Quick identification of Type I restriction enzyme isoschizomers using newly developed pTypeI and reference plasmids

Although DNA-recognition sequences are among the most important characteristics of restriction enzymes and their corresponding methylases, determination of the recognition sequence of a Type-I restriction enzyme is a complicated procedure. To facilitate this process we have previously developed plasmid R-M tests and the computer program RM search. To specifically identify Type-I isoschizomers, we engineered a pUC19 derivative plasmid, pTypeI, which contains all of the 27 Type-I recognition sequences in a 248-bp DNA fragment. Furthermore, a series of 27 plasmids (designated ‘reference plasmids’), each containing a unique Type-I recognition sequence, were also constructed using pMECA, a derivative of pUC vectors. In this study, we tried those vectors on 108 clinical E. coli strains and found that 48 strains produced isoschizomers of Type I enzymes. A detailed study of 26 strains using these ‘reference plasmids’ revealed that they produce seven different isoschizomers of the prototypes: EcoAI, EcoBI, EcoKI, Eco377I, Eco646I, Eco777I and Eco826I. One strain EC1344 produces two Type I enzymes (EcoKI and Eco377I)

Four new type I restriction enzymes identified in Escherichia coli clinical isolates

Using a plasmid transformation method and the RM search computer program, four type I restriction enzymes with new recognition sites and two isoschizomers (EcoBI and Eco377I) were identified in a collection of clinical Escherichia coli isolates. These new enzymes were designated Eco394I, Eco826I, Eco851I and Eco912I. Their recognition sequences were determined to be GAC(5N)RTAAY, GCA(6N)CTGA, GTCA(6N)TGAY and CAC(5N)TGGC, respectively. A methylation sensitivity assay, using various synthetic oligonucleotides, was used to identify the adenines that prevent cleavage when methylated (underlined). These results suggest that type I enzymes are abundant in E.coli and many other bacteria, as has been inferred from bacterial genome sequencing projects

Impact of target site distribution for Type I restriction enzymes on the evolution of methicillin-resistant Staphylococcus aureus (MRSA) populations.

A limited number of Methicillin-resistant Staphylococcus aureus (MRSA) clones are responsible for MRSA infections worldwide, and those of different lineages carry unique Type I restriction-modification (RM) variants. We have identified the specific DNA sequence targets for the dominant MRSA lineages CC1, CC5, CC8 and ST239. We experimentally demonstrate that this RM system is sufficient to block horizontal gene transfer between clinically important MRSA, confirming the bioinformatic evidence that each lineage is evolving independently. Target sites are distributed randomly in S. aureus genomes, except in a set of large conjugative plasmids encoding resistance genes that show evidence of spreading between two successful MRSA lineages. This analysis of the identification and distribution of target sites explains evolutionary patterns in a pathogenic bacterium. We show that a lack of specific target sites enables plasmids to evade the Type I RM system thereby contributing to the evolution of increasingly resistant community and hospital MRSA

Type I restriction enzymes and their relatives

Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the enzymes and of their S subunits. Finally, we describe examples of Type II restriction–modification systems that have features in common with Type I enzymes, with emphasis on the varied Type IIG enzymes

Highlights of the DNA cutters:a short history of the restriction enzymes

In the early 1950’s, ‘host-controlled variation in bacterial viruses’ was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine

A Novel Method to Detect Bacterial Restriction-Modification (R-M) Systems

Analysis of recently sequenced microbial genomes has revealed many DNA sequences that code for previously unknown restriction endonucleases and their corresponding methyltransferases. These findings show that numerous restriction enzymes abundant in bacteria have yet to be discovered. Traditionally, restriction enzymes have been discovered by the classical restriction and modification (R-M) phenomena of bacteriophages (type I and III enzymes), or by direct enzyme assays (type II enzymes). To avoid the limitations of these traditional approaches, a quantitative R-M test based on plasmid transformation efficiency (Plasmid R-M Test) was established using DNA fragments derived from the E. coli bacteriophage lambda. This test is similar to traditional “efficiency of plating” (EOP) assays but measures “efficiency of transformation” (EOT). To determine the feasibility of using plasmid transformation to detect restriction activity, five known R-M systems were tested, including: type I (EcoBI, EcoAl, Eco124I), type II (Hindlll), and type III (EcoP1I). To test the hypothesis that this methodology could be used to locate recognition sequences, we applied this methodology to determine the DNA recognition sequence for KpnAl, which was found to be GAA(6N)TGCC. For this, the computer program, RM Search was developed to analyze positive and negative DNA sequence data. In addition, a simple method was designed and used to identify the modification sites for the KpnAI methyltransferase. This method employs the concept of restriction enzyme sensitivity to the methylation status of double-stranded DNA. The recognition sequences for three previously characterized Salmonella R-M systems, StySEAI, StySENI, and StySGI were found to be ACA(6N)TYCA, CGA(6N)TACC, and TAAC(7N)RTCG, respectively. In addition, this project identified R-M systems in clinical E. coli strains EC826, EC851, and EC912. The recognition sequences for these systems respectively are GCA(6N)CTGA, GTCA(6N)TGAY, and CAC(5N)TGGC. Because plasmid transformation methods are available for many bacteria and enzyme purification is not required, this model system can be extended to further bacterial species to search for new R-M systems. Combined with RM Search, a newly developed computer program, this new test may become one of the standard methods used to find new restriction enzymes, and to predict their recognition sequences