Cloning of pectate lyase gene pel from Pseudomonas fluorescens and detection of sequences homologous to pel in Pseudomonas viridiflava and Pseudomonas putida

Abstract

Pectate lyase (PL) depolymerizes pectin and other polygalacturonates (PGAs) and is thought to play a role in bacterial invasion of plants. Production of PL by the soft-rotting pathogen Pseudomonas fluorescens CY091 is regulated by Ca2+. In the presence of Ca2+, this bacterium constitutively synthesizes PL in media containing glucose, glycerol, or PGA and excretes over 87% of total PL into culture fluids. In the absence of Ca2+, the organism fails to use PGA as a carbon source and produces very low levels of PL in media containing glucose or glycerol. Of the small amount of PL produced by the bacterium in Ca(2+)-deficient media, over 78% was detected within the cells, indicating that Ca2+ is critical not only for the production but also for the secretion of PL. The pel gene, encoding an alkaline PL (pI 10.0, Mr 41,000) was cloned and located on the overlapping region of a 4.3-kb SalI and a 7.1-kb EcoRI fragment. The 7.1-kb EcoRI fragment appears to contain a promoter for pel gene expression. A 1.7-kb SalI-XhoI subfragment of the 4.3-kb SalI fragment was cloned into pUC18 to give pROTM2. Escherichia coli cells carrying pROTM2 produce 50 to 100 times more PL than do cells carrying other pectolytic constructs. Production of PL by E. coli (pROTM2) was not affected by carbon sources or by Ca2+. The pI and Mr of PL from E. coli corresponded to values for its counterpart from P. fluorescens. A 0.7-kb BglII-ClaI fragment encoding the pel structural sequence was used to detect pel homologs in various species of fluorescent pseudomonads. Homologous sequences were observed in 10 of 11 strains of P. fluorescens, P. viridiflava, and P. putida. The pel gene in fluorescent pseudomonads is well conserved and may exist and remain repressed in certain strains or species which exhibit nonpectolytic phenotypes under laboratory conditions.