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The BsaHI restriction-modification system: cloning, sequencing and analysis of conserved motifs.

ABSTRACT: BACKGROUND: Restriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length. Understanding the mechanism of this recognition represents a significant challenge that we begin to address for the BsaHI restriction-modification system, which recognises the six base sequence GRCGYC. RESULTS: The DNA sequences of the genes for the BsaHI methyltransferase, bsaHIM, and restriction endonuclease, bsaHIR, have been determined (GenBank accession #EU386360), cloned and expressed in E. coli. Both the restriction endonuclease and methyltransferase enzymes share significant similarity with a group of 6 other enzymes comprising the restriction-modification systems HgiDI and HgiGI and the putative HindVP, NlaCORFDP, NpuORFC228P and SplZORFNP restriction-modification systems. A sequence alignment of these homologues shows that their amino acid sequences are largely conserved and highlights several motifs of interest. We target one such conserved motif, reading SPERRFD, at the C-terminal end of the bsaHIR gene. A mutational analysis of these amino acids indicates that the motif is crucial for enzymatic activity. Sequence alignment of the methyltransferase gene reveals a short motif within the target recognition domain that is conserved among enzymes recognising the same sequences. Thus, this motif may be used as a diagnostic tool to define the recognition sequences of the cytosine C5 methyltransferases. CONCLUSION: We have cloned and sequenced the BsaHI restriction and modification enzymes. We have identified a region of the R. BsaHI enzyme that is crucial for its activity. Analysis of the amino acid sequence of the BsaHI methyltransferase enzyme led us to propose two new motifs that can be used in the diagnosis of the recognition sequence of the cytosine C5-methyltransferases.

SUBMITTER: Neely RK

PROVIDER: S-EPMC2413257 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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The BsaHI restriction-modification system: cloning, sequencing and analysis of conserved motifs.

Neely Robert K RK Roberts Richard J RJ

BMC molecular biology 20080514

<h4>Background</h4>Restriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length. Understanding the mechanism of this recognition represents a significant challenge that we begin to address for the BsaHI restriction-modification system, which recognises the six base sequence GRCGYC.<h4>Results</h4>The DNA sequences of the genes for the BsaHI methyltransferase, bsaHIM, and restriction endonuclease, bsaHIR, have been determined (GenBank acces ...[more]

PMID: 18479503

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Project description:Although prokaryotic DNA methylation investigations have long focused on immunity against exogenous DNA, it has been recently recognized that DNA methylation impacts gene expression and phase variation in Streptococcus pneumoniae and Streptococcus suis. A comprehensive analysis of DNA methylation is lacking for beta-hemolytic streptococci, and thus we sought to examine DNA methylation in the major human pathogen group A Streptococcus (GAS). Using a database of 224 GAS genomes encompassing 80 emm types, we found that nearly all GAS strains encode a type I restriction modification (RM) system that lacks the hsdS' alleles responsible for impacting gene expression in S. pneumoniae and S. suis. The GAS type I system is located on the core chromosome, while sporadically present type II orphan methyltransferases were identified on prophages. By combining single-molecule real-time (SMRT) analyses of 10 distinct emm types along with phylogenomics of 224 strains, we were able to assign 13 methylation patterns to the GAS population. Inactivation of the type I RM system, occurring either naturally through phage insertion or through laboratory-induced gene deletion, abrogated DNA methylation detectable via either SMRT or MinION sequencing. Contrary to a previous report, inactivation of the type I system did not impact transcript levels of the gene (mga) encoding the key multigene activator protein (Mga) or Mga-regulated genes. Inactivation of the type I system significantly increased plasmid transformation rates. These data delineate the breadth of the core chromosomal type I RM system in the GAS population and clarify its role in immunity rather than impacting Mga regulon expression. IMPORTANCE The advent of whole-genome approaches capable of detecting DNA methylation has markedly expanded appreciation of the diverse roles of epigenetic modification in prokaryotic physiology. For example, recent studies have suggested that DNA methylation impacts gene expression in some streptococci. The data described herein are from the first systematic analysis of DNA methylation in a beta-hemolytic streptococcus and one of the few analyses to comprehensively characterize DNA methylation across hundreds of strains of the same bacterial species. We clarify that DNA methylation in group A Streptococcus (GAS) is primarily due to a type I restriction modification (RM) system present in the core genome and does not impact mga-regulated virulence gene expression, but does impact immunity against exogenous DNA. The identification of the DNA motifs recognized by each type I RM system may assist with optimizing methods for GAS genetic manipulation and help us understand how bacterial pathogens acquire exogenous DNA elements.

Dataset Information

The BsaHI restriction-modification system: cloning, sequencing and analysis of conserved motifs.

Publications

The BsaHI restriction-modification system: cloning, sequencing and analysis of conserved motifs.

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