Structure of two pectate lyase genes from Erwinia chrysanthemi EC16 and their high-level expression in Escherichia coli.
The pelB and pelE genes from Erwinia chrysanthemi EC16, which encode different pectate lyase enzymes, were sequenced and expressed at a high level in Escherichia coli. The genes possessed little similarity to each other in 5' signal regions, signal peptide sequences, coding sequences, or 3' noncoding regions. Both genes contained their own promoters as well as sequences 3' to the coding regions with considerable secondary structure which may function as rho-independent transcriptional termination signals. High-level expression plasmids were constructed with both genes, which led to 20% or more of E. coli cellular protein. The pectate lyases were secreted efficiently to the periplasm and, to a lesser extent, the culture medium. The mature proteins in E. coli periplasmic fractions were obtained in milligram amounts and high purity with a single-column affinity purification method. E. coli cells which produced high amounts of the pelE protein macerated potato tuber tissue as efficiently as E. chrysanthemi EC16 cells but cells producing high amounts of the pelB protein were less effective. Thus, the pelE gene product is an important pathogenicity factor which solely enables E. coli to cause a soft-rot disease on potato tuber tissue under laboratory conditions.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004885:Erwinia / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D011133:Polysaccharide-Lyases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011401:Promoter Regions, Genetic
D011133:Polysaccharide-Lyases; C023911:pectate lyase