Functional Sduties Of Four Genes Predicted To Be Involved In NAD~+ Synthesis In Xanthomonas Campestris Pathovar Campestris

Nicotinamide adenine dinucleotide(NAD+or coenzyme ?)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(?,? and ?)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 ?,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 ? by converting NaMN to NaAD but also in the salvage pathways ? and ? by converting NMN to NAD+.Deletion of nadVXcc,which encodes a nicotinamide phosphoribosyltransferase(NadV)catalyzing Nm to NMN in salvage pathway ?,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 ? but not ? to synthesize NAD+.Whether it has salvage pathway ? needs to be further studied.The growth of nadEXcc-deleted,nadMXcc-deleted and nadVXcc-deleted mutants(?nadEXcc,?nadMXcc 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,?nadDXcc 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.?nadDXcc,?nadEXcc,?nadMXcc and AnadVXcc displayed reduced antioxidant capacity and decreased resistance to the SDS.Furthermore,?nadDXcc exhibited a severe reduction in resistance to metal ions and osmotic stress as well as in virulence and hypersensitive response induction.The virulence of ?nadEXcc 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 ? but not ? to syntheze NAD+.This work expands our comprehensive understanding of the synthetic pathways of the key coenzyme NAD+in Xcc.