Cloning, expression and characterization of the Sau3AI restriction and modification genes in Staphylococcus carnosus TM300.

The genes encoding the restriction enzyme (ENase) and modification enzyme (MTase) of Staphylococcus aureus 3A (recognition sequence 5'-GATC-3') have been cloned in Staphylococcus carnosus TM300 using the vector pCA44. Clones carrying both genes were isolated from DNA libraries prepared with MboI + BamHI. The DNA region encoding M.Sau3AI was subcloned on a 3.66-kb EcoRV fragment in vector pT181mcs. Plasmids purified from the clones were resistant to digestion with Sau3AI, indicating that the sau3AIM gene was expressed and the product was functional in S. carnosus. Cell lysates of clones with both activities encoded on plasmid pSEM7, cut DNA with the same pattern as Sau3AI, showing that the sau3AIR gene was also expressed and the ENase was functional in S. carnosus. Sequence analysis shows that both genes are transcribed in the same direction and encode polypeptides with calculated Mrs of 56,477 for R.Sau3AI and 47,300 for M.Sau3AI. Efforts to clone one or both genes in Escherichia coli have so far failed.