茄科作物青枯菌NADH脱氢酶F亚基在细胞运动和致病性中的功能研究

NADH dehydrogenase subunit F is required for cell motility and pathogenicity in Ralstonia solanacearum

摘要:

[目的]劳尔氏菌（Ralstonia solanacearum）在茄科作物上引起严重的细菌性青枯病，本研究旨在发掘青枯劳尔氏菌与致病相关的基因。[方法]利用Tn5转座子构建随机插入突变体，分析生物膜形成、细胞运动和致病性；对有表型变化的突变体，运用TAIL-PCR方法鉴定Tn5插入位点，确定所突变的基因。[结果]以模式菌株GMI000为出发菌，总共获得了400个突变体，其中2个突变体不能形成生物膜，在软琼脂平板上的运动能力下降；接种感病番茄植物，这2个突变体都不能引起萎焉症状。TAIL-PCR结果显示，2个突变体的Tn5插入位点都在NADH脱氢酶F亚基（nuoF）中，距离翻译起始位点分别为103-bp和225-bp。ripAY基因启动子推动的nuoF基因互补载体，完全恢复了2个突变体的表型。[结论]NADH脱氢酶复合物是微生物呼吸电子传递链中的第一步催化酶。我们的结果表明，NADH脱氢酶复合物对R.solanacearum生物膜形成、细胞运动和致病性也有重要作用。

Abstract:

[Objective] Ralstonia solanacearum is the causative agent of a devastating bacterial wilt disease in solanaceous plants. The purpose of this work was to identify genes involved in the pathogenesis of R. solanacearum. [Methods] We used a Tn5-based mutagenesis strategy to generate random insertion mutants that were then assayed for biofilm formation, cell motility and pathogenicity. Thermal asymmetric interlaced PCR (TAIL-PCR) was performed to identify the Tn5 insertion site of mutants with altered phenotypes. [Results] A total of 400 mutants were generated in the model strain GMI1000. Two mutants were found to be unable to form biofilms and had reduced swimming and swarming motility on soft agar media. When inoculated into tomato plants, both mutants failed to cause wilting disease symptoms. Both mutants carried a Tn5 insertion within the NADH dehydrogenase subunit F gene (nuoF), and the insertion sites were at 103 bp and 225 bp from the translational start site. A complemented strain expressing nuoF under the control of the ripAY promoter fully restored the wild-type phenotype. [Conclusion] The NADH dehydrogenase complex is the first enzyme in the respiratory electron transport chain in microbes. Our data here indicated that NADH dehydrogenase plays a role in biofilm formation, cell motility and pathogenicity in R. solanacearum.