野油菜黄单胞菌中LipB-LipA是唯一的硫辛酰化途径

LipB-LipA is the only lipoyl acylation pathway in Xanthomonas campestris

【目的】硫辛酸是细胞内重要的辅因子,参与多种基础代谢过程。野油菜黄单胞菌(Xcc)是十字花科植物黑腐病的病原菌,在全球范围内引起植物病害,引起重大经济损失。为此研究Xcc中硫辛酸的合成途径,为防治黑腐病提供新思路。【方法】利用大肠杆菌硫辛酸合成关键酶LipA和LipB序列,同源比对发现Xcc基因组中XC0713(XccLipA)和XC0712(XccLipB)具有较高的同源性。采用PCR方法分别扩增XccLipA和XccLipB基因,并连入表达载体pBAD24M后分别互补大肠杆菌突变株,并检测转化子生长表型。利用同源重组方法,获得替换突变株,分析其生长性状,并利用剪叶法检测替换突变株对寄主植物甘蓝的致病力。【结果】XcclipA和XcclipB能分别恢复大肠杆菌lipA和lipB突变株在基础培养上生长。XcclipA和XcclipB都是菌体生长的必需基因,不能直接被敲除。但导入pSRK-EclplA后,成功分别获得XcclipA和XcclipB敲除突变株。两种EclplA替换后的敲除突变株在基础培养上都不能生长,添加硫辛酸后能恢复生长表型。在丰富培养基上,XcclipB敲除突变株能正常生长,而XcclipA敲除突变株不能生长,添加硫辛酸后生长也能恢复。分别测定不同培养条件下生长曲线,也得到同样的结果。寄主植物侵染结果显示,与野生菌相比,XcclipA敲除突变株致病性几乎丧失,而XcclipB敲除突变株的致病性与野生菌无显著性差异。【结论】Xcc中lipA编码硫辛酸合成酶,lipB编码辛酰转移酶,两者都是必需基因。Xcc中LipB-LipA途径是唯一的硫辛酰化途径,而没有外源性的硫辛酸途径。lipA敲除后显著影响Xcc的致病性,可作为抗菌药物筛选的靶点。

[Objective]Cofactor lipoic acid participates in many basic cellular metabolic processes.Xanthomonas campestris pv.campestris(Xcc)is the pathogen of black rot of cruciferous plants,causing plant disease all over the world.Studies on the synthetic mechanism of lipoic acid in Xcc will facilitate the discovery of novel methods to control the disease.[Methods]XC0713(XccLipA)and XC0712(XccLipB)in Xcc were found based on the sequence alignments with E.coli LipA and LipB,the key enzymes for lipoic acid synthesis.For complementation analysis,XcclipA and XcclipB were amplified and ligated into expression vector pBAD24 M,which were further transferred into E.coli mutants,respectively.The growth of transformants was analyzed.With the aid of E.coli lplA expression,XcclipA and XcclipB were deleted in the genome by homologous recombination,respectively.The growth and virulence of the two mutants were analyzed.[Results]XcclipA and XcclipB complemented the growth of E.coli fabA and fabB mutants on the minimal media,respectively.Both XcclipA and XcclipB are essential genes,which cannot be deleted directly.However,XcclipA and XcclipB could be deleted when pSRK-EclplA was introduced.The two mutants could not grow on the minimal medium,but grew well when lipoic acid supplemented.XcclipB mutant could grow on the nutritional medium.XcclipA mutant almost abolished the virulence to the host plant,but XcclipB mutant showed similar virulence with wild-type strain.[Conclusion]XcclipA codes lipoyl synthase,and XcclipB codes octanoyl transferrase,both are essential genes for Xcc growth.The LipB-LipA is the only lipoic acylation pathway in Xcc.XcclipA deletion mutant showed no virulence to host plant,indicating XccLipA is a promising target for anti-bacteria agent selection.