scholarly journals Hyperthermophilic DNA Methyltransferase M.PabI from the Archaeon Pyrococcus abyssi

2006 ◽  
Vol 72 (8) ◽  
pp. 5367-5375 ◽  
Author(s):  
Miki Watanabe ◽  
Harumi Yuzawa ◽  
Naofumi Handa ◽  
Ichizo Kobayashi
Keyword(s):  
Dna Methyltransferase ◽  
Dna Methyltransferases ◽  
Type I ◽  
Type Ii ◽  
Gene Complex ◽  
Sequence Comparisons ◽  
Novel Structure ◽  
Dna Engineering ◽  
Pyrococcus Abyssi ◽  
Restriction Modification

ABSTRACT Genome sequence comparisons among multiple species of Pyrococcus, a hyperthermophilic archaeon, revealed a linkage between a putative restriction-modification gene complex and several large genome polymorphisms/rearrangements. From a region apparently inserted into the Pyrococcus abyssi genome, a hyperthermoresistant restriction enzyme [PabI; 5′-(GTA/C)] with a novel structure was discovered. In the present work, the neighboring methyltransferase homologue, M.PabI, was characterized. Its N-terminal half showed high similarities to the M subunit of type I systems and a modification enzyme of an atypical type II system, M.AhdI, while its C-terminal half showed high similarity to the S subunit of type I systems. M.PabI expressed within Escherichia coli protected PabI sites from RsaI, a PabI isoschizomer. M.PabI, purified following overexpression, was shown to generate 5′-GTm6AC, which provides protection against PabI digestion. M.PabI was found to be highly thermophilic; it showed methylation at 95°C and retained at least half the activity after 9 min at 95°C. This hyperthermophilicity allowed us to obtain activation energy and other thermodynamic parameters for the first time for any DNA methyltransferases. We also determined the kinetic parameters of k cat, Km , DNA, and Km , AdoMet. The activity of M.PabI was optimal at a slightly acidic pH and at an NaCl concentration of 200 to 500 mM and was inhibited by Zn2+ but not by Mg2+, Ca2+, or Mn2+. These and previous results suggest that this unique methyltransferase and PabI constitute a type II restriction-modification gene complex that inserted into the P. abyssi genome relatively recently. As the most thermophilic of all the characterized DNA methyltransferases, M.PabI may help in the analysis of DNA methylation and its application to DNA engineering.

scholarly journals A DNA Methyltransferase Can Protect the Genome from Postdisturbance Attack by a Restriction-Modification Gene Complex

2002 ◽  
Vol 184 (22) ◽  
pp. 6100-6108 ◽  
Author(s):  
Noriko Takahashi ◽  
Yasuhiro Naito ◽  
Naofumi Handa ◽  
Ichizo Kobayashi
Keyword(s):  
Dna Methyltransferase ◽  
Dna Methyltransferases ◽  
Cell Killing ◽  
Gene Complex ◽  
Prokaryotic Genomes ◽  
A Cell ◽  
Gene Complexes ◽  
Potential Mobility ◽  
Restriction Modification

ABSTRACT In prokaryotic genomes, some DNA methyltransferases form a restriction-modification gene complex, but some others are present by themselves. Dcm gene product, one of these orphan methyltransferases found in Escherichia coli and related bacteria, methylates DNA to generate 5′-CmCWGG just as some of its eukaryotic homologues do. Vsr mismatch repair function of an adjacent gene prevents C-to-T mutagenesis enhanced by this methylation but promotes other types of mutation and likely has affected genome evolution. The reason for the existence of the dcm-vsr gene pair has been unclear. Earlier we found that several restriction-modification gene complexes behave selfishly in that their loss from a cell leads to cell killing through restriction attack on the genome. There is also increasing evidence for their potential mobility. EcoRII restriction-modification gene complex recognizes the same sequence as Dcm, and its methyltransferase is phylogenetically related to Dcm. In the present work, we found that stabilization of maintenance of a plasmid by linkage of EcoRII gene complex, likely through postsegregational cell killing, is diminished by dcm function. Disturbance of EcoRII restriction-modification gene complex led to extensive chromosome degradation and severe loss of cell viability. This cell killing was partially suppressed by chromosomal dcm and completely abolished by dcm expressed from a plasmid. Dcm, therefore, can play the role of a “molecular vaccine” by defending the genome against parasitism by a restriction-modification gene complex.

scholarly journals Low-level expression of the Type II restriction–modification system confers potent bacteriophage resistance in Escherichia coli

DNA Research ◽  
2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Karolina Wilkowska ◽  
Iwona Mruk ◽  
Beata Furmanek-Blaszk ◽  
Marian Sektas
Keyword(s):  
Dna Methyltransferase ◽  
Host Bacterium ◽  
Type Ii ◽  
Potential Conflict ◽  
Modification System ◽  
Biological Assays ◽  
Restriction Modification System ◽  
Highly Sensitive ◽  
System Maintenance ◽  
Restriction Modification

Abstract Restriction–modification systems (R–M) are one of the antiviral defense tools used by bacteria, and those of the Type II family are composed of a restriction endonuclease (REase) and a DNA methyltransferase (MTase). Most entering DNA molecules are usually cleaved by the REase before they can be methylated by MTase, although the observed level of fragmented DNA may vary significantly. Using a model EcoRI R–M system, we report that the balance between DNA methylation and cleavage may be severely affected by transcriptional signals coming from outside the R–M operon. By modulating the activity of the promoter, we obtained a broad range of restriction phenotypes for the EcoRI R–M system that differed by up to 4 orders of magnitude in our biological assays. Surprisingly, we found that high expression levels of the R–M proteins were associated with reduced restriction of invading bacteriophage DNA. Our results suggested that the regulatory balance of cleavage and methylation was highly sensitive to fluctuations in transcriptional signals both up- and downstream of the R–M operon. Our data provided further insights into Type II R–M system maintenance and the potential conflict within the host bacterium.

scholarly journals Systematic analysis of REBASE identifies numerous Type I restriction-modification systems that contain duplicated, variable hsdS specificity genes that randomly switch methyltransferase specificity by recombination

2020 ◽  
Author(s):  
John M. Atack ◽  
Chengying Guo ◽  
Thomas Litfin ◽  
Long Yang ◽  
Patrick J. Blackall ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Bacterial Species ◽  
Dna Methyltransferases ◽  
Inverted Repeats ◽  
Global Changes ◽  
Length Variation ◽  
Type I ◽  
Phase Variable ◽  
Global Methylation ◽  
Restriction Modification

AbstractN6-adenine DNA methyltransferases associated with some Type I and Type III restriction-modification (R-M) systems are able to randomly switch expression by variation in the length of locus-encoded simple sequence repeats (SSRs). SSR tract-length variation causes ON/OFF switching of methyltransferase expression, resulting in genome-wide methylation differences, and global changes in gene expression. These epigenetic regulatory systems are called phasevarions, phase-variable regulons, and are widespread in bacteria. A distinct switching system has also been described in Type I R-M systems, based on recombination-driven changes in hsdS genes, which dictate the DNA target site. In order to determine the prevalence of recombination-driven phasevarions, we generated a program called RecombinationRepeatSearch to interrogate REBASE and identify the presence and number of inverted repeats of hsdS downstream of Type I R-M loci. We report that 5.9% of Type I R-M systems have duplicated variable hsdS genes containing inverted repeats capable of phase-variation. We report the presence of these systems in the major pathogens Enterococcus faecalis and Listeria monocytogenes, which will have important implications for pathogenesis and vaccine development. These data suggest that in addition to SSR-driven phasevarions, many bacteria have independently evolved phase-variable Type I R-M systems via recombination between multiple, variable hsdS genes.ImportanceMany bacterial species contain DNA methyltransferases that have random on/off switching of expression. These systems called phasevarions (phase-variable regulons) control the expression of multiple genes by global methylation changes. In every previously characterised phasevarion, genes involved in pathobiology, antibiotic resistance, and potential vaccine candidates are randomly varied in their expression, commensurate with methyltransferase switching. A systematic study to determine the extent of phasevarions controlled by invertible Type I R-M systems has never before been performed. Understanding how bacteria regulate genes is key to the study of physiology, virulence, and vaccine development; therefore it is critical to identify and characterize phase-variable methyltransferases controlling phasevarions.

scholarly journals Systematic Analysis of REBASE Identifies Numerous Type I Restriction-Modification Systems with Duplicated, Distinct hsdS Specificity Genes That Can Switch System Specificity by Recombination

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
John M. Atack ◽  
Chengying Guo ◽  
Thomas Litfin ◽  
Long Yang ◽  
Patrick J. Blackall ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Phase Variation ◽  
Dna Methyltransferases ◽  
Inverted Repeats ◽  
Global Changes ◽  
Type I ◽  
Phase Variable ◽  
Global Methylation ◽  
Content Type ◽  
Restriction Modification

ABSTRACT N6-Adenine DNA methyltransferases associated with some Type I and Type III restriction-modification (R-M) systems are able to undergo phase variation, randomly switching expression ON or OFF by varying the length of locus-encoded simple sequence repeats (SSRs). This variation of methyltransferase expression results in genome-wide methylation differences and global changes in gene expression. These epigenetic regulatory systems are called phasevarions, phase-variable regulons, and are widespread in bacteria. A distinct switching system has also been described in Type I R-M systems, based on recombination-driven changes in hsdS genes, which dictate the DNA target site. In order to determine the prevalence of recombination-driven phasevarions, we generated a program called RecombinationRepeatSearch to interrogate REBASE and identify the presence and number of inverted repeats of hsdS downstream of Type I R-M loci. We report that 3.9% of Type I R-M systems have duplicated variable hsdS genes containing inverted repeats capable of phase variation. We report the presence of these systems in the major pathogens Enterococcus faecalis and Listeria monocytogenes, which could have important implications for pathogenesis and vaccine development. These data suggest that in addition to SSR-driven phasevarions, many bacteria have independently evolved phase-variable Type I R-M systems via recombination between multiple, variable hsdS genes. IMPORTANCE Many bacterial species contain DNA methyltransferases that have random on/off switching of expression. These systems, called phasevarions (phase-variable regulons), control the expression of multiple genes by global methylation changes. In every previously characterized phasevarion, genes involved in pathobiology, antibiotic resistance, and potential vaccine candidates are randomly varied in their expression, commensurate with methyltransferase switching. Our systematic study to determine the extent of phasevarions controlled by invertible Type I R-M systems will provide valuable information for understanding how bacteria regulate genes and is key to the study of physiology, virulence, and vaccine development; therefore, it is critical to identify and characterize phase-variable methyltransferases controlling phasevarions.

Genome-wide analysis of restriction-modification system in unicellular and filamentous cyanobacteria

2006 ◽  
Vol 24 (3) ◽  
pp. 181-190 ◽  
Author(s):  
Fangqing Zhao ◽  
Xiaowen Zhang ◽  
Chengwei Liang ◽  
Jinyu Wu ◽  
Qiyu Bao ◽  
...  
Keyword(s):  
Positive Selection ◽  
Draft Genome ◽  
Gene Clusters ◽  
Pcr Analysis ◽  
Type I ◽  
Type Ii ◽  
Filamentous Cyanobacteria ◽  
Modification System ◽  
Wide Range ◽  
Restriction Modification

Cyanobacteria are an ancient group of gram-negative bacteria with strong genome size variation ranging from 1.6 to 9.1 Mb. Here, we first retrieved all the putative restriction-modification (RM) genes in the draft genome of Spirulina and then performed a range of comparative and bioinformatic analyses on RM genes from unicellular and filamentous cyanobacterial genomes. We have identified 6 gene clusters containing putative Type I RMs and 11 putative Type II RMs or the solitary methyltransferases (MTases). RT-PCR analysis reveals that 6 of 18 MTases are not expressed in Spirulina, whereas one hsdM gene, with a mutated cognate hsdS, was detected to be expressed. Our results indicate that the number of RM genes in filamentous cyanobacteria is significantly higher than in unicellular species, and this expansion of RM systems in filamentous cyanobacteria may be related to their wide range of ecological tolerance. Furthermore, a coevolutionary pattern is found between hsdM and hsdR, with a large number of site pairs positively or negatively correlated, indicating the functional importance of these pairing interactions between their tertiary structures. No evidence for positive selection is found for the majority of RMs, e.g., hsdM, hsdS, hsdR, and Type II restriction endonuclease gene families, while a group of MTases exhibit a remarkable signature of adaptive evolution. Sites and genes identified here to have been under positive selection would provide targets for further research on their structural and functional evaluations.

Dimeric/oligomeric DNA methyltransferases: an unfinished story

2009 ◽  
Vol 390 (9) ◽  
Author(s):  
Ernst G. Malygin ◽  
Alexey A. Evdokimov ◽  
Stanley Hattman
Keyword(s):  
Experimental Data ◽  
Dna Methyltransferases ◽  
Structure And Function ◽  
Type I ◽  
Type Ii ◽  
Kinetic Characteristics ◽  
Biological Substrate ◽  
Oligomeric Dna ◽  
And Function

Abstract DNA methyltransferases (MTases) are enzymes that carry out post-replicative sequence-specific modifications. The initial experimental data on the structure and kinetic characteristics of the EcoRI MTase led to the paradigm that type II systems comprise dimeric endonucleases and monomeric MTases. In retrospect, this was logical because, while the biological substrate of the restriction endonuclease is two-fold symmetrical, the in vivo substrate for the MTase is generally hemi-methylated and, hence, inherently asymmetric. Thus, the paradigm was extended to include all DNA MTases except the more complex bifunctional type I and type III enzymes. Nevertheless, a gradual enlightenment grew over the last decade that has changed the accepted view on the structure of DNA MTases. These results necessitate a more complex view of the structure and function of these important enzymes.

scholarly journals Combination of Multiplex PCRs for Staphylococcal Cassette Chromosome mec Type Assignment: Rapid Identification System for mec, ccr, and Major Differences in Junkyard Regions

2006 ◽  
Vol 51 (1) ◽  
pp. 264-274 ◽  
Author(s):  
Yoko Kondo ◽  
Teruyo Ito ◽  
Xiao Xue Ma ◽  
Shinya Watanabe ◽  
Barry N. Kreiswirth ◽  
...  
Keyword(s):  
Epidemiological Studies ◽  
Rapid Identification ◽  
Identification System ◽  
Type I ◽  
Type Ii ◽  
Gene Complex ◽  
Multiplex Pcrs ◽  
Type Assignment ◽  
Mrsa Strains ◽  
Staphylococcal Cassette Chromosome

ABSTRACT Staphylococcal cassette chromosome mec (SCCmec) typing, in combination with genotyping of the Staphylococcus aureus chromosome, has become essential for defining methicillin-resistant S. aureus (MRSA) clones in epidemiological studies. We have developed a convenient system for SCCmec type assignment. The system consists of six multiplex PCRs (M-PCRs) for identifying the ccr gene complex (ccr), the mec gene complex (mec), and specific structures in the junkyard (J) regions: M-PCR with primer set 1 (M-PCR 1) identified five types of ccr genes; M-PCR 2 identified class A to class C mec; M-PCRs 3 and 4 identified specific open reading frames in the J1 regions of type I and IV and of type II, III, and V SCCmec elements, respectively; M-PCR 5 identified the transposons Tn554 and ΨTn554 integrated into the J2 regions of type II and III SCCmec elements; and M-PCR 6 identified plasmids pT181 and pUB110 integrated into J3 regions. The system was validated with 99 MRSA strains carrying SCCmec elements of different types. The SCCmec types of 93 out of the 99 MRSA strains could be assigned. The SCCmec type assignments were identical to those made with a PCR system that uses numerous primer pairs to identify genes or gene alleles. Our system of six M-PCRs is thus a convenient and reliable method for typing SCCmec elements.

The Type IIB restriction endonucleases

2010 ◽  
Vol 38 (2) ◽  
pp. 410-416 ◽  
Author(s):  
Jacqueline J.T. Marshall ◽  
Stephen E. Halford
Keyword(s):  
Current Knowledge ◽  
Restriction Enzymes ◽  
Protein Assembly ◽  
Gene Organization ◽  
Type I ◽  
Type Ii ◽  
Dna Strands ◽  
Restriction Modification ◽  
Restriction Modification Systems ◽  
Do So

The endonucleases from the Type IIB restriction–modification systems differ from all other restriction enzymes. The Type IIB enzymes cleave both DNA strands at specified locations distant from their recognition sequences, like Type IIS nucleases, but they are unique in that they do so on both sides of the site, to liberate the site from the remainder of the DNA on a short duplex. The fact that these enzymes cut DNA at specific locations mark them as Type II systems, as opposed to the Type I enzymes that cut DNA randomly, but in terms of gene organization and protein assembly, most Type IIB restriction–modification systems have more in common with Type I than with other Type II systems. Our current knowledge of the Type IIB systems is reviewed in the present paper.

scholarly journals Cloning, crystallization and preliminary X-ray diffraction analysis of an intact DNA methyltransferase of a type I restriction–modification enzyme fromVibrio vulnificus

2014 ◽  
Vol 70 (4) ◽  
pp. 489-492
Author(s):  
Ly Huynh Thi Yen ◽  
Suk-Youl Park ◽  
Jeong-Sun Kim
Keyword(s):  
Dna Methyltransferase ◽  
Vibrio Vulnificus ◽  
Single Gene ◽  
Monoclinic Space Group ◽  
Type I ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
And Function ◽  
Restriction Modification

Independently of the restriction (HsdR) subunit, the specificity (HsdS) and methylation (HsdM) subunits interact with each other, and function as a methyltransferase in type I restriction–modification systems. A single gene that combines the HsdS and HsdM subunits inVibrio vulnificusYJ016 was expressed and purified. A crystal suitable for X-ray diffraction was obtained from 25%(w/v) polyethylene glycol monomethylether 5000, 0.1 MHEPES pH 8.0, 0.2 Mammonium sulfate at 291 K by hanging-drop vapour diffusion. Diffraction data were collected to a resolution of 2.31 Å using synchrotron radiation. The crystal belonged to the primitive monoclinic space groupP21, with unit-cell parametersa= 93.25,b= 133.04,c= 121.49 Å, β = 109.7°. With four molecules in the asymmetric unit, the crystal volume per unit protein weight was 2.61 Å3 Da−1, corresponding to a solvent content of 53%.

scholarly journals Contribution of RecFOR machinery of homologous recombination to cell survival after loss of a restriction–modification gene complex

Microbiology ◽  
2009 ◽  
Vol 155 (7) ◽  
pp. 2320-2332 ◽  
Author(s):  
Naofumi Handa ◽  
Asao Ichige ◽  
Ichizo Kobayashi
Keyword(s):  
Homologous Recombination ◽  
Cell Survival ◽  
Restriction Enzymes ◽  
Host Cells ◽  
Type Ii ◽  
Gene Complex ◽  
Recbcd Enzyme ◽  
Mutant Forms ◽  
Host Killing ◽  
Restriction Modification

Loss of a type II restriction–modification (RM) gene complex, such as EcoRI, from a bacterial cell leads to death of its descendent cells through attack by residual restriction enzymes on undermethylated target sites of newly synthesized chromosomes. Through such post-segregational host killing, these gene complexes impose their maintenance on their host cells. This finding led to the rediscovery of type II RM systems as selfish mobile elements. The host prokaryote cells were found to cope with such attacks through a variety of means. The RecBCD pathway of homologous recombination in Escherichia coli repairs the lethal lesions on the chromosome, whilst it destroys restricted non-self DNA. recBCD homologues, however, appear very limited in distribution among bacterial genomes, whereas homologues of the RecFOR proteins, responsible for another pathway, are widespread in eubacteria, just like the RM systems. In the present work, therefore, we examined the possible contribution of the RecFOR pathway to cell survival after loss of an RM gene complex. A recF mutation reduced survival in an otherwise rec-positive background and, more severely, in a recBC sbcBC background. We also found that its effect is prominent in the presence of specific non-null mutant forms of the RecBCD enzyme: the resistance to killing seen with recC1002, recC1004, recC2145 and recB2154 is severely reduced to the level of a null recBC allele when combined with a recF, recO or recR mutant allele. Such resistance was also dependent on RecJ and RecQ functions. UV resistance of these non-null recBCD mutants is also reduced by recF, recJ or recQ mutation. These results demonstrate that the RecFOR pathway of recombination can contribute greatly to resistance to RM-mediated host killing, depending on the genetic background.

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