Shevchik V E, Kester H C, Benen J A, Visser J, Robert-Baudouy J, Hugouvieux-Cotte-Pattat N
Laboratoire de Génétique Moléculaire des Microorganismes, UMR-CNRS 5577, INSA, F-69621 Villeurbanne Cedex, France.
J Bacteriol. 1999 Mar;181(5):1652-63. doi: 10.1128/JB.181.5.1652-1663.1999.
Erwinia chrysanthemi 3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-alpha-D-galacturonosidase, PehX, have been previously identified in other E. chrysanthemi strains. Using a genomic bank of a 3937 mutant with the major pel genes deleted, we cloned a pectinase gene identified as pelX, encoding the exopolygalacturonate lyase. The deduced amino acid sequence of the 3937 PelX is very similar to the PelX of another E. chrysanthemi strain, EC16, except in the 43 C-terminal amino acids. PelX also has homology to the endo-pectate lyase PelL of E. chrysanthemi but has a N-terminal extension of 324 residues. The transcription of pelX, analyzed by gene fusions, is dependent on several environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, nitrogen starvation, and catabolite repression. Regulation of pelX expression is dependent on the KdgR repressor, which controls almost all the steps of pectin catabolism, and on the global activator of sugar catabolism, cyclic AMP receptor protein. In contrast, PecS and PecT, two repressors of the transcription of most pectate lyase genes, are not involved in pelX expression. The pelX mutant displayed reduced pathogenicity on chicory leaves, but its virulence on potato tubers or Saintpaulia ionantha plants did not appear to be affected. The purified PelX protein has no maceration activity on plant tissues. Tetragalacturonate is the best substrate of PelX, but PelX also has good activity on longer oligomers. Therefore, the estimated number of binding subsites for PelX is 4, extending from subsites -2 to +2. PelX and PehX were shown to be localized in the periplasm of E. chrysanthemi 3937. PelX catalyzed the formation of unsaturated digalacturonates by attack from the reducing end of the substrate, while PehX released digalacturonates by attack from the nonreducing end of the substrate. Thus, the two types of exo-degrading enzymes appeared complementary in the degradation of pectic polymers, since they act on both extremities of the polymeric chain.
菊欧文氏菌3937分泌多种果胶分解酶，其中已鉴定出8种具有内切模式的果胶酸裂解酶同工酶（PelA、PelB、PelC、PelD、PelE、PelI、PelL和PelZ）。先前在其他菊欧文氏菌菌株中已鉴定出两种外切酶，即外多聚半乳糖醛酸裂解酶PelX和外多聚-α-D-半乳糖苷酶PehX。利用一个缺失主要pel基因的3937突变体的基因组文库，我们克隆了一个被鉴定为pelX的果胶酶基因，该基因编码外多聚半乳糖醛酸裂解酶。3937 PelX推导的氨基酸序列与另一菊欧文氏菌菌株EC16的PelX非常相似，只是在C端的43个氨基酸不同。PelX也与菊欧文氏菌的内切果胶酸裂解酶PelL具有同源性，但有一个324个残基的N端延伸。通过基因融合分析，pelX的转录依赖于多种环境条件。它由果胶分解代谢产物诱导，并受生长阶段、氧限制、氮饥饿和分解代谢物阻遏的影响。pelX表达的调控依赖于KdgR阻遏物，它控制果胶分解代谢的几乎所有步骤，还依赖于糖分解代谢的全局激活剂环AMP受体蛋白。相比之下，大多数果胶酸裂解酶基因转录的两种阻遏物PecS和PecT不参与pelX的表达。pelX突变体在菊苣叶片上的致病性降低，但其对马铃薯块茎或非洲紫罗兰植株的毒力似乎未受影响。纯化的PelX蛋白对植物组织没有浸解活性。四聚半乳糖醛酸是PelX的最佳底物，但PelX对更长的寡聚物也有良好活性。因此，PelX结合亚位点的估计数量为4个，从亚位点-2延伸到+2。已证明PelX和PehX定位于菊欧文氏菌3937的周质中。PelX通过从底物的还原端攻击催化形成不饱和二聚半乳糖醛酸，而PehX通过从底物的非还原端攻击释放二聚半乳糖醛酸。因此，这两种外切降解酶在果胶聚合物的降解中似乎具有互补性，因为它们作用于聚合物链的两端。
