide or dinucleotide repeats in an ORF make it prone to frequent length changes as a consequence of slipped strand mispairing mediated mutagenesis. Interestingly, R-M genes comprise a significant percentage of H. pylori strain-specific genes and are more prevalent in H. pylori than in other bacterial species whose genomes have been fully sequenced. R-M systems in H. pylori have been identified on the basis of sequence similarity to known restriction endonucleases and methyltransferases, genetic organization, and specific enzyme isolation and characterization. Analysis of genome sequences of H. pylori strains 26695, J99, HPAGI and 26 others has revealed the presence of more than 20 R-M systems in each stain, which are far more than detected in any other bacterial genome sequence till date. hp1369 and hp1370 are two ORFs in stain 26695 coding for hypothetical proteins. hp 1369 has a stretch of poly-G repeats, thus making hp1369-hp1370, a candidate of phase variation. hpag1_1313 is homolog of hp1369-hp1370 which got up-regulated, in a person suffering from acute gastritis, thus making these genes an interesting subject of investigation. This study was therefore initiated with the following objectives: 1. Cloning, over-expression and purification of Type III MTase (ORF- hp1369- hp1370) and its cognate restriction enzyme (hp1371). 2. Biochemical characterization of MTase (HP1369-HP1370): Determination of oligomeric status, kinetic properties, binding affinities for AdoMet and DNA. Sequence analysis shows the presence of a poly-G track (10 Gs) at 3’-end of hp1369 which is a signature sequence for phase variation. Addition of a single nucleotide can place both hp1369 and hp1370 in-frame, which could code for a single polypeptide. hp1369 and hp1370 in H. pylori strain 26695 alone do not code for any functional protein but with the fusion of hp1369 and hp1370 can code for a protein with all the nine motifs of a DNA MTase. Interestingly, on the basis of arrangement of Motifs, it is probably the first example of ε type of methyltransferase. By site-directed mutagenesis a single G nucleotide was inserted in the poly-G track and both the ORFs (hp1369 and hp1370 ) became in-frame, coding for fully functional HP1369-HP1370 MTase. Kinetic parameters for functional HP1369-HP1370 MTase were determined, and has shown that there was substrate inhibition in methylation reaction at higher concentrations of AdoMets. When preincubation studies were done, enzyme-DNA complex was found to be more competent than enzyme-AdoMet complex. HP1369-HP1370 MTase exists as dimer in solution, having affinity for duplex DNA and does not bind to single-stranded DNA. Binding affinity for ligand (AdoMet) was determined by Isothermal Titration Calorimetry method. H. pylori has evolving restriction-modification systems. It is capable of taking new R-M systems from the environment in the form of DNA released from other bacteria or other Helicobacter strains. H. pylori genome is dynamic with high mutation rates. Random mutations in R-M genes can result in a non-functional R-M systems or R-M systems with new properties. The dynamics of R-M system plays a vital role in shaping up the genome.

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Enzyme DNA methylation is an important biochemical process that imprints DNA with additional information. DNA methylation is catalyzed by S-adenosyl-L-methionine (AdoMet)-dependent methyltraferases (MTases). Prokaryotic DNA MTases are usually components of restriction-modification(R-M) systems that enable cells to resist propagation of foreign genomes that would otherwise kill them. Based on the position methyl group transfer on the bases in DNA, MTases are classified into two groups-exocyclic or amino MTases and endocyclic or ring MTases. The amino MTases methylate exocyclic amino nitrogen to form either N6-methyladenine or n4-methycytosine. N6-methyaladenine is mostly found in the genomes of bacteria, archaea protists and fungi. Helicobacter pylori is a gram-negative, flagellated, fastidious bacterium that colonizes the highly acidic environment of the gastric mucosa. Frequently and persistence of H.paylori infection in humans make it attractive model for studying the host- pathogen interaction mechanisms. Analysis of the genome sequence of H.pylori strains 26695, J99.HPAGI, and G27 revealed an abundance of restriction-modification (R-M) systems. Most of the R-M system genes are either conserved among the strains or specific to each strain. Strain specific genes are responsible for different phenotypes in several host adapted pathogens such as H.pylori. Many of the R-M gene homologues exhibit different usages of condon bias and lower G+C content from the average genes suggesting horizontal transfer of the R-M system genes in H. Pylori. Genome analysis of strain 26695 showed the presence of three putative type III R-M systems and hp0592-hp0593 constitutes one such type III R-M system. Based on the conserved motif arrangements, HP0593 MTases belongs to the subgroups of MTases. The amino acid sequence of HP0593 MTases has 38% sequence identity to Ecop11 MTases and EcoP151 MTase, both of which belongs to type IIIR-M systems therefore, it was important to study in detail previously unexplored role of this putative type III DNA MTase (HP0593) in H. Pylori. Investigation of methyltransferease activity and sequence specifically of putative DNA adenine MTase (HP0593) HP0593 (N6-adenine) - DNA MTase is a member of a type III R-M system in H. pylori strain 26695. HP0593 MTase has been cloned, over expressed and purified heterologously in Escherichia coli. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was carried out with purified HP0593 and profile showed a single peak with expected molecular mass of 70.6kDa. The protein was determined as-5.8. HP0593 MTase exits predominantly as monomer and a small fraction as dimer in solution as determined by size exclusion chromatography and glutaraldehyde cross-linking studies. The recognition sequence of the purified MTase was determined as 5’GCAG-3’ and the target base of methylation is adenine. Dot-blot assay using antibodies that reacted specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine specific MTase. Exocyclic MTase have a conserved catalytic motif (D/N/S/SPPY/F/W). Most interestingly, the amino acid sequence analysis of HP0593 MTase revealed the presence of a PCQ-like motif, which is the catalytic motif for C5-cytosine MTase in addition to DPPY motif. In order to check the role of both these MTase by glycine. HP0593 –Y107G and C54G mutant proteins were purified to near homogeneity. It was found that the Y107G mutant protein was catalytically inactive as compared to wild-type HP0593 MTase. On the other hand the C54G mutant protein was found to be as active as the wild-type HP0593 MTase indicating that HP0593 MTase is an adenine MTase and not a C5- cytosine MTase. Kinetic and catalytic properties of HP0593 DNA adenine methyltransferase DNA binding studies were carried out by electrophoretic mobility shift assay using DNA having cognate site and either in absence or presence of AdoHcy or sinefungin. In all the three cases two different DNA-protein complexes were observed-a fast running complex I and a slow running complex 2. It can be surmised that the fast running complex could be HP0593 monomer-DNA and the slow running complex could be a HP0593 dimer-DNA complex. With non specific DNA (lacking 5’-GCAG-3’ sequences) no complexes were formed even in the presence of cofactors. Based on the above observations it is suggested that a specific interactions of HP0593 MTase with DNA occurs on cognate recognition site. The activity of HP0593 MTase is optional at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. When initial velocities were plotted against varying concentrations of duplex DNA having a single 5’GCAG-3’ site a rectangular hyperbola was obtained confirming that HP0593 MTase obeys michaelis menten kinetics. From non-linear regression analysis of the plot of initial velocity versus DNA concentration Km (DNA) and kcat were calculated. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of methylation activity on enzyme concentration indicated that more than one molecule of enzyme is required for its activity. Metal ion cofactors such as CO 2, Mn2+ and Mg2+ stimulated the HP09593 MTase activity. As Mn2+ showed maximum stimulation of methyaltion activity compared to other metal ions, surface plasmon resonance spectroscopy was used to determine the kinetics of DNA binding by HP0593 MTase in the absence and presence of Mn2+. In the presence of Mn2+, HP0593 MTase showed~1000-fold increase in affinity to duplex DNA. DNA MTase bind substrates in random or sequential order. Preincubation study demonstrated that the preformed enzyme-DNA complex is competent than the preformed enzyme-AdoMet complex. This suggests that MTase binds to DNA first followed by AdoMet. Isotope partitioning analysis indicated that HP0593 MTase shows a distributive mechanism of methylation DNA having more than one recognition site. Effects of inactivation of HP0593 DNA MTase in Helicobacter pylori 26695 strain and its functional role. DNA dot-blot assay using hp0593 gene specific primer showed that this gene is present in 25.15% of the clinical strains checked suggesting that hp0593 is strain-specific gene. Strain-specific genes in many host-adapted pathogene impart strain specific phenotype. Wild-type 26695 strain grew slightly faster at the initial phase of growth in PH 4.5 compared to pH 7.4. A~5-fold enhanced level of hp0593 mRNA expression was growth under acidic condition HP0593 MTase could play an important role in H. pylori physiology through methylation. To elucidate the possible role(s) played by the MTase in H.pylori physiology, an hp0593 knock-out in 26695 strain was generated by chloramphenecol cassette mediated insertional gene inactivation. Growth kinetic study was carried out with both wild-type and hp0593 knock-out strain at pH7.4, the growth of the hp0593 strain. At pH 4.5 no major differences were observed in the growth compared to the wild-type hp0593 knock-out strain. To further investigate the effect of the knock-out, cell-morphology study was carried out after growing the strains at pH 7.4 till mid-exponential phase. Transmission electron microscopy studies reveled changes in cell shape, presence of sheathed structure and production of outer membrane vesicles (OMVs) in the hp0593 knock out strain. OMVs contain effectors molecules during infection helps in pathogenicity caused by H.pylori.This is the first report where inactivation of DNA MTase causes shedding of vesicles. OMVs are also known to modulate the production of IL-8 by gastic epitheial cells. To check weather H.pylori strains could produce IL-8, both wild-type and hp0593 knock-out strains were co-cultured with AGS cell infected with the hp0593 knock out strain. This was further confirmed by semi-quantitative RT-PCR analysis. To analyze the different phenotypes observed in the hp0593 knock-out strain, transcriptome profile were compared by microarray and RT-PCR analysis. In thehp0593 knock-out strain peptidologlycan and murein synthesis genes like pbp2, murC and neu4 showed upregulation which could be responsible for the changes in cell shape presence of sheathed structure and OMVs production. The RT-PCR data showed ~9-fold down-regulation of dank chaperone which might play a key role in slow growth phenotype in the hp0593 knock-out strain. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes such as dank, neuA, murC, fliH, filP and cag5, the results presented in this study provide impetus for exploring the role of HP0593 DNA MTase in the cellular processes of H.pylori. However, R-M systems are not absolutely essential, but different methylation patterns may contribute to strain-specific epigenetic gene regulation and may contribute to variability among the strains.

Recently, non-cellular viral agents became the focus of a large number of scientific groups. A prominent and widespread group of these viruses are flaviviruses, which include, for example, Zika virus, Dengue fever virus, tick-borne encephalitis virus and West Nile virus. There is a considerable diversity among these viruses, however, highly conserved proteins can be found throughout this viral genus. The largest and most conserved protein encoded by flaviviruses is the nonstructural NS5 protein. Its N-terminal domain bears the methyltransferase (MTase) activity. Thanks to the methylation of its genome, it allows the virus to initiate translation and at the same time mask it from the host's immune system. By blocking the active site of this enzyme with a small molecule, viral infection could be stopped not only in one flavivirus, but, due to the high conservation of MTases, in all other flaviviruses. This diploma thesis deals with the aforementioned MTase domain of the NS5 protein, specifically of the West Nile virus (WNV). After designing an insert encoding the WNV MTase domain, amplifying it and ligating it into the vector, the MTase domain was prepared by a recombinant expression, followed by purification. Subsequently, complexes of the protein with small molecules (MTase ligands) were formed, in...