Molecular cloning and expression in Escherichia coli of genes encoding pectate lyase and pectin methylesterase activities from Bacteroides thetaiotaomicron

Abstract

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.

Cultured Representatives of Two Major Phylogroups of Human Colonic Faecalibacterium prausnitzii Can Utilize Pectin, Uronic Acids, and Host-Derived Substrates for Growth

“…Pectin is extensively fermented in the human colon (7,55), but the ability to utilize pectin for growth has been reported for relatively few groups of human colonic bacteria. Salyers et al (43,44) showed that pectin utilization was relatively common among Bacteroides spp., occurring in 47% of 188 isolates surveyed and prompting subsequent studies on B. thetaiotaomicron (9,54). In contrast, of the 154 strains of Grampositive anaerobes tested, which included five strains reported as Fusobacterium prausnitzii, only Eubacterium eligens was previously found to utilize pectin or polygalacturonic acid (43) ( Table 3).…”

Degradation of Pectins with Different Degrees of Esterification by Bacteroides thetaiotaomicron Isolated from Human Gut Flora

“…Sometimes, the fermentation of pectin-containing complex substrates, like apple fiber, by human fecal bacteria was studied (19). Recently, Tierny et al (47) found pectate lyase, pectin esterase, and polygalacturonase activities in B. thetaiotaomicron 217 grown on pectin as the sole carbon source. These authors investigated molecular cloning and expression of genes encoding the pectate lyase and pectin esterase activities in E. coli.…”

Environmental and Gut Bacteroidetes: The Food Connection

“…Noteworthy, the genomes of Bacteroidetes species have revealed that they are champions with respect to the diversity and number of CAZymes they contain, reflecting the molecular strategies evolved by this microbial community to differentiate, capture, and degrade complex glycans. Consequently and as a result of this ability to degrade host and plant glycans, cultured (environmental or gut) species are often used to isolate specific enzymes for polysaccharide degradation (Berg et al, 1980; Tierny et al, 1994; Bernardet and Nakagawa, 2006; Reichenbach, 2006). …”