Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung 406, Taiwan, Republic of China.
J Agric Food Chem. 2010 Feb 10;58(3):1653-63. doi: 10.1021/jf903637s.
Exopolysaccharide and several extracellular enzymes of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, are important virulence determinants. It is known that Clp (cAMP receptor protein-like protein) and RpfF (an enoyl-CoA hydratase homologue required for the synthesis of diffusible signal factor, DSF) regulate the production of these determinants. Addition of DSF or Xcc extracellular protein containing partially purified mannanase (EC 3.2.1.78, encoded by manA) can disperse the cell aggregates formed by rpfF mutant. In this study, nucleotide G 64 nt upstream of the manA translation start codon was determined as the transcription initiation site by the 5' RACE technique. Transcriptional fusion assays showed that manA transcription is positively regulated by Clp and RpfF and induced by locust bean gum. The manA coding region was cloned and expressed in E. coli as recombinant ManA (rManA). The rManA was purified by affinity chromatography, and its biochemical properties were characterized. The rManA had a pH optimum at 7.0 (0.1 M Hepes) and a temperature optimum at about 37 degrees C. Sequence and mutational analyses demonstrated that Xcc manA encodes the major mannanase, a member of family 5 of glycosyl hydrolases. This study not only extends previous work on Clp and RpfF regulation by showing that they both influence the expression of manA in Xcc, but it also for the first time characterizes Xanthomonas mannanase at the protein level.
黄单胞菌野油菜致病变种（Xcc）的胞外多糖和几种胞外酶是黑腐病的重要毒力决定因子，这些物质是由 cAMP 受体蛋白样蛋白（Clp）和 RpfF（一种烯酰辅酶 A 水合酶同源物，对合成扩散信号因子 DSF 是必需的）调控产生的。DSF 或含有部分纯化甘露聚糖酶（EC 3.2.1.78，由 manA 编码）的 Xcc 胞外蛋白的添加可以分散 rpfF 突变体形成的细胞聚集体。在这项研究中，通过 5'RACE 技术确定了 manA 翻译起始密码子上游 64 个核苷酸的 G 为转录起始位点。转录融合测定表明，manA 转录受 Clp 和 RpfF 的正调控，并受罗望子胶诱导。克隆并在大肠杆菌中表达了 manA 编码区作为重组 ManA（rManA）。通过亲和层析纯化 rManA，并对其生化特性进行了表征。rManA 的 pH 最适值为 7.0（0.1M Hepes），温度最适值约为 37 摄氏度。序列和突变分析表明，Xcc manA 编码主要的甘露聚糖酶，它是糖苷水解酶家族 5 的成员。这项研究不仅通过表明 Clp 和 RpfF 都影响 Xcc 中 manA 的表达来扩展以前关于 Clp 和 RpfF 调控的工作，而且还首次在蛋白质水平上对黄单胞菌甘露聚糖酶进行了表征。
