Function Analysis Of Kdpg Aldolase In Two Kinds Of Pectobacterium

Pectobacterium carotovorum subsp.carotovorum(Pcc)and P.atrosepticum(Pba)are important plant pathogens in Pectobacterium.Due to a rich diversity of species and a wide range of host plants in Pectobacterium,there is still a lack of effective prevention and control measures in production.Therefore,the study of the pathogenic mechanism of soft rot pathogens such as Pcc and Pba can provide a scientific basis for the development of effective prevention and control of bacterial soft rot and blackleg disease,and has important theoretical and practical significance.The ED(Entner-Doudoroff)pathway is an important carbohydrate metabolic.The main metabolic substrate is gluconic acid,and 2-keto-3-deoxy-6-phosphogluconate(KDPG)aldolase(Eda)and 6-phosphogluconate dehydratase(Edd)are the key enzymes of the ED pathway.Previous studies in the laboratory found that the ED pathway is not essential for the pathogenicity of Pcc and Pba,while Eda in the ED pathway is related to pathogenicity.The effect of Eda on the pathogenicity of Pba is mainly through the influence of Pba on the utilization of pectin during the infection process.To further elucidate the function of the ED pathway in Pcc and Pba,this study has performed biological analyses of edd and eda in the ED pathway.We found that Pcc has an incomplete ED pathway due to the lack of edd and Pba has a complete ED pathway through genomic analysis.To investigate the effect of Eda and Edd on the use of carbon sources in Pcc and Pba.Determination of SCRI 1039,SCRI 1039?edd,PccSl,PccS1(eddPba)and PccS1(pBBR)growth in normal medium(MM+0.2%gluconate and MM+0.2%pyruvate),and phenotypic tests on BIOLOG's 94 different nutrients for PccSl,PccS1?eda,SCRI 1039,SCRI 1039?eda and SCRI 1039?edd.We can conclude that SCRI 1039 can use gluconic acid,PccS1 is unable to use gluconate,and SCRI 1039?edd utilization ability is poor,but,PccSl(eddPba)can partially restores its ability to use gluconic acid.It is proved that edd plays an important role in the utilization of gluconic acid in Pcc and Pba.The utilization of 71 carbon sources in BIOLOG was different among eda-related strains.Among them,there was a significant difference in the utilization of three carbon sources of pectin,D-galacturonic acid and D-gluconate,PccS1?eda and SCRI 1039?eda have a reduced ability to use pectin and D-gluconate,making it impossible to use almost D-galacturonic acid,indicating that eda plays an important role in the metabolism of pectin for Pcc and Pba.In the PROSITE family of proteins and domain databases,we found that Eda has two domains and one active site is at the end of the domain.In order to further study the pathogenicity-related functions of Eda's different domains.In this study,two domains of Eda in the strain PccS1 were constructed to complement PccS1 ?eda by means of the complementarity of the vector,and the strains PccS1?eda(eda1-FLAG)and PccS1?eda(eda2-FLAG)were obtained.Western blot results showed that the complemented Eda single domain can be normally expressed.However,the pathogenicity assays in chinese cabbage and calla lily revealed that the pathogenicity was not restored,while the virulence of the ?eda(eda)complementary strain was restored.It was proved that both domains of Eda played an important role in the pathogenic process in Pcc.In order to further explore how Eda affects the regulatory mechanisms of the pathogenicity in Pectobacterium.In this study,transcriptome sequencing analysis was performed on PccS1,PccS1 ?eda,SCRI 1039 and SCRI 1039?eda.The results showed that 11 genes were up-regulated and 6 genes were down-regulated in PccS1?eda compared with PccSl.The differentially expressed genes were mainly concentrated in carbon metabolism,pyruvate metabolism,pentose phosphate pathway and dicarboxylic acid metabolism.Compared with SCRI 1039,SCRI 1039?eda had 407 genes up-regulated and 316 genes down-regulated.A large number of differentially expressed genes in carbon metabolism,flagellar assembly,starch and sucrose metabolism,and citric acid cycle.It can be found that eda plays an important role on the carbon metabolic in PccS1 and SCRI 1039.Gene mutations in hexR,a transcriptional regulator associated with carbon metabolism,were analyzed in the results.The results of the pathogenicity test showed that AhexR had no effect on the pathogenicity.In summary,the ED pathway is the only way to metabolize gluconic acid in Pectobacterium,eda is associated with pectin metabolism and utilization of gluconic acid in Pcc and Pba.At the same time,Eda's pathogenic regulatory network is closely related to carbon metabolism.