| The watermelon industry and the seed production industry have suffered severely from Bacterial Fruit Blotch(BFB)caused by Acidovorax citrulli,one of the important cash crops in the world.At present,the application of copper-containing agents is the main method to prevent and control BFB.With the widespread application of copper-containing bactericides,heavy metals in soil accumulate in large quantities,leading to increasingly serious heavy metal pollution.In addition,copper resistant strains are present in the pathogenic bacteria,making copper preparation basically ineffective in preventing and controlling BFB.Therefore,studying the anti-copper gene of bacteria and exploring its anti-copper mechanism can provide new ideas and theoretical basis for the research and development of new drug targets for the effective prevention and control of BFB.Therefore,based on the genome database of Acidovorax citrulli FC440 strain accumulated by the previous research team and the transcriptome database under copper stress,this study screened 12 candidate copper resistance genes by means of bioinformatics.The homologous recombination technique was used to construct these gene deletion mutant strains,and two copper sensitive mutants were obtained through the detection of copper resistance ability.The complementary strains with gene function were further constructed to verify the function of genes,and two genes related to the copper resistance mechanism of Acidovorax citrulli were identified.The main results are as follows:Phenotypic results of the growth ability of 12 candidate copper-resistant mutants at different copper concentrations showed that deletion of cueR(gene code GE00648)and copZ(gene code GE00647)of CueR and CopZ led to reduced bacterial copper resistance,and the two mutants were respectively named FC440(ΔcueR)and FC440(ΔcopZ).Bioinformatics analysis showed that the CueR-like protein of Acidovorax citrulli was similar to the CueR structure of other 4 types of bacteria,that is,the tertiary structure was mainly composed of Helix and irregular coil,and contained helix-turn-helix(HTH)domain that could be combined with DNA.The CueR-like sequences of Acidovorax citrulli and Pseudomonas aeruginosa were the most similar.The interaction prediction showed that the CueR of the Acidovorax citrulli could interact with the P-type ATPase(CopA)and CueO of the bacteria,and there was a palinic structure in the CopA promoter sequence(5’-TGACCTN7ATGTCA-3’,gray part showed asymmetric bases)that could bind to the CueR.Bioinformatics analysis showed that CueR protein of Acidovorax citrulli has similar structure and function with CueR,a transcription regulator in Escherichia coli.Compared with the wild-type strain,the mutant strain FC440(ΔcueR)is significantly more sensitive to copper stress and its growth ability is significantly reduced with the increase of copper concentration,while the gene complementary strain FC440(△cueR-cueR)has similar performance to the wild-type strain,namely,its copper tolerance is restored.Fluorescence quantitative PCR results showed that under copper stress,the expressions of cueR,copA and cueO were significantly up-regulated in FC440(WT),while those of copA and cueO in FC440(ΔcueR)were significantly down-regulated.The above results prove that cueR-like genes that are resistant to copper are present in Acidovorax citrulli.The swimming ability of the mutant strain FC440(ΔcueR)has been significantly enhanced,and its swimming performance of the gene complementary strain FC440(ΔcueR-cueR)is similar to that of the wild type,indicating that the cueR-like gene is negatively correlated with its swimming ability.The mutation of cueR-like gene did not affect the pathogenicity,extracellular cellulase activity,extracellular protease activity,extracellular polysaccharide secretion and biofilm formation ability of Acidovorax citrulli except for the resistance to copper and swimming,indicating that cueR-like bacteria did not participate in these physiological activities.Bioinformatics analysis showed that CopZ-like proteins were mainly composed of helices and folds,with CxxC motif and metal binding sites binding cysteine residues.The CopZ-like sequences of Acidovorax citrulli and Enterococcus hirae were the most similar.Bioinformatics analysis showed that CopZ protein of Acidovorax citrulli and CopZ protein of Escherichia coli had similar structure and function.Compared with the wild-type strain,the mutant strain FC440(ΔcopZ)has a significant sensitivity to copper stress,and its growth ability significantly decreases with the increase of copper concentration,but the phenotype of the complementary strain FC440(△copZ-copZ)is consistent with that of the mutant strain,that is,its copper tolerance has not recovered.The growth capacity of the single gene complementary strain FC440(△copZ-cueR)and the double gene complementary strain FC440(△copZ-copZ-cueR)on the copper containing medium is restored to the wild type,that is,its copper tolerance is restored.Fluorescence quantitative PCR results show that under copper stress,the expressions of copZ,cueR,copA and cueO are significantly up-regulated in wild type FC440;in FC440(ΔcopZ),the expressions of cueR,copA and cueO are significantly down-regulated;in FC440(ΔcueR),the expressions of copZ,copA and cueO are significantly down-regulated.The above results showed that copZ mutation inhibited the expression of adjacent downstream genes cueR at the same time,and the copper-resistant phenotype of copZ mutation was caused by cueR deficiency.However,copZ expression responded to copper stress and was regulated by cueR,and this gene was involved in the copper-resistant mechanism of bacterium,which was a copper-resistant related gene of Acidovorax citrulli.Compared to the wild-type strain,the swimming ability of the mutant strain FC440(ΔcopZ)is significantly enhanced,while the performance of the complementary strain FC440(ΔcopZ-copZ)is similar to that of the wild-type strain,indicating that the copZ-like gene is negatively correlated with its swimming ability.The mutation of copZ-like gene did not affect the pathogenicity,extracellular cellulase activity,extracellular protease activity,extracellular polysaccharide secretion and biofilm formation ability of Acidovorax citrulli except the resistance to copper and swimming,indicating that copZ-like gene did not participate in these physiological activities of the bacteria. |
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