MOLECULAR-CLONING AND EXPRESSION IN ESCHERICHIA-COLI OF GENES ENCODING PECTATE LYASE AND PECTIN METHYLESTERASE ACTIVITIES FROM BACTEROIDES-THETAIOTAOMICRON

Bacteroides thetaiotaomicron strain 217 can use pectins as a sole carb on source. Preliminary characterization of the pectinolytic enzymes re vealed three complementary activities in this strain: a pectin methyle sterase (PME), a pectate lyase (PL) and a polygalacturonase (PG), whic h were all inducible by pectin or polygalacturonate. Use of the lambdo id phage replacement vector lambda EMBL3 allowed a 13.2 kb insert medi ating both PL and PME activities to be isolated. Subcloning of two Eco RI fragments in pBR325 led to the separate isolation of the pel and pm e genes. They were expressed constitutively in Escherichia coli HB101, as proved by the activities observed even in mineral medium supplemen ted only with glucose. In addition, the pme gene was expressed in both orientations. These results suggest that each gene represents an indi vidual transcriptional unit. Several properties of the cloned PL were different from those of the original strain: it was mainly associated to the outer membrane, its optimum pH was higher, and its stability at 50 degrees C was lost but partially preserved by CaCl2. In addition, the apparent specific PL activity in the E. coli membrane fraction was about 30-fold higher. On the other hand, most of the properties of th e cloned PME were similar to those of the original. Despite an enhance d thermostability, the apparent specific activity of the cloned PME wa s about 6-fold lower, and was independent of the insert orientation.