Genetic and biochemical characterization of an exopolygalacturonase and a pectate lyase from Yersinia enterocolitica

Yersinia enterocolitica, an invasive foodborne human pathogen, degrades pol ypectate by producing two depolymerizing enzymes, pectate lyase (PL) and po lygalacturonase (PG). The gene encoding the PG activity, designated pehY, w as located in a 3-kb genomic fragment of E enterocolitica ATCC 49397. The c omplete nucleotide sequence of this 3-kb fragment was determined and an ope n reading frame consisting of 1803 bp was predicted to encode a PG protein with an estimated M-r of 66 kDa and pi of 6.3. The amino acid sequence of p rePG showed 59 and 43% identity to that of the exopolygalacturonase (exoPG) of Erwinia chrysanthemi and Ralstonia solanacearum, respectively. The I: e nterocolitica PG overproduced in Escherichia coli was purified to near homo geneity using perfusion cation exchange chromatography. Analysis of the PG depolymerization products by high performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD) revealed the exolytic nature of this enzyme. The Y. enterocolitica PL overproduced in E. coli was also partially purified and the M-r and pi were estimated to be 55 kDa and 5.2, respectively. HPAEC-PAD analysis of the PL depolymerization products indica ted the endolytic nature of this enzyme. Southern hybridization analyses re vealed that pehY and pel genes of Y. enterocolitica are possibly encoded in the chromosome rather than in the plasmid. Purified exopolygalacturonase ( over 10 activity units) was unable to macerate plant tissues.