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Josep Casadesús - One of the best experts on this subject based on the ideXlab platform.
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Envelope instability in DNA adenine Methylase mutants of Salmonella enterica.
Microbiology (Reading England), 2020Co-Authors: M. Graciela Pucciarelli, Josep Casadesús, Ana I Prieto, Francisco Garcı A-del PortilloAbstract:Mutants of Salmonella enterica serovar Typhimurium lacking DNA adenine (Dam) Methylase show reduced secretion of invasion effectors encoded in the Salmonella-pathogenicity island 1 (SPI-1). Concomitant with this alteration, a high number and quantity of extracellular proteins are detected in cultures of Dam(-) mutants. This study shows by subcellular fractionation analysis that the presence of numerous extracellular proteins in cultures of Dam(-) mutants is linked to an exacerbated release of membrane particulate material. The membrane 'leaky' phenotype and the impaired functionality of type III secretion systems were, however, unrelated since exacerbated release of proteins to the medium was evident in Dam(-) strains carrying mutations in either SPI-1 (invA, invJ) or flagellar (flhD) genes. This result supports the view that Dam methylation controls a plethora of cellular processes. Electron microscopy analysis demonstrated that the accumulation of membrane particulate material occurs preferentially as vesicles in stationary cultures of Dam(-) strains. In addition, a reduction in the relative amount of peptidoglycan-associated lipoprotein (PAL), TolB, OmpA and murein lipoprotein (Lpp) bound to peptidoglycan was observed in actively growing Dam(-) mutants. The existence of an envelope defect was further confirmed by the increased sensitivity to deoxycholate exhibited by Dam(-) mutants, mostly during exponential growth. Unexpectedly, lack of Dam methylation neither increased envelope instability nor impaired the association of PAL-Tol-Lpp proteins to the peptidoglycan in Escherichia coli. Accordingly, E. coli Dam(-) mutants did not show sensitivity to deoxycholate. Altogether, these results indicate that, besides its role in modulating the secretion of effectors by the SPI-1-encoded type III apparatus, Dam methylation controls cell envelope integrity in S. enterica.
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roles of dna adenine methylation in host pathogen interactions mismatch repair transcriptional regulation and more
Fems Microbiology Reviews, 2009Co-Authors: Martin Marinus, Josep CasadesúsAbstract:The DNA adenine methyltransferase (Dam Methylase) of Gammaproteobacteria and the cell cycle-regulated methyltransferase (CcrM) Methylase of Alphaproteobacteria catalyze an identical reaction (methylation of adenosine moieties using S -adenosyl-methionine as a methyl donor) at similar DNA targets (GATC and GANTC, respectively). Dam and CcrM are of independent evolutionary origin. Each may have evolved from an ancestral restriction-modification system that lost its restriction component, leaving an ‘orphan’ Methylase devoted solely to epigenetic genome modification. The formation of 6-methyladenine reduces the thermodynamic stability of DNA and changes DNA curvature. As a consequence, the methylation state of specific adenosine moieties can affect DNA–protein interactions. Well-known examples include binding of the replication initiation complex to the methylated oriC , recognition of hemimethylated GATCs in newly replicated DNA by the MutHLS mismatch repair complex, and discrimination of methylation states in promoters and regulatory DNA motifs by RNA polymerase and transcription factors. In recent years, Dam and CcrM have been shown to play roles in host–pathogen interactions. These roles are diverse and have only partially been understood. Especially intriguing is the evidence that Dam methylation regulates virulence genes in Escherichia coli, Salmonella , and Yersinia at the posttranscriptional level.
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DNA Adenine Methylation Regulates Virulence Gene Expression in Salmonella enterica Serovar Typhimurium
Journal of Bacteriology, 2006Co-Authors: Roberto Balbontín, Francisco Garciadel Portillo, Gary Rowley, M. Graciela Pucciarelli, Javier López-garrido, Ym Wormstone, Sacha Lucchini, Jay C. D. Hinton, Josep CasadesúsAbstract:Transcriptomic analyses during growth in Luria-Bertani medium were performed in strain SL1344 of Salmonella enterica serovar Typhimurium and in two isogenic derivatives lacking Dam Methylase. More genes were repressed than were activated by Dam methylation (139 versus 37). Key genes that were differentially regulated by Dam methylation were verified independently. The largest classes of Dam-repressed genes included genes belonging to the SOS regulon, as previously described in Escherichia coli, and genes of the SOS-inducible Salmonella prophages ST64B, Gifsy-1, and Fels-2. Dam-dependent virulence-related genes were also identified. Invasion genes in pathogenicity island SPI-1 were activated by Dam methylation, while the fimbrial operon std was repressed by Dam methylation. Certain flagellar genes were repressed by Dam methylation, and Dam− mutants of S. enterica showed reduced motility. Altered expression patterns in the absence of Dam methylation were also found for the chemotaxis genes cheR (repressed by Dam) and STM3216 (activated by Dam) and for the Braun lipoprotein gene, lppB (activated by Dam). The requirement for DNA adenine methylation in the regulation of specific virulence genes suggests that certain defects of Salmonella Dam− mutants in the mouse model may be caused by altered patterns of gene expression.
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Epigenetic Gene Regulation in the Bacterial World
Microbiology and Molecular Biology Reviews, 2006Co-Authors: Josep CasadesúsAbstract:Like many eukaryotes, bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions. Unlike eukaryotes, however, bacteria use DNA adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal. DNA adenine methylation plays roles in the virulence of diverse pathogens of humans and livestock animals, including pathogenic Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella. In Alphaproteobacteria, methylation of adenine at GANTC sites by the CcrM Methylase regulates the cell cycle and couples gene transcription to DNA replication. In Gammaproteobacteria, adenine methylation at GATC sites by the Dam Methylase provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage genomes, transposase activity, and regulation of gene expression. Transcriptional repression by Dam methylation appears to be more common than transcriptional activation. Certain promoters are active only during the hemimethylation interval that follows DNA replication; repression is restored when the newly synthesized DNA strand is methylated. In the E. coli genome, however, methylation of specific GATC sites can be blocked by cognate DNA binding proteins. Blockage of GATC methylation beyond cell division permits transmission of DNA methylation patterns to daughter cells and can give rise to distinct epigenetic states, each propagated by a positive feedback loop. Switching between alternative DNA methylation patterns can split clonal bacterial populations into epigenetic lineages in a manner reminiscent of eukaryotic cell differentiation. Inheritance of self-propagating DNA methylation patterns governs phase variation in the E. coli pap operon, the agn43 gene, and other loci encoding virulence-related cell surface functions.
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N^6-methyl-adenine: an epigenetic signal for DNA–protein interactions
Nature Reviews Microbiology, 2006Co-Authors: Didier Wion, Josep CasadesúsAbstract:N^6-methyl-adenine is a common DNA modification in bacterial genomes, which is catalysed by two classes of DNA adenine methyltransferases: those associated with restriction–modification (R–M) systems and 'solitary' methyltransferases that do not have a restriction-enzyme companion. R–M systems protect bacteria from the invasion of foreign DNA (for example, phages). Each R–M system is made up of a restriction enzyme and a modification enzyme, which both recognize the same DNA target. Some modification enzymes are DNA adenine methyltransferases, whereas others are DNA cytosine methyltransferases. In γ-proteobacteria, methylation of the adenine moiety at GATC sites by the Dam Methylase provides signals for chromosome replication, nucleoid organization and segregation, mismatch repair, transposition of insertion elements, phase variation, bacterial conjugation and packaging of phage DNA. Furthermore, Dam methylation is required for virulence in Salmonella, Haemophilus, Yersinia and Vibrio species. In uropathogenic Escherichia coli , phase variation in Pap fimbriae is regulated at the transcriptional level by Dam methylation and the leucine-responsive regulatory protein (Lrp). Synthesis of Prf, S, Afa, K88 and CS31a fimbriae is also regulated by Dam methylation and Lrp. In turn, phase variation in the E. coli Agn43 antigen is regulated by Dam and OxyR. In Salmonella , Dam methylation regulates the expression of genes involved in invasion of epithelial cells (SPI-1 genes), synthesis of fimbrial adhesins (Pef and Std), envelope proteins (Braun lipoprotein), flagella and chemotaxis. Also, Dam methylation is required for bile resistance. Dam methylation regulates conjugal transfer of the Salmonella virulence plasmid and other F-like plasmids. In Caulobacter and other α-proteobacteria, methylation of the adenine moiety at GANTC sites by the CcrM Methylase regulates the cell cycle and is essential for viability. Lack of CcrM methylation attenuates virulence in Brucella . DNA adenine methylation is found in the genomes of certain protists and fungi, and might exist in other eukaryotes. The authors review the funDamental roles of N^6-methyl-adenine in bacteria. In γ-proteobacteria, Dam methylation facilitates DNA–protein interactions involved in chromosome segregation, mismatch repair, transposition, and the epigenetic control of gene expression. In α-proteobacteria, the CcrM Methylase is an important cell-cycle regulator. N^6-methyl-adenine is found in the genomes of bacteria, archaea, protists and fungi. Most bacterial DNA adenine methyltransferases are part of restriction–modification systems. Certain groups of Proteobacteria also harbour solitary DNA adenine methyltransferases that provide signals for DNA–protein interactions. In γ-proteobacteria, Dam methylation regulates chromosome replication, nucleoid segregation, DNA repair, transposition of insertion elements and transcription of specific genes. In Salmonella , Haemophilus , Yersinia and Vibrio species and in pathogenic Escherichia coli , Dam methylation is required for virulence. In α-proteobacteria, CcrM methylation regulates the cell cycle in Caulobacter , Rhizobium and Agrobacterium , and has a role in Brucella abortus infection.
W Guschlbauer - One of the best experts on this subject based on the ideXlab platform.
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Dam Methylase from escherichia coli kinetic studies using modified dna oligomers hemimethylated substrates
Nucleic Acids Research, 1995Co-Authors: Stephane Marzabal, Stephen Dubois, Vera Thielking, Angeles Cano, Ramon Eritja, W GuschlbauerAbstract:We have measured steady-state kinetics of the N6-adenine methyltransferase Dam Mtase using as substrates non-selfcomplementary tetradecamer duplexs (d[GCCGGATCTAGACG]-d[CGTCTAGATCC-GGC]) containing the hemimethylated GATC target sequence in one or the other strand and modifications in the GATC target sequence of the complementary strands. Modifications included substitution of guanine by hypoxanthine (I), thymine by uracil (U) or 5-ethyl-uracil (E) and adenine by 2,6-diamino-purine (D). Thermodynamic parameters were obtained from the concentration dependence of the melting temperature (Tm) of the duplexes. Large differences in DNA methylation of duplexes containing single dI for dG substitution of the Dam recognition site were observed compared with the canonical substrate, if the substitution involved the top strand (on the G.C rich side). Substitution in either strand by uracil (dU) or 5-ethyluracil (dE) resulted in small perturbation of the methylation patterns. When 2,6-diamino-purine (dD) replaced the adenine to be methylated, small, but significant methylation was observed. The kinetic parameters of the methylation reaction were compared with the thermodynamic free energies and significant correlation was observed.
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the role of the preserved sequences of Dam Methylase
Nucleic Acids Research, 1993Co-Authors: Jeanbaptiste Guyot, Jacques Grassi, Ulrich Hahn, W GuschlbauerAbstract:Abstract We have undertaken a site directed mutational analysis of two of the preserved regions in the amino acid sequence of Dam Methylase in order to characterize their role. Mutations in region IV (sequence DPPY) abolish catalytic activity and greatly affect AdoMet crosslinking. Mutants in region III display a lowered specific activity with an unchanged AdoMet crosslinking capacity. We have also made a series of deletions both at the N and C terminal parts of the protein, which have been found to provide inactive enzyme. We discuss the significance of these results for the understanding of the functional properties of the enzyme.
Deog Su Hwang - One of the best experts on this subject based on the ideXlab platform.
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interaction of seqa and Dam Methylase on the hemimethylated origin of escherichia coli chromosomal dna replication
Journal of Biological Chemistry, 1999Co-Authors: Sukhyun Kang, Deog Su HwangAbstract:Abstract Preferential binding of SeqA protein to hemimethylated oriC, the origin of Escherichia coli chromosomal replication, delays methylation by Dam Methylase. Because the SeqA-oriC interaction appears to be essential in timing of chromosomal replication initiation, the biochemical functions of SeqA protein and Dam Methylase at the 13-mer L, M, and R region containing 4 GATC sequences at the left end oforiC were examined. We found that SeqA protein preferentially bound hemimethylated 13-mers but not fully nor unmethylated 13-mers. Regardless of strand methylation, the binding of SeqA protein to the hemimethylated GATC sequence of 13-mer L was followed by additional binding to other hemimethylated GATC sequences of 13-mer M and R. On the other hand, Dam Methylase did not discriminate binding of 13-mers in different methylation patterns and was not specific to GATC sequences. The binding specificity and higher affinity of SeqA protein over Dam Methylase to the hemimethylated 13-mers along with the reported cellular abundance of this protein explains the dominant action of SeqA protein over Dam Methylase to the newly replicated oriC for the sequestration of chromosomal replication. Furthermore, SeqA protein bound to hemimethylated 13-mers was not dissociated by Dam Methylase, and most SeqA protein spontaneously dissociated 10 min after binding. Also, SeqA protein delayed thein vitro methylation of hemimethylated 13-mers by Dam Methylase. These in vitro results suggest that the intrinsic binding instability of SeqA protein results in release of sequestrated hemimethylated oriC.
Wilhelm Guschlbauer - One of the best experts on this subject based on the ideXlab platform.
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Small is beautiful": major modifications in DNA structure or dynamics by small substituents or ligands.
Acta Biochimica Polonica, 1996Co-Authors: Wilhelm GuschlbauerAbstract:: This short review assembles the contributions of the author's laboratory to the structural aspects of DNA. DNA was modified by small ligands and/or substituents. There are three aspects to this work: a) Protonation of guanosine and DNA and the formation of triple- and quadruple-strands of guanosine, its nucleotides, their polymers and DNA. b) Substitution of the 2'-position of deoxyribose by the most polar atom, fluorine: studies on 2'-deoxy-2'-fluro-nucleosides, -nucleotides and their polymers, studied both by structural and biological methods. c) The effect of introducing the methyl group in the large groove of DNA: NMR studies of oligonucleotides containing N6-methylated adenine residues, and enzymatic and molecular biology work on Dam Methylase are reported.
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Dam methyltransferase from Escherichia coLi: sequence of a peptide segment involved in S-adenosyl-methionine binding
Nucleic Acids Research, 1993Co-Authors: Caroline Wenzel, Wilhelm GuschlbauerAbstract:Abstract DNA adenine methyltransferase (Dam Methylase) has been crosslinked with its cofactor S-adenosyl methionine (AdoMet) by UV irradiation. About 3% of the enzyme was radioactively labelled after the crosslinking reaction performed either with (methyl-3H)-AdoMet or with (carboxy-14C)-AdoMet. Radiolabelled peptides were purified after trypsinolysis by high performance liquid chromatography in two steps. They could not be sequenced due to radiolysis. Therefore we performed the same experiment using non-radioactive AdoMet and were able to identify the peptide modified by the crosslinking reaction by comparison of the separation profiles obtained from two analytical control experiments performed with 3H-AdoMet and Dam Methylase without crosslink, respectively. This approach was possible due to the high reproducibility of the chromatography profiles. In these three experiments only one radioactively labelled peptide was present in the tryptic digestions of the crosslinked enzyme. Its sequence was found to be XA-GGK, corresponding to amino acids 10-14 of Dam Methylase. The non-identified amino acid in the first sequence cycle should be a tryptophan, which is presumably modified by the crosslinking reaction. The importance of this region near the N-terminus for the structure and function of the enzyme was also demonstrated by proteolysis and site-directed mutagenesis experiments.
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Crosslinking of Dam methyltransferase with S-adenosyl-methionine
FEBS Letters, 1991Co-Authors: Caroline Wenzel, Maxime Moulard, Anders Løbner-olesen, Wilhelm GuschlbauerAbstract:Highly purified DNA-adenine methyltransferase was irradiated in the presence of different concentrations of radiolabelled S-adenosyl-methionine (AdoMet) with a conventional Mineralight UV-lamp from several minutes up to 1 h while incubating in ice. Incorporation of radioactivity was monitored by electrophoresis of the crosslink between S-adenosyl-methionine and Dam Methylase on SDS-polyacrylamide gels followed by fluorography. Crosslinking reached a maximum in presence of 10 μM S-adenosyl-methionine; it was inhibited in the presence of substates which competitively inhibit methylation of DNA by Dam Methylase, like sinefungin or S-adenosyl-homocysteine, but not in the presence of non-inhibitors like ATP or S-isobutyl-adenosine. The crosslink obtained was resistant against a wide range of even drastic conditions commonly used in protein and peptide chemistry. Proteins which do not bind S-adenosyl-methionine, as well as heat activated Dam Methylase were not photolabelled. After limited proteolysis the radioactive label appeared only in certain of the peptides obtained. From Western blots carried out with polyclonal antibodies produced against a synthetic peptide corresponding in its sequence to amino acids 92-106 of the Dam Methylase, the crosslinking of AdoMet could be tentatively mapped at a position after amino acid 106.
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the double role of methyl donor and allosteric effector of s adenosyl methionine for Dam Methylase of e coli
Nucleic Acids Research, 1990Co-Authors: Agnes Bergerat, Wilhelm GuschlbauerAbstract:Abstract The turnover of DNA-adenine-Methylase of E. coli strongly decreases when the temperature is lowered. This has allowed us to study the binding of Dam Methylase on 14 bp DNA fragments at 0 degrees C by gel retardation in the presence of Ado-Met, but without methylation taking place. The enzyme can bind non-specific DNA with low affinity. Binding to the specific sequence occurs in the absence of S-adenosyl-methionine (Ado-Met), but is activated by the presence of the methyl donor. The two competitive inhibitors of Ado-Met, sinefungin and S-adenosyl-homocysteine, can neither activate this binding to DNA by themselves, nor inhibit this activation by Ado-Met. This suggests that Ado-Met could bind to Dam Methylase in two different environments. In one of them, it could play the role of an allosteric effector which would reinforce the affinity of the enzyme for the GATC site. The analogues can not compete for such binding. In the other environment Ado-Met would be in the catalytic site and could be exchanged by its analogues. We have also visualized conformational changes in Dam Methylase induced by the simultaneous binding of Ado-Met and the specific target sequence of the enzyme, by an anomaly of migration and partial resistance to proteolytic treatment of the ternary complex Ado-Met/Dam Methylase/GATC.
Francisco Garciadel Portillo - One of the best experts on this subject based on the ideXlab platform.
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DNA Adenine Methylation Regulates Virulence Gene Expression in Salmonella enterica Serovar Typhimurium
Journal of Bacteriology, 2006Co-Authors: Roberto Balbontín, Francisco Garciadel Portillo, Gary Rowley, M. Graciela Pucciarelli, Javier López-garrido, Ym Wormstone, Sacha Lucchini, Jay C. D. Hinton, Josep CasadesúsAbstract:Transcriptomic analyses during growth in Luria-Bertani medium were performed in strain SL1344 of Salmonella enterica serovar Typhimurium and in two isogenic derivatives lacking Dam Methylase. More genes were repressed than were activated by Dam methylation (139 versus 37). Key genes that were differentially regulated by Dam methylation were verified independently. The largest classes of Dam-repressed genes included genes belonging to the SOS regulon, as previously described in Escherichia coli, and genes of the SOS-inducible Salmonella prophages ST64B, Gifsy-1, and Fels-2. Dam-dependent virulence-related genes were also identified. Invasion genes in pathogenicity island SPI-1 were activated by Dam methylation, while the fimbrial operon std was repressed by Dam methylation. Certain flagellar genes were repressed by Dam methylation, and Dam− mutants of S. enterica showed reduced motility. Altered expression patterns in the absence of Dam methylation were also found for the chemotaxis genes cheR (repressed by Dam) and STM3216 (activated by Dam) and for the Braun lipoprotein gene, lppB (activated by Dam). The requirement for DNA adenine methylation in the regulation of specific virulence genes suggests that certain defects of Salmonella Dam− mutants in the mouse model may be caused by altered patterns of gene expression.
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increased excision of the salmonella prophage st64b caused by a deficiency in Dam Methylase
Journal of Bacteriology, 2005Co-Authors: Ana Alonso, Graciela M Pucciarelli, Nara Figueroabossi, Francisco Garciadel PortilloAbstract:Salmonella enterica mutants defective in Dam Methylase are strongly attenuated in virulence and release a large amount of proteins to the extracellular medium. The extent to which these two phenotypes are linked is unknown. Using a proteomic approach, we identified Sb6, Sb13, and Sb36 as proteins present in larger amounts in culture supernatants of an S. enterica serovar Typhimurium Dam mutant than in those of the wild-type strain. These three proteins are encoded in the Salmonella prophage ST64B. Higher amounts of ST64B phage DNA and tailless viral capsids were also detected in supernatant extracts of the Dam mutant, suggesting that Dam methylation negatively regulates the excision of ST64B. Reverse transcription-PCR analysis revealed that the expression of two ST64B genes encoding a putative antirepressor and a phage replication protein increases in the Dam mutant. The SOS response also augments the excision of ST64B. Infection assays performed with phage-cured strains demonstrated that ST64B does not carry genes required for virulence in the mouse model. Evidence was also obtained discarding a relationship between the high excision of ST64B and the envelope instability or virulence attenuation phenotype. Taken together, these data indicate that ST64B excises at a high rate in Dam mutants due to the loss of repression exerted by Dam on phage genes and induction of the SOS response characteristic of these mutants. The exacerbated excision of ST64B does not however contribute to the incapacity of Dam mutants to cause disease.
