Cloning of a pair of genes encoding isoschizomeric restriction endonucleases from bacillus species: the BspEI and BspMII restriction and modification systems

The respective genes ( R− M) encoding restriction and modification systems from two Bacillus species which recognise the same nucleotide sequence, 5′-TCCGGA, have been cloned and expressed in Escherichia coli. The BspEI R-M genes were cloned on a 3.6-kb HindIII fragment, whereas the BspMII R-M genes were cloned on three contiguous HindIII fragments totalling 9.8 kb. Upon thermal induction, E. coli carrying the bspEIR clones under the control of the phage λ PL promoter, express high levels of R·BspEI (10 6 units/g wet cell paste). The bspMIIR gene, on the other hand, is only poorly expressed (about 4 × 10 3 units/g wet cell paste) following induction. Although the enzymes of both R-M systems recognize the same sequence and the restriction endonucleases (ENases) cleave DNA at the same position, the modification specified by the methyltransferases (MTases) differ. The internal cytosine is the site for M·BspMII modification (TC meCGGA), whereas the external cytosine is modified by M·BspEI (T meCCGGA). The two R-M systems probably evolved independently.