Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N6-adenine DNA methyltransferases of Neisseria meningitidis

Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N6-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N6-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5′-CGYm6AG-3′), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5′-ACm6ACC-3′) and ModD1 (exemplified by M.Nme579I) (5′-CCm6AGC-3′). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5′-CGYm6AG-3′. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5′-GCGCm6AGG-3′ sites, to 100% at 5′-ACGTm6AGG-3′ sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching.

[1]  Richard J. Roberts,et al.  REBASE—a database for DNA restriction and modification: enzymes, genes and genomes , 2009, Nucleic Acids Res..

[2]  Alexander N Gorban,et al.  A random six-phase switch regulates pneumococcal virulence via global epigenetic changes , 2014, Nature Communications.

[3]  Tyson A. Clark,et al.  ModM DNA methyltransferase methylome analysis reveals a potential role for Moraxella catarrhalis phasevarions in otitis media , 2014, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[4]  K. Seib,et al.  Phasevarions Mediate Epigenetic Regulation of Antimicrobial Susceptibility in Neisseria meningitidis , 2014, Antimicrobial Agents and Chemotherapy.

[5]  J. Korlach,et al.  The complex methylome of the human gastric pathogen Helicobacter pylori , 2013, Nucleic acids research.

[6]  David T. F. Dryden,et al.  Type III restriction-modification enzymes: a historical perspective , 2013, Nucleic acids research.

[7]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[8]  Zhenyu Zhu,et al.  Fidelity Index Determination of DNA Methyltransferases , 2013, PloS one.

[9]  B. Schulz,et al.  Dual Pili Post-translational Modifications Synergize to Mediate Meningococcal Adherence to Platelet Activating Factor Receptor on Human Airway Cells , 2013, PLoS pathogens.

[10]  Nick Kamps-Hughes,et al.  Massively parallel characterization of restriction endonucleases , 2013, Nucleic acids research.

[11]  Matthew K Waldor,et al.  Entering the era of bacterial epigenomics with single molecule real time DNA sequencing. , 2013, Current opinion in microbiology.

[12]  V. Nagaraja,et al.  Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense , 2013, Microbiology and Molecular Reviews.

[13]  Tyson A. Clark,et al.  Comprehensive Methylome Characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at Single-Base Resolution , 2013, PLoS genetics.

[14]  Tyson A. Clark,et al.  Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing , 2012, Nature Biotechnology.

[15]  Dan S. Tawfik,et al.  Evolutionary transitions to new DNA methyltransferases through target site expansion and shrinkage , 2012, Nucleic acids research.

[16]  Richard J. Roberts,et al.  The methylomes of six bacteria , 2012, Nucleic acids research.

[17]  S. Turner,et al.  Going beyond five bases in DNA sequencing. , 2012, Current opinion in structural biology.

[18]  S. Beatson,et al.  Origin of the Diversity in DNA Recognition Domains in Phasevarion Associated modA Genes of Pathogenic Neisseria and Haemophilus influenzae , 2012, PloS one.

[19]  Richard J. Roberts,et al.  Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing , 2011, Nucleic acids research.

[20]  J. Bujnicki,et al.  Novel non-specific DNA adenine methyltransferases , 2011, Nucleic acids research.

[21]  Sean M. Grimmond,et al.  Phasevarion Mediated Epigenetic Gene Regulation in Helicobacter pylori , 2011, PloS one.

[22]  R. Rappuoli,et al.  A novel epigenetic regulator associated with the hypervirulent Neisseria meningitidis clonal complex 41/44 , 2011, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[23]  Julie C Dunning Hotopp,et al.  Neisseria meningitidis is structured in clades associated with restriction modification systems that modulate homologous recombination , 2011, Proceedings of the National Academy of Sciences.

[24]  R. Rappuoli,et al.  Influence of serogroup B meningococcal vaccine antigens on growth and survival of the meningococcus in vitro and in ex vivo and in vivo models of infection. , 2010, Vaccine.

[25]  M. Jennings,et al.  The phasevarion: phase variation of type III DNA methyltransferases controls coordinated switching in multiple genes , 2010, Nature Reviews Microbiology.

[26]  A. Piekarowicz,et al.  Characterization of the NgoAXP: phase-variable type III restriction-modification system in Neisseria gonorrhoeae. , 2009, FEMS microbiology letters.

[27]  S. Grimmond,et al.  Phasevarions Mediate Random Switching of Gene Expression in Pathogenic Neisseria , 2009, PLoS pathogens.

[28]  Zhenyu Zhu,et al.  The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases , 2008, Nucleic acids research.

[29]  A. Erwin,et al.  Haemophilus influenzae phasevarions have evolved from type III DNA restriction systems into epigenetic regulators of gene expression , 2007, Nucleic acids research.

[30]  B. Barrell,et al.  Meningococcal Genetic Variation Mechanisms Viewed through Comparative Analysis of Serogroup C Strain FAM18 , 2006, PLoS genetics.

[31]  Richard Moxon,et al.  Bacterial contingency loci: the role of simple sequence DNA repeats in bacterial adaptation. , 2006, Annual review of genetics.

[32]  D. Wion,et al.  N6-methyl-adenine: an epigenetic signal for DNA–protein interactions , 2006, Nature Reviews Microbiology.

[33]  S. Grimmond,et al.  The phasevarion: a genetic system controlling coordinated, random switching of expression of multiple genes. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[34]  D. Hood,et al.  Involvement of genes of genome maintenance in the regulation of phase variation frequencies in Neisseria meningitidis. , 2004, Microbiology.

[35]  A. Kiss,et al.  Changing the recognition specificity of a DNA-methyltransferase by in vitro evolution. , 2004, Nucleic acids research.

[36]  Dan S. Tawfik,et al.  Altering the sequence specificity of HaeIII methyltransferase by directed evolution using in vitro compartmentalization. , 2004, Protein engineering, design & selection : PEDS.

[37]  J. Heitman,et al.  A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. , 2003, Nucleic acids research.

[38]  K. Seib,et al.  Phase variable restriction-modification systems in Moraxella catarrhalis. , 2002, FEMS immunology and medical microbiology.

[39]  A. van der Ende,et al.  NmeSI Restriction-Modification System Identified by Representational Difference Analysis of a HypervirulentNeisseria meningitidis Strain , 2001, Infection and Immunity.

[40]  Kim Rutherford,et al.  Artemis: sequence visualization and annotation , 2000, Bioinform..

[41]  M. Achtman,et al.  Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[42]  D. Ferguson,et al.  Opc‐ and pilus‐dependent interactions of meningococci with human endothelial cells: molecular mechanisms and modulation by surface polysaccharides , 1995, Molecular microbiology.

[43]  T. Bickle,et al.  Biology of DNA restriction. , 1993, Microbiological reviews.

[44]  A. Barlow,et al.  Point mutation in meningococcal por A gene associated with increased endemic disease , 1991, The Lancet.

[45]  T. Bickle,et al.  Type III DNA restriction and modification systems EcoP1 and EcoP15. Nucleotide sequence of the EcoP1 operon, the EcoP15 mod gene and some EcoP1 mod mutants. , 1988, Journal of molecular biology.

[46]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[47]  P. Modrich,et al.  EcoRI methylase. Physical and catalytic properties of the homogeneous enzyme. , 1977, The Journal of biological chemistry.