Biological Functions Of BioC And Bio In Xanthomonas Campestris Pv.Campestris

Xanthomonas campestris pv.campestris(Xcc)is the pathogen of cruciferous black rot,because of its serious harm and complicated pathogenesis,it is urgent to develop new anti-black rot drugs.Biotin is an enzyme co-factor required by all three domains of life,participating in many metabolic pathways,it is essential for the normal growth and physiological activities of organisms.In some pathogens,the synthesis of biotin is closely related to its pathogenicity,so the biotin synthesis pathway has become a potential target for new antibacterial drugs.Therefore,this paper studied the mechanism of biotin synthesis in Xanthomonas campestris and the physiological functions of malonyl-ACP methyltransferase(Bio C)and pimeloyl-ACP methyl esterase(Bio H).The first part of this research was about the identification and functional study of bio C and bio H in Xanthomonas campestris.Xcc0383 and Xcc1678 were annotated as malonyl-ACP methyltransferase(Bio C),Xcc0385 were annotated as pimeloyl-ACP methyl esterase(Bio H).These three genes were cloned into an expression vector and transferred into Escherichia coli bio C and bio H mutants,respectively.The results showed that Xcc0383 could restored the biotin auxotrophy of ?Ecbio C mutant,Xcc0385 also restored the biotin auxotrophy of ?Ecbio H mutant.Thses results indicated that Xcc0383 and Xcc0385 had the similar function with Ec Bio C and Ec Bio H,respectively.The knockout mutants of Xcc0383,Xcc1678 and Xcc0385 were constructed.The mutant of Xcc0383 and Xcc0385 was biotin auxotrophy,with the mutant of Xcc1678 had no effect on the growth of Xcc.Subsequently,by constructing a complemented strain,it was confirmed that Xcc0383 and Xcc0385 encoded Bio C and Bio H in Xcc,respectively,and Xcc1678 does not participate in biotin synthesis.The protein of Xcc0385 was expressed and purified to construct the enzymatic reaction in vitro.The results showed that Xcc0385 could demethylate the methyl pimeloyl-ACP to form pimeloyl-ACP,further indicated that Xcc0385 had Bio H activity.The pathogenicity,pathogenicity-related factors and stress resistance of the mutant strain were examined.After knocking out Xcc0383 and Xcc0385,the ability of Xcc to infect cabbage was decreased.In the mutant of Xcc0383,the secretion of extracellular protease was decreased and the stress resistance was significantly less than Xcc wild type.These results indicated that,by blocking the synthesis of biotin in Xcc,it can reduce its virulence,and Xcc0383 affects the secretion of extracellular protease and stress resistance through mechanisms other than biotin synthesis.Based on the above results,Xcc0383 and Xcc0385 in Xcc encode Bio C and Bio H to participate in the biotin synthesis pathway;after Xcc0383 and Xcc0385 are knocked out,Xcc cannot synthesize biotin by itself,and the pathogenicity decreases;Xcc0383 is associated with the productivity of pathogenic factors and stress resistance.The second part of this research was about the relationship among the bio C,fab G and bio H in Xcc.Xcc0384,located between the Xcc0383 and Xcc0385,was annotated as3-ketoacyl-ACP reductase(Fab G).The physiological function of Xcc0384 is still unknown,and this three-gene cluster also emerged in other bacteria in Xanthomonas.Therefore,the research on Xcc0383,Xcc0384 and Xcc0385 was carried out to explore the relationship among them.Xcc0384 was cloned into an expression vector and founded that Xcc0384 was failed to restored the growth of Escherichia coli fab G temperature-sensitive mutant CL104 at 42 ?.The protein of Xcc0384 was purified and in vitro enzymatic reaction revealed that Xcc0384 could not catalyze the initiation reaction and elongation reaction of fatty acid synthesis,which indicated that Xcc0384 does not have the activity of 3-ketoacyl-ACP reductase.The ?Xcc0383?0384,?Xcc0384?0385 and ?Xcc0383?0384?0385 mutant strains were constructed,and the growth of ?Xcc0383?0384 was found to be significantly slower than Xcc wild-type strain and other mutant strains.In the basal medium,?Xcc0383?0384can not utilize exogenous biotin to restore growth,but can restore growth by using exogenous pimelic acid;?Xcc0383?0384 had slight antibacterial effect on Xcc.By transferring both Xcc0383 and Xcc0385 into ?Xcc0384?0385,the growth of complement stain was better than transferred single Xcc0385 into ?Xcc0384?0385.In the Xcc0384 mutant,the pathogenic phenotype of had no obvious change.Based on the above results,the following hypothesis is proposed.In Xcc,in addition to biotin synthesis,Xcc0385 is involved in other metabolic pathways,catalyzing the production of certain toxic substances,while Xcc0384 is responsible for reducing and degrading this toxic substances;Xcc0384 is associated with the absorption and utilization of exogenous pimelic acid and is independent of the pathogenic phenotype.