Functional Analysis Of Copper Resistance Genes CusABC And ZneB In Acidovorax Citrulli

Acidovorax citrulli（Ac）is the pathogen of Bacterial Fruit Blotch（BFB）in cucurbits,which poses a serious threat to cucurbitaceous crops worldwide.At present,copper-containing chemicals are mainly used for bactericidal and antibacterial.Long-term use increases the copper tolerance of the bacteria,so that the copper-containing bactericidal and antibacterial agent has a poor control effect on BFB disease.In order to explore the anti-copper mechanism of the bacteria,this study used homologous recombination technology to obtain four copper-sensitive mutant strains FC440（?cus A）,FC440（?cus B）,FC440（?cus C）,FC440（?zneB）,,respectively Construct corresponding functionally complementary strains,and analyze related biological characteristics and related gene expression;use RNA-Seq technology to analyze the transcriptome level of FC440 under copper stress.The verification results are as follows:（1）The study of CusA-like protein found that:CusA-like protein has a conservative ACR-tran domain physicochemical properties and interaction mode similar to Escherichia coli in structure.Compared with the FC440 wild strain,the growth ability of FC440（?cus A）in the copper stress medium was weakened,and the functional complement strain FC440（?cus A-cus A）was restored.Compared with the wild type,the mutant FC440（?cus A）has weaker motility and recovers after gene function complementation.The loss-of-function mutation of this gene enhanced the pathogenicity of the strain without affecting other biological characteristics.Fluorescence quantitative PCR results showed that the pathogenicity-related gene hrc N and the mobility-related gene fli C in FC440（?cus A）were down-regulated.（2）The study on CusB-like protein found that:the membrane fusion protein CusB has the Hlyd-D23domain,and its physical and chemical properties are similar to E.coli CusB.The mutant FC440（?cus B）has a significantly weakened growth ability in a copper-stressed medium.The cellulase secretion capacity of FC440（?cus B）is lower than that of wild type.FC440（?cus B）has an enhanced pathogenic phenotype compared with wild type.The cus B-like mutation did not change other biological characteristics.Fluorescence quantitative PCR results showed that the expression of cus A-like in FC440（?cus B）was down-regulated,and the expression of cus C-like and fli C was up-regulated.（3）The study on CusC-like found that the CusC-like conservative OEP outer membrane efflux protein structure,physical and chemical properties and other aspects are similar to the model strain E.coli.After the cus C-like mutation,the growth ability of the strain in the copper stress medium is weakened,and the gene function complementation construction is completed and the recovery is consistent with the wild-type strain.The loss of cus C-like function did not affect other biological characteristics.Fluorescence quantitative results showed that the copper-resistant gene zneB and flagellar gene fli C were down-regulated in the cus C-like mutant.（4）The research on Zne B-like has found that Zne B-like has the domain of Hly D-23,and its physical and chemical properties are similar to copper-resistant bacteria.Under the stress of Cu²⁺and Zn²⁺,the growth ability of acidophilus cucumis was weaker than that of the wild type.The zneB-like mutants have weaker swimming ability than wild-type,increased secretion of extracellular vitaminase,and enhanced pathogenicity,which has nothing to do with other biological characteristics.Fluorescence quantitative PCR results showed that in FC440（ΔzneB）,the expression of flagellar gene fli C was down-regulated,and the expression of cus C-like gene was up-regulated.（5）Analysis of transcriptome data under copper stress showed that differentially expressed genes were significantly enriched in about 9 major pathways such as transmembrane transport of the ABC transport system,signal transduction of the two-component system pathway,and amino acid metabolism;the results of q PCR analysis reflected The gene expression trend is consistent with the trend of transcriptome sequencing data.The above results indicate that cus A-like,cus B-like,cus C-like,and zneB-like are involved in the copper resistance of Ac,and zneB-like is also related to zinc resistance,but these genes are related to the extracellular polysaccharides and cytoplasm of Ac.Exoprotease,induction of allergic necrosis,and the ability to form biofilms have nothing to do.Among them,cus A-like and zneB-like are positively correlated with motility,and cus B-like and zneB-like are related to extracellular secretion activity.q PCR shows that these genes can affect the expression of flagellar gene fli C.In addition,q PCR in FC440（?cus A）shows that the pathogenicity-related gene hrc N is closely related to cus A,and q PCR in FC440（?cus B）shows that cus A and cus C are related to The class cus B is closely related.The amino acid metabolism of glutamate and glutathione under copper stress plays an important role in the resistance to copper stress of Ac;the two-component system participates in the response of Ac to copper ion stress and is negatively affected.There may be anti-copper mechanisms in this bacteria including transmembrane transport of ABC transporters,redox reactions,and intracellular binding precipitation.The results of this study have accumulated basic data for further analysis of the molecular mechanism of copper tolerance of Ac.