Mäe A, Heikinheimo R, Palva E T
Department of Molecular Genetics, Uppsala Genetic Center, Swedish University of Agricultural Sciences, Sweden.
Mol Gen Genet. 1995 Apr 10;247(1):17-26. doi: 10.1007/BF00425817.
The celV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 bp and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of CelV1 was highly similar to that of CelV of another E. c. subsp. carotovora strain SCR1193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. CelV1 mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.
编码胡萝卜软腐欧文氏菌胡萝卜软腐亚种SCC3193分泌型纤维素酶（CelV1）的celV1基因被克隆并测定了其核苷酸序列。该基因含有一个1511 bp的开放阅读框，编码一个由504个氨基酸组成的分泌蛋白。预测的CelV1氨基酸序列与另一株胡萝卜软腐欧文氏菌亚种SCR1193的CelV高度相似，但与菌株SCC3193先前鉴定的纤维素酶CelS完全不同。利用与lacZ报告基因的基因融合来表征celV1和celS的调控。这两个基因均以生长阶段依赖性方式协同诱导，并受到分解代谢物阻遏。植物提取物刺激celV1而非celS的表达。在所有测试条件下，celS基因的表达水平均远低于celV1。通过标记交换使胡萝卜软腐欧文氏菌亚种SCC3193中的celV1基因失活，结果表明celV1编码菌株SCC3193的主要纤维素酶，因为所得突变菌株SCC6001没有纤维素酶活性。CelV1突变体的毒力降低，这表明CelV1虽然不是致病性绝对必需的，但增强了菌株SCC3193浸解植物组织的能力。celV1突变体中celS基因的失活并未导致毒力进一步降低。
