Abstract
The genes for two of several pectate lyase isozymes produced by the phytopathogenic enterobacterium Erwinia chrysanthemi 1237 were subcloned and compared by DNA-DNA hybridization, and the encoded proteins were analyzed. The borders of the genes were located on a restriction map by incremental exonuclease III deletions. DNA-DNA hybridization studies revealed a low percentage of mismatch (7 to 17%) between pelB and pelC. No homology was detected between pelC and other regions of the E. chrysanthemi 1237 chromosome, in which three other isozyme genes apparently reside. The pectate lyase isozymes were readily purified by chromatofocusing or granulated-gel bed isoelectric focusing from the periplasmic shock fluids of Escherichia coli subclones. The molecular weights of PLb and PLc were 30,000 and 33,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their isoelectric points were 7.6 and 8.1, respectively, as determined by equilibrium isoelectric focusing in ultrathin polyacrylamide gels. The Km values for PLb and PLc were 0.20 and 0.32 mg/ml, respectively, with polygalacturonate as a substrate. Thin-layer chromatography of reaction products and viscometric assays revealed little difference between the two isozymes. All our data indicate that the genes are duplicates and that the proteins are isofunctional.