Localization of the type I restriction-modification enzyme EcoKI in the bacterial cell

To localise the type I restriction-modification (R-RI) enzyme EcoKI within the bacterial cell, the Hsd subunits present in subcellular fractions were analysed using immunoblotting techniques. The endonuclease (ENase) as well as the methylase (MTase) were found to be associated with the cytoplasmic m embrane. HsdR and HsdM: subunits produced individually were soluble, cytopl asmic polypeptides and only became membrane-associated when coproduced with the insoluble HsdS subunit. The release of enzyme from the membrane fracti on following benzonase treatment indicated a role for DNA in this interacti on. Trypsinization of spheroplasts revealed that the HsdR subunit in the as sembled ENase was accessible to protease, while HsdM and HsdS, in both ENas e and MTase complexes, were fully protected against digestion. me postulate that the R-M enzyme EcoKI is associated with the cytoplasmic membrane in a manner that allows access of HsdR to the periplasmic space, while the MTas e components are localised on the inner side of the plasma membrane. (C) 20 00 Academic Press.