【作者】 彭琼；

【导师】 唐东阶；

【作者基本信息】 广西大学 ， 生物学， 2021， 硕士

【摘要】 烟酰胺腺嘌呤二核苷酸(Nicotinamide adenine dinucleotide,简称 NAD+,也称辅酶Ⅰ)是生物体新陈代谢中必需的辅酶。所有细菌都需要合成NAD+。除了从头合成之外,已报道了三条补救合成NAD+的途径(Ⅰ、Ⅱ和Ⅲ)。然而,植物病原细菌有关NAD+的合成机理还未有详细报道。十字花科黑腐病菌(Xanthomonas campestris pathovar campestris,简称Xcc)是革兰氏阴性细菌。基因组注释显示,Xcc含有参与NAD+从头合成和三条补救途径的绝大多数基因。本工作通过突变分析对推测参与这些途径的4个基因(nadDXcc、nadEXcc、nadMXcc和nadVXcc)的功能进行了较详细的研究。nadEXcc编码NAD+合成酶,该酶在NAD+从头合成及补救途径Ⅰ的最后一步将烟酸腺嘌呤二核苷酸(NaAD)转化成NAD+。缺失nadEXcc会明显减少细胞中的NAD+含量。nadDXcc编码烟酸单核苷酸腺苷酸转移酶(NadD),该酶不仅能催化烟酸单核苷酸(NaMN)生成NaAD,还能催化烟酰胺单核苷酸(NMN)生成NAD+。nadMXcc编码烟酰胺核苷酸腺苷酸转移酶(NadM)。与NadD类似,NadM也能分别催化NaMN和NMN生成NaAD和NAD+。缺失nadDXcc会导致NAD+的合成完全丧失,但缺失nadMXcc不会明显影响NAD+合成。说明NadD对Xcc NAD+的合成极为重要,而NadM不重要。NadD不仅在NAD+从头合成及补救途径Ⅰ通过将NaMN转化成NaAD起重要作用,而且在补救途径Ⅱ和Ⅲ中通过将NMN催化成NAD+起重要作用。nadVXcc编码烟酰胺磷酸核糖转移酶(NadV),该酶在补救途径Ⅱ中催化烟酰胺(Nm)生成NMN。缺失nadVXcc会明显减少NMN的含量。突变实验结果表明nadVXcc和nadEXcc不能都缺失。RNA印迹结果表明缺失nadEXcc会显著增强nadVXcc的表达,但缺失nadVXcc不会改变nadEXcc的表达水平。这些结果说明,除了从头合成途径之外,Xcc还具有合成NAD+的补救途径Ⅱ,但没有补救途径Ⅲ。至于是否具有补救途径Ⅰ还需要进一步研究。缺失nadEXcc、nadMXcc或nadVXcc的突变体(ΔnadEXcc、ΔnadMXcc、AnadVXcc)在丰富培养基和基本培养基的生长都不受影响,但nadDXcc的缺失突变体(ΔnadDXcc)的生长严重减弱。特别是AnadDXcc在基本培养基几乎不能繁殖;然而,加入2 mM NAD+能使它的生长达到野生型水平,说明NAD+是Xcc生长必须的。ΔnadVXcc、ΔnadMXcc、ΔnadDXcc 和ΔnadEXcc 突变体抗氧化和抗 SDS 能力减弱。另外,ΔnadDXcc抗渗透压和金属离子的能力以及致病和引起超敏反应的能力都严重降低;ΔnadEXcc的致病力也明显降低。但所有这些突变体胞外酶和胞外多糖的产量正常。综上所述,通过突变分析,本研究揭示了 NAD+是Xcc生长、致病和抗逆所必须的以及nadDXcc、nadEXcc和nadVXcc在NAD+合成中起重要作用。此外,除了从头合成,Xcc还使用补救途径Ⅱ合成NAD+,但不采用补救途径Ⅲ。本工作拓展了我们对Xcc的关键辅酶NAD+的合成途径的全面了解。更多还原

【Abstract】 Nicotinamide adenine dinucleotide(NAD+or coenzyme Ⅰ)is an essential coenzyme in the metabolism of organisms.All bacteria need to synthesize NAD+.It has been reported that in addition to de novo synthesis there are three salvage pathways(Ⅰ,Ⅱ and Ⅲ)to synthesize NAD+in bacteria.However,the synthesis mechnism of NAD+in phytopathogenic bacteria has not been reported in details.The crucifer black rot pathogen Xanthomonas campestris pathovar campestris(Xcc)is a Gram-negative bacterium.Genome annotation revealed that Xcc possesses the overwhelming majority of the genes involved in the NAD+de novo synthesis pathway and the three salvage pathways.In this work,four of the genes(nadVXcc,nadMXcc,nadDXcc and nadEXcc)predicted to be involved in the pathways were functionally studied in some details by mutation analysis.Deletion of nadEXcc,which encodes a NAD+synthetase catalyzing nicotinic acid adenine dinucleotide(NaAD)to NAD+in the last step of the NAD+de novo synthesis and salvage pathway Ⅰ,significantly reduced the production of NAD+.nadDXcc encodes a nicotinic acid mononucleotide adenylyltransferase(NadD)which can catalyze not only nicotinic acid mononucleotide(NaMN)to NaAD,but also nicotinamide mononucleotide(NMN)to NAD+.nadMXcc encodes a nicotinamide-nucleotide adenylyltransferase(NadM).Similar to NadD,NadM can also catalyze NaMN and NMN to form NaAD and NAD+,respectively.Deletion of nadDXcc resulted in a complete loss of NAD+production but deletion of nadMXcc did not significantly affect NAD+production,suggesting that NadD but not NadM is critical for NAD+synthesis in Xcc.NadD plays a key role not only in the NAD+de novo synthesis and salvage pathway Ⅰ by converting NaMN to NaAD but also in the salvage pathways Ⅱ and Ⅲ by converting NMN to NAD+.Deletion of nadVXcc,which encodes a nicotinamide phosphoribosyltransferase(NadV)catalyzing Nm to NMN in salvage pathway Ⅱ,resulted in a significant reduction of NMN.Mutation analysis revealed that nadVXcc and nadEXcc could not be deleted together.Northern blot showed that deletion of nadEXcc significantly increased the expression of nadVXcc,but deletion of nadVXcc did not alter the expression of nadEXcc.Taken together,these results indicate that in addition to the de novo pathway Xcc possesses salvage pathway Ⅱ but not Ⅲ to synthesize NAD+.Whether it has salvage pathway Ⅰ needs to be further studied.The growth of nadEXcc-deleted,nadMXcc-deleted and nadVXcc-deleted mutants(ΔnadEXcc,ΔnadMXcc and AnadVXcc)was not affected in either nutrient rich medium(NYG)or minimal medium(MMX),while the growth of nadDXcc-deleted mutant was reduced severely.In particular,ΔnadDXcc could hardly propagate in MMX;however,its growth could reach the wild-type level when 2 mM NAD+was added in MMX,indicating that NAD+is essential for Xcc growth.ΔnadDXcc,ΔnadEXcc,ΔnadMXcc and AnadVXcc displayed reduced antioxidant capacity and decreased resistance to the SDS.Furthermore,ΔnadDXcc exhibited a severe reduction in resistance to metal ions and osmotic stress as well as in virulence and hypersensitive response induction.The virulence of ΔnadEXcc was also reduced significantly.However,all of these mutants gave a wild-type production in extracellular enzymes and exopolysaccharides.In conclusion,by mutation analyses this work revealed that NAD+is essential for Xcc growth,virulence as well as stress tolerance,and that nadDXcc,nadEXcc and nadVXcc play important roles in NAD+ synthesis.In addition to de novo synthesis,Xcc uses salvage pathway Ⅱ but not Ⅲ to syntheze NAD+.This work expands our comprehensive understanding of the synthetic pathways of the key coenzyme NAD+in Xcc.更多还原