THE TSP45I RESTRICTION-MODIFICATION SYSTEM IS PLASMID-BORNE WITHIN ITS THERMOPHILIC HOST

Thermus species YS45 harbors two small cryptic plasmids of 5.8 (pTsp45 s) and approximately 12 kb (pTsp45I). Plasmid pTsp45s has been entirel y sequenced, revealing three significant ORFs. In addition to a previo usly reported thermophilic plasmid-encoded replication protein (Rep), pTsp45s contains two genes for the Tsp45I methyltransferase (M.Tsp45I) and restriction endonuclease (Tsp45I). These two converging genes (ts p45IM and tsp45IR) overlap by 4 bp at their stop codons within an XbaI site. M.Tsp45I (413 aa, 47.0 kDa, recognizing 5'-GTSAC-3') is highly homologous to other m(6)A-methyltransferases, especially M.EcaI (recog nizing 5'-GGTNACC-3'). Tsp45I (332 aa, 37.4 kDa, cleaving 5'-down arro w GTSAC-3') is not homologous to M.Tsp45I, or to other restriction end onucleases. Recombinant Tsp45I is stably produced in E. coli, and clea ves DNA at 65 degrees C with the same specificity as the native enzyme . Therefore, the thermophilic Tsp45I restriction-modification system i s plasmid-borne within its native host. (C) 1997 Elsevier Science B.V.