CLONING AND ANALYSIS OF THE PLASMID-BORNE GENES ENCODING THE BSP6I RESTRICTION AND MODIFICATION ENZYMES

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 t he genes for restriction endonuclease (ENase; bsp6IR) and DNA methyltr ansferase (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 m(5)C-MTases. In addition, its vari able region exhibits a slight similarity to the 5'-GCNGC-3'-specific t arget-recognition domain (TRD) from M . phi 3T. No aa sequence similar ity 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-meth ylated Escherichia coli hosts. The results indicate that pre-methylati on increases the efficiency of establishment of the complete R-M syste m. In addition, we have obtained orientation-dependent differences in transformation efficiency.