Schoedel C, Collmer A
J Bacteriol. 1986 Jul;167(1):117-23. doi: 10.1128/jb.167.1.117-123.1986.
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.
对植物致病肠道细菌菊欧文氏菌1237产生的几种果胶酸裂解酶同工酶中的两种基因进行了亚克隆，并通过DNA-DNA杂交进行比较，同时对编码的蛋白质进行了分析。通过核酸外切酶III逐步缺失法确定了基因在限制酶切图谱上的边界。DNA-DNA杂交研究表明，pelB和pelC之间的错配率较低（7%至17%）。在pelC与菊欧文氏菌1237染色体的其他区域之间未检测到同源性，而其他三种同工酶基因显然位于该染色体的其他区域。通过色谱聚焦或颗粒凝胶床等电聚焦法，很容易从大肠杆菌亚克隆的周质休克液中纯化果胶酸裂解酶同工酶。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，PLb和PLc的分子量分别为30,000和33,000。通过在超薄聚丙烯酰胺凝胶中进行平衡等电聚焦测定，它们的等电点分别为7.6和8.1。以聚半乳糖醛酸为底物时，PLb和PLc的Km值分别为0.20和0.32 mg/ml。反应产物的薄层色谱分析和粘度测定表明，这两种同工酶之间几乎没有差异。我们所有的数据表明，这些基因是重复的，并且这些蛋白质是同功能的。
