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Cloning and analysis of the plasmid-borne genes encoding the Bsp6I restriction and modification enzymes
1Institute of Biotechnology FERMENTAS, Vilnius, Lithuania.
Abstract:
The Bsp6I restriction and modification (R-M) system has been localized on the plasmid pXH13, naturally occurring in the Bacillus sp. strain RFL6. The genes coding for the Bsp6I R-M system, a Fnu4HI isoschizomer recognizing the sequence GCNGC, have been cloned in Escherichia coli by two steps. The nucleotide sequence of a 2126-bp region containing the genes for restriction endonuclease (ENase; bsp6IR) and DNA methyltransferase (MTase; bsp6IM) has been determined. The genes are separated by 99 bp and are arranged tandemly with bsp6IR preceding bsp6IM. The DNA sequence predicts an ENase of 174 amino acids (aa) (19.9 kDa) and a MTase of 315 aa (36.3 kDa). M.Bsp6I contains all the conserved aa sequence motifs characteristic for m5C-MTases. In addition, its variable region exhibits a slight similarity to the 5'-GCNGC-3'-specific target-recognition domain (TRD) from M.phi 3T. No aa sequence similarity was found between R.Bsp6I and M.Bsp6I, nor among R.Bsp6I and other known ENases. We have tested recombinant plasmids carrying the complete R-M system for their ability to transform native and pre-methylated Escherichia coli hosts. The results indicate that pre-methylation increases the efficiency of establishment of the complete R-M system. In addition, we have obtained orientation-dependent differences in transformation efficiency.
Insights
The Bsp6I restriction-modification system genes were cloned from Bacillus sp. into E. coli. Pre-methylation of E. coli hosts enhanced the establishment of this DNA modifying system.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The Bsp6I restriction and modification (R-M) system, an isoschizomer of Fnu4HI, recognizes the GCNGC sequence.
- This R-M system is naturally found on plasmid pXH13 in Bacillus sp. strain RFL6.
Purpose of the Study:
- To clone and characterize the genes encoding the Bsp6I R-M system.
- To determine the nucleotide sequence of the Bsp6I restriction endonuclease (bsp6IR) and DNA methyltransferase (bsp6IM) genes.
- To investigate the transformation efficiency of the cloned R-M system in Escherichia coli.
Main Methods:
- Cloning of the Bsp6I R-M system genes into Escherichia coli.
- DNA sequencing of a 2126-bp region containing the bsp6IR and bsp6IM genes.
- Bioinformatic analysis of predicted protein sequences for restriction endonuclease and DNA methyltransferase.
- Transformation experiments using native and pre-methylated E. coli hosts.
Main Results:
- The nucleotide sequence revealed tandemly arranged bsp6IR and bsp6IM genes, separated by 99 bp.
- Predicted proteins: a 174 amino acid restriction endonuclease (19.9 kDa) and a 315 amino acid DNA methyltransferase (36.3 kDa).
- The methyltransferase (M.Bsp6I) contains conserved motifs for m5C-MTases and shows slight similarity to M.phi 3T's target recognition domain.
- No significant amino acid sequence similarity was found between the restriction endonuclease (R.Bsp6I) and other known enzymes.
- Pre-methylation of E. coli hosts significantly increased the transformation efficiency of the complete R-M system.
- Orientation-dependent differences in transformation efficiency were observed.
Conclusions:
- The Bsp6I R-M system genes have been successfully cloned and sequenced.
- The characterization provides insights into the structure and function of this specific restriction-modification system.
- Host pre-methylation is a critical factor for efficient establishment of the Bsp6I R-M system in E. coli.