Liu Y, Jiang G, Cui Y, Mukherjee A, Ma W L, Chatterjee A K
Plant Sciences Unit, University of Missouri, Columbia, Missouri 65211, USA.
J Bacteriol. 1999 Apr;181(8):2411-21. doi: 10.1128/JB.181.8.2411-2421.1999.
Erwinia carotovora subsp. carotovora produces extracellular pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The concerted actions of these enzymes largely determine the virulence of this plant-pathogenic bacterium. E. carotovora subsp. carotovora also produces HarpinEcc, the elicitor of the hypersensitive reaction. We document here that KdgREcc (Kdg, 2-keto-3-deoxygluconate; KdgR, general repressor of genes involved in pectin and galacturonate catabolism), a homolog of the E. chrysanthemi repressor, KdgREch and the Escherichia coli repressor, KdgREco, negatively controls not only the pectinases, Pel and Peh, but also Cel, Prt, and HarpinEcc production in E. carotovora subsp. carotovora. The levels of pel-1, peh-1, celV, and hrpNEcc transcripts are markedly affected by KdgREcc. The KdgREcc- mutant is more virulent than the KdgREcc+ parent. Thus, our data for the first time establish a global regulatory role for KdgREcc in E. carotovora subsp. carotovora. Another novel observation is the negative effect of KdgREcc on the transcription of rsmB (previously aepH), which specifies an RNA regulator controlling exoenzyme and HarpinEcc production. The levels of rsmB RNA are higher in the KdgREcc- mutant than in the KdgREcc+ parent. Moreover, by DNase I protection assays we determined that purified KdgREcc protected three 25-bp regions within the transcriptional unit of rsmB. Alignment of the protected sequences revealed the 21-mer consensus sequence of the KdgREcc-binding site as 5'-G/AA/TA/TGAAA[N6]TTTCAG/TG/TA-3'. Two such KdgREcc-binding sites occur in rsmB DNA in a close proximity to each other within nucleotides +79 and +139 and the third KdgREcc-binding site within nucleotides +207 and +231. Analysis of lacZ transcriptional fusions shows that the KdgR-binding sites negatively affect the expression of rsmB. KdgREcc also binds the operator DNAs of pel-1 and peh-1 genes and represses expression of a pel1-lacZ and a peh1-lacZ transcriptional fusions. We conclude that KdgREcc affects extracellular enzyme production by two ways: (i) directly, by inhibiting the transcription of exoenzyme genes; and (ii) indirectly, by preventing the production of a global RNA regulator. Our findings support the idea that KdgREcc affects transcription by promoter occlusion, i.e., preventing the initiation of transcription, and by a roadblock mechanism, i.e., by affecting the elongation of transcription.
胡萝卜软腐欧文氏菌胡萝卜软腐亚种可产生胞外果胶酸裂解酶（Pel）、多聚半乳糖醛酸酶（Peh）、纤维素酶（Cel）和蛋白酶（Prt）。这些酶的协同作用在很大程度上决定了这种植物病原菌的毒力。胡萝卜软腐欧文氏菌胡萝卜软腐亚种还可产生过敏反应激发子HarpinEcc。我们在此证明，KdgREcc（Kdg，2-酮-3-脱氧葡萄糖酸；KdgR，参与果胶和半乳糖醛酸分解代谢的基因的通用阻遏物），即菊欧文氏菌阻遏物KdgREch和大肠杆菌阻遏物KdgREco的同源物，不仅对果胶酶Pel和Peh，而且对胡萝卜软腐欧文氏菌胡萝卜软腐亚种中Cel、Prt和HarpinEcc的产生具有负调控作用。pel-1、peh-1、celV和hrpNEcc转录本的水平受到KdgREcc的显著影响。KdgREcc突变体比KdgREcc+亲本的毒力更强。因此，我们的数据首次确立了KdgREcc在胡萝卜软腐欧文氏菌胡萝卜软腐亚种中的全局调控作用。另一个新发现是KdgREcc对rsmB（以前称为aepH）转录的负面影响，rsmB是一种控制胞外酶和HarpinEcc产生的RNA调节因子。rsmB RNA的水平在KdgREcc突变体中高于KdgREcc+亲本。此外，通过DNase I保护试验，我们确定纯化的KdgREcc保护了rsmB转录单元内的三个25bp区域。对受保护序列的比对揭示了KdgREcc结合位点的21聚体共有序列为5'-G/AA/TA/TGAAA[N6]TTTCAG/TG/TA-3'。rsmB DNA中有两个这样的KdgREcc结合位点彼此紧邻，位于核苷酸+79和+139之间，第三个KdgREcc结合位点位于核苷酸+207和+231之间。对lacZ转录融合体的分析表明，KdgR结合位点对rsmB的表达有负面影响。KdgREcc还结合pel-1和peh-1基因的操纵子DNA，并抑制pel1-lacZ和peh1-lacZ转录融合体的表达。我们得出结论，KdgREcc通过两种方式影响胞外酶的产生：（i）直接通过抑制胞外酶基因的转录；（ii）间接通过阻止全局RNA调节因子的产生。我们的发现支持了KdgREcc通过启动子阻遏（即阻止转录起始）和通过路障机制（即通过影响转录延伸）来影响转录的观点。
