|
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.
|
