西瓜食酸菌RND蛋白家族外排转运体cusB基因抗铜功能研究

Functional analysis of a RND family efflux transporter component-cusB gene associated with copper resistance in Acidovorax citrulli

摘要:

【目的】研究RND外排泵中cusB基因突变对西瓜食酸菌抗铜性的影响。【方法】采用Tn5转座子随机插入基因组制备筛选得到突变体，通过双亲杂交的方法构建功能互补菌株，并从西瓜食酸菌抗铜性、胞外纤维素酶和胞外蛋白酶分泌、胞外多糖产生、生物膜形成、致病性及过敏性反应等方面阐明RND外排泵中MFP蛋白亚基对西瓜食酸菌的影响。【结果】突变体ΔcusB在含有1.25 mmol/L或2.5 mmol/L CuSO4的KMB平板上不能生长，cusB基因的突变导致西瓜食酸菌的胞外多糖分泌和生物膜形成与野生型有差异，但不影响胞外纤维素酶、胞外蛋白酶、致病性及过敏性反应。【结论】RND外排泵相关基因cusB的突变会影响西瓜食酸菌的某些生物学特性，并导致病菌对铜十分敏感。研究以RND外排泵转运重金属为导向初步解析了西瓜食酸菌的抗铜机制。

Abstract:

[Objective] we test the influence of RND family efflux transporter, MFP subunit cusB gene, on the copper resistance for Acidovorax citrulli. [Methods] Transposon (Tn5) was inserted into the genome randomly screened to prepare mutant strains constructed by functional complementation biparents mating and discussed on the effect of RND family efflux transporter MFP subunit cusB gene on the copper resistance for A. citrulli on basis of the copper resistance for A. citrulli, extracellular cellulase and extracellular protease secreted extracellular polysaccharide production, biofilm formation, virulence and motility etc. [Results] The mutant ΔcusB can’t be grown when it contained the 1.25 mmol/L CuSO4 or 2.5 mmol/L flat on KMB. The structure of cusB extracellular polysaccharide secretion and biofilm formation A. citrulli can be different from wild-type due to the effect of the mutant gene. Whatever, the mutant gene can’t directly affect the extracellular cellulase enzymes, extracellular protease, pathogenicity and allergic reactions also be obtained in our investigation. [Conclusion] The biological characteristics of A. citrulli can be affected by cusB associated mutations. Meanwhile, cusB associated mutations affect the copper resistance for A. citrulli.