Metabolic Mechanisms Of 3-Hydroxybenzoate And 4-Hydroxybenzoate In Xanthomonas

Xanthomonas belongs to a large genus of Gram-negative phytopathogenic bacteria,which can produce yellow pigments called xanthomonadins.It's known that xanthomonadins biosynthesis is dependent primarily on a gene cluster named pig cluster.It has been recognized that the one gene of pig cluster,xanB2,is responsible for the production of a diffusible factor(DF),which is capable of regulating pigments formation.Our previous results has showed that DF is 3-hydroxybenzoate(3-HBA),which is involved in xanthomonadins biosynthesis.Here,we utilize Xanthomonas campestris pv.campestris(Xcc)as a model organism to study the enzymatic mechanism of XanB2 and the metabolic mechanism of 3-HBA and 4-HBA,the products of XanB2 catalyzed reaction.Deletion of xanB2 impaired the production of 3-HBA and 4-HBA in comparison to the wild-type strain,but this effect could be fully restored by over-expressing xanB2.By incubating purified XanB2 with chorismate,we demonstrated that XanB2 was a unique chorismate lyase which could catalyse chorismate to yield not only 3-HBA but also 4-HBA.Addition of 4-HBA to the culture media,the reporter strain xanB2 deletion mutant ?xanB2 could restore their production of respiratory chain component Coenzyme Q8(CoQ8).In contrast,3-HBA couldn't repair the CoQ8 biosynthesis of ?xanB2.These results suggested that 3-HBA and 4-HBA were required for the biosynthesis of xanthomonadin and CoQ8,respectively.Through the domain analysis and point mutations,we found that XanB2 catalysed 3-HBA and 4-HBA formation via a mechanism with the N-terminal region I conferring activity for 4-HBA biosynthesis and the C-terminal region III responsible for the production of 3-HBA.Mutations of the middle region II reduced the enzymatic activity for the yield of these two compounds.Thus,our work showed that XanB2 served as a key enzyme,which linked the shikimate pathway to CoQ and xanthomonadin biosynthetic pathway in Xcc.Xcc could rapidly degrade 4-HBA rather than 3-HBA in XOLN minimal medium.The bioinformatics analysis revealed that there was a superoperonic cluster for 4-HBA degradation in Xcc chromosome.4-HBA was catalysed by 4-hydroxybenzoate 3-monooxygenase,which was encoded by XCC0356,to form the central intermediate protocatechuate(PCA).Deletion of XCC0356 resulted in high level accumulation of 4-HBA compared with the wild-type strain,but it didn't affect on the degradation of PCA.Xcc showed reduced virulence to plant host in the absence of XCC0356.When the cells exposed to 4-HBA,the XCC0356 transcriptional level was significantly elevated.However,its transcription wasn't affected during Xcc growth on PCA.PCA was then mineralized via ortho-cleavage pathway by XCC0364-XCC0371.Through disruption of key genes of this region,we found that XCC0364 and XCC0365 encoded a special form of ?-ketoadipate succinyl-coenzyme A(CoA)transferase.XCC0367 and XCC0368 encoded a protocatechuate 3,4-dioxygenase.qRT-PCR analysis indicated that the transcriptional levels of these four genes were significantly induced after addition of 4-HBA or PCA.And their deletion mutants also exhibited a reduction of virulence in plant host.Moreover,XCC0355 and XCC0373 were identified as positive regulators in 4-HBA degradation by integrated bioinformatic analysis and targeted deletion/complementation methods.The comparative genomic analysis and molecular biological data suggested that there were two efflux pumps involved in responding to 3-HBA and 4-HBA in Xcc.They were encoded by four genes Xcc4168-Xcc4171 and three genes Xcc1398-Xcc1400,respectively.Xcc lacking XCC4168-XCC4171 couldn't grow at wild-type rates on media containing 2 mM 3-HBA or 1.5 mM 4-HBA.Deletion of XCC1398-XCC1400 didn't cause a similar growth defect.However,disruption of XCC1398-XCC1400 in the background of XCC4168-XCC4171 deletion mutant ?4168-71 led to increase sensitivity to 3-HBA or 4-HBA compared with the mutant ?4168-71.These results suggested that XCC1398-XCC1400 efflux system also contributed to expulsion of 4-HBA and 3-HBA,though it didn't play a leading role.In fact,these two efflux pumps were involved in tolerance of other chemicals,such as fusaric acid,salicylate,benzoate and as forth.In the plant virulence test,the deletion mutants of these two pumps exhibited a reduction of virulence in Chinese radish.Furthermore,according to the bioinformatic and transcriptional analysis,we inferred that Xcc contained two transporters,which were involved in 3-HBA and 4-HBA uptake.Since the detail of xanthomonadin biosynthetic mechanism remains unclear,except for the only one gene of pig cluster,xanB2,we also checked other genes in the cluster to determine whether they were involved in xanthomonadin biosynthesis as well.Mutagenesis of Xcc pig cluster was performed.After observing the production of pigments and 3-HBA/4-HBA in eighteen single-gene deletion mutants besides ?xanB2,we found that thirteen genes were required for normal levels of xanthomonadin biosynthesis.And nine of the deletion mutants couldn't display the similar 3-HBA/4-HBA levels as that of wild-type strain.In addition,all the mutants showed reduced virulence to the tested plants.Taken together,this study provided not only a framework of 3-HBA and 4-HBA biosynthesis,degradation and transport system but also a brief analysis of xanthomonadin biosynthetic gene cluster in Xcc,which would throw light on the mechanisms of stress tolerance and xanthomonadin biosynthesis.