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 carbon source. Preliminary characterization of the pectinolytic enzymes revealed three complementary activities in this strain: a pectin methylesterase (PME), a pectate lyase (PL) and a polygalacturonase (PG), which were all inducible by pectin or polygalacturonate. Use of the lambdoid phage replacement vector lambda EMBL3 allowed a 13.2 kb insert mediating both PL and PME activities to be isolated. Subcloning of two EcoRI fragments in pBR325 led to the separate isolation of the pel and pme genes. They were expressed constitutively in Escherichia coli HB101, as proved by the activities observed even in mineral medium supplemented only with glucose. In addition, the pme gene was expressed in both orientations. These results suggest that each gene represents an individual 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 the cloned PME were similar to those of the original. Despite an enhanced thermostability, the apparent specific activity of the cloned PME was about 6-fold lower, and was independent of the insert orientation.
Tierny
Y
Y
Laboratoire de Microbiologie, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
Béchet
M
M
Joncquiert
J C
JC
Dubourguier
H C
HC
Guillaume
J B
JB
D001439:Bacteroides / Q000201:enzymology / Q000235:genetics*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D013104:Spheroplasts / Q000378:metabolism; D013696:Temperature
D002413:Cations, Divalent; D003470:Culture Media; D004269:DNA, Bacterial; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase; D011133:Polysaccharide-Lyases; C023911:pectate lyase