儿茶酚铁载体对贝莱斯芽孢杆菌B9601-Y2的生物膜形成及抑菌活性的影响

Effects of bacillibactin on the biofilm formation and biocontrol activity of Bacillus velezensis B9601-Y2

摘要:

背景 嗜铁素是一种能特异性螯合三价铁离子的小分子，不仅能维持细菌胞内外铁离子平衡，还可通过竞争、抗生作用抑制病原菌。目的 贝莱斯芽孢杆菌(Bacillus velezensis) B9601-Y2 (Y2)的嗜铁素基因的功能尚不清楚，因此我们构建儿茶酚铁载体dhbA突变体，研究其对Y2的表型、拮抗玉米大斑病菌大斑病凸脐蠕孢(Exserohilum turcicum)能力的影响，为菌株Y2的生物膜形成及生防活性调控提供新的理论依据。方法 通过敲除儿茶酚铁载体生物合成基因簇基因成员dhbA，观测突变体的菌落表型、生长曲线、群集运动性能及生物膜形成能力；采用Chrome azurol S (CAS)嗜铁素检测液定量测定野生型和ΔdhbA突变体的嗜铁素相对产量；应用平板对峙和离体接种法，探究野生型和ΔdhbA突变体抑制玉米大斑病菌菌落生长能力的差异。结果 敲除菌株Y2的儿茶酚铁载体生物合成基因簇成员基因dhbA后，其菌落颜色明显变白，群集能力下降了51.9%，生物膜形成能力下降了19.5%，嗜铁素产量下降了93.5%，菌体细胞及其脂肽类粗提物对玉米大斑病菌的抑菌带宽度分别下降了31.9%、42.5%，对玉米大斑病菌侵染玉米离体叶片的防效也显著下降了75%。结论 dhbA是菌株Y2发挥生防功能不可或缺的基因之一，其缺失对菌株Y2的正常生长、群集能力、生物膜形成、嗜铁素合成和抑菌活性都造成了负面的影响。

Abstract:

Background Siderophores are small molecules that can specifically chelate trivalent iron ions, which not only maintain the balance of intra- and extracellular iron ions of bacteria but also inhibit pathogenic microbes through competitive and antibiotic effects.Objective The function of the siderophore gene in Bacillus velezensis B9601-Y2 (Y2) remains unclear. Therefore, we constructed a dhbA mutant of bacillibactin to study the effects of bacillibactin on the phenotype and biocontrol activity (against Exserohilum turcicum) of Y2. The results are expected to provide a new theoretical basis for regulating the biofilm formation and biocontrol activity of Y2.Methods The biosynthetic gene cluster gene member dhbA of bacillibactin was knocked out, and the phenotype, growth curve, swarming motility, and biofilm formation of the mutant ΔdhbA were observed and measured quantitatively. Chrome azurol S was used to measure the relative siderophore production of the wild type (WT) and ΔdhbA. Plate confrontation and in vitro inoculation methods were employed to explore the inhibitory effects of WT and ΔdhbA on the colony growth of E. turcicum.Results After the knockout of dhbA, B. velezensis B9601-Y2 showed whitening colonies, with the swarming motility, biofilm formation, and siderophore production decreasing by 51.9%, 19.5%, and 93.5%, respectively. In addition, the diameters of the inhibition zones of the cells and lipopeptide crude extract of ΔdhbA against E. turcicum decreased by 31.9% and 42.5%, respectively, compared with those of WT. Furthermore, the knockout of dhbA decreased the control efficacy on E. turcicum of detached maize leaves by 75%.Conclusion This study proved that dhbA was one of the indispensable genes for the biocontrol function of B. velezensis B9601-Y2, and the knockout of this gene negatively affected the normal growth, swarming motility, biofilm formation, siderophore synthesis, and biocontrol activity of Y2.