Identification And Functional Analysis Of Copper Resistance Genes TolC And GntR In Acidovorax Citrulli

Bacterial fruit blotch（BFB）is a kind of seed-borne bacterial disease which seriously damages cucurbits,BFB is caused by Acidovorax citrulli.Since the outbreak of BFB,it has spread rapidly all over the world,causing heavy losse to the cucurbits crops.At present,the use of copper containing preparations is the main measure to prevent and control BFB in the field,but the excessive use of copper leads to not only the soil pollution,but also the emergence of copper-tolerant strains of Acidovorax citrulli.Therefore,exploring the mechanism of copper resistance can provide a theoretical basis for the research and development of new methods and measures for effective prevention and control of BFB.Firstly,effects of two types of expression vectors on the biological behaviors of Acidovorax citrulli were analyzed to provide a theoretical reference for studying the gene function.The results showed that vector p BBR1MCS-5 had no significant effect on the activities of extracellular cellulase and extracellular protease,content of extracellular polysaccharide,motility function,growth rate,copper resistance,pathogenicity on host and non-host,etc.But vector p HC60 significantly inhibited the growth rate and motility of the strain.Our results suggest that the expression vector itself may affect some biological characteristics of Acidovorax citrulli,so an appropriate expression vector is necessarily to be chosen while exploring the function of a gene.In the present study,p BBR1MCS-5 was used as expression vector in next steps.Secondly,two mutants with reduced copper resistance were obtained by screening a Tn5 insertion mutants library of Acidovorax citrulli strain FC440.The mutation sites of strain FC440 were homologous to genes Aave-1811 and Aave-2798 in strain AAC00-1.Bioinformatics analysis showed that the genes Aave-1811 and Aave-2798 encoded TolC outer membrane protein and GntR family transcription regulator,respectively.The tolC gene in strains AAC00-1,FC440,XJL12 and AW0601 all contain 1488 bp,but the sequences were different between them.Among them,strains FC440 and XJL12,or AAC00-1 and AW0601 had identical sequence.The structural prediction showed that TolC protein can secrete a signal peptide with a size of about 18～19 amino acids.The protein was highly hydrophobic at sites located at 20～30 amino acid residues,and there was a transmembrane region and subcellular location in the periplasmic space.The phylogenetic tree based on TolC protein showed that the relationship between Acidovorax citrulli and Acidovorax avenae was closer,and the relationship of Escherichia coli and Salmonella typhimurium was closer.The mutant FC440（?tolC）lost its growth ability on the KMB medium containing 3.75 mmol/L Cu SO₄,and its gene functional complementary strain was partially restored copper resistance on the KMB medium containing 3.75 mmol/L Cu SO₄,which suggests that the TolC protein can contribute to the copper resistance of Acidovorax citrulli.The mutation of the tolC did not affect other biological characteristics except for copper resistance,which indicates that TolC is not involved in these physiological activities of the bacteria.The gnt R gene in strain FC440 contains 1041 bp.The putative amino acid sequence of gnt R had no signal peptide,and there was a high hydrophobic region between 185～195 amino acid residues and a cross membrane domain in it.The physicochemical properties showed that the protein was unstable and located in the cytoplasm.Phylogenetic analysis showed that GntR of Acidovorax citrulli were closely related to Agrobacterium tumefaciens and had a distant genetic relationship with Escherichia coli.The growth of mutant FC440（?gnt R）was inhibited on KMB medium containing 3.75 mmol/L Cu SO₄,and its gene functional complementation strain was recovered completely on medium with 5.625 mmol/L Cu SO₄.It was proved that the GntR had a contribution to the copper resistance of Acidovorax citrulli.The mutation of gnt R gene did not affect other biological characteristics except for the copper resistance,which suggests that the GntR may have no significant correlation with those physiological activities of the bacteria.In conclusion,our results suggest that TolC and GntR are involved in the mechanisms of copper resistance in Acidovorax citrulli.