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New restriction enzymes discovered from Escherichia coli clinical strains using a plasmid transformation method
Julie K A Kasarjian1, Masatake Iida, Junichi Ryu
1Division of Microbiology and Molecular Genetics, Department of Biochemistry and Microbiology, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA.
Abstract:
The presence of restriction enzymes in bacterial cells has been predicted by either classical phage restriction-modification (R-M) tests, direct in vitro enzyme assays or more recently from bacterial genome sequence analysis. We have applied phage R-M test principles to the transformation of plasmid DNA and established a plasmid R-M test. To validate this test, six plasmids that contain BamHI fragments of phage lambda DNA were constructed and transformed into Escherichia coli strains containing known R-M systems including: type I (EcoBI, EcoAI, Eco124I), type II (HindIII) and type III (EcoP1I). Plasmid DNA with a single recognition site showed a reduction of relative efficiency of transformation (EOT = 10(-1)-10(-2)). When multiple recognition sites were present, greater reductions in EOT values were observed. Once established in the cell, the plasmids were subjected to modification (EOT = 1.0). We applied this test to screen E.coli clinical strains and detected the presence of restriction enzymes in 93% (14/15) of cells. Using additional subclones and the computer program, RM Search, we identified four new restriction enzymes, Eco377I, Eco585I, Eco646I and Eco777I, along with their recognition sequences, GGA(8N)ATGC, GCC(6N)TGCG, CCA(7N)CTTC, and GGA(6N)TATC, respectively. Eco1158I, an isoschizomer of EcoBI, was also found in this study.
Insights
A new plasmid-based test effectively detects bacterial restriction enzymes, crucial for understanding DNA modification. This method identified four novel enzymes and confirmed existing ones in clinical E. coli strains.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Restriction-modification (R-M) systems are vital bacterial defense mechanisms.
- Traditional methods for R-M system identification include phage tests, in vitro assays, and genomic analysis.
Purpose of the Study:
- To develop and validate a novel plasmid-based assay for detecting restriction enzyme activity in bacteria.
- To screen clinical Escherichia coli strains for the presence of R-M systems.
Main Methods:
- Adapted phage R-M test principles for plasmid DNA transformation.
- Constructed plasmids with specific BamHI fragments of phage lambda DNA.
- Transformed plasmids into E. coli strains with known R-M systems and screened clinical isolates.
Main Results:
- The plasmid R-M test demonstrated reduced transformation efficiency for unmodified plasmid DNA (EOT = 10(-1)-10(-2)).
- Established plasmids underwent modification, restoring transformation efficiency (EOT = 1.0).
- Detected restriction enzymes in 93% of screened E. coli clinical strains.
- Identified four new restriction enzymes (Eco377I, Eco585I, Eco646I, Eco777I) and an isoschizomer (Eco1158I).
Conclusions:
- The plasmid R-M test is a reliable method for identifying active restriction enzymes in bacterial populations.
- The study expands the known repertoire of bacterial restriction enzymes and their recognition sequences.
- This assay facilitates the discovery of novel R-M systems in clinical and environmental bacteria.