鸭疫里氏杆菌CH-1株B739_RS03695基因的功能鉴定

Functional identification of B739_RS03695 in Riemerella anatipestifer CH-1

摘要:

【背景】鸭疫里默氏杆菌是一种可引起鸭传染性浆膜炎的革兰氏阴性病原菌。对于该菌的铁离子转运系统已有大量研究，但有关该菌铁硫簇装配基因的功能知之甚少。【目的】序列分析表明，鸭疫里默氏杆菌CH-1株B739_RS03695编码铁硫结合蛋白，但其具体功能未知。本研究旨在鉴定鸭疫里默氏杆菌CH-1株B739_RS03695基因在抗氧化应激及耐药中的功能。【方法】构建B739_RS03695缺失株，通过测定生长曲线、氧化应激存活率探究其在抗氧化应激中的功能；通过最小抑菌浓度、杀菌试验探究其在耐药中的功能。【结果】与亲本株相比，RA CH-1ΔB739_RS03695在GCB培养基中生长不受影响，但在铁限制性培养基中的生长受到明显抑制；与亲本株相比，RA CH-1ΔB739_RS03695对H₂O₂的敏感性增加；与亲本株相比，RA CH-1ΔB739_RS03695对庆大霉素的抵抗力显著增加；荧光定量PCR结果表明，与对照组相比，B739_RS03695基因的转录水平在铁限制性条件和存在过氧化氢条件下显著上调。【结论】B739_RS03695基因缺失后会损害RA CH-1在铁限制性培养基中生长，B739_RS03695基因参与RA CH-1对过氧化氢的抵抗并促进庆大霉素对RA CH-1的杀灭作用。

关键词:鸭疫里默氏杆菌;铁硫结合蛋白;氧化应激;耐药

Abstract:

[Background] Riemerella anatipestifer (RA) is a Gram-negative bacterium that causes infectious serositis in ducks. Despite the extensive studies about the iron transport system of this bacterium, little is known about the function of iron-sulfur cluster assembly gene. [Objective] Sequence analysis shows that B739_RS03695 encodes an iron sulfur binding protein in R. anatipestifer CH-1, while the function of this gene remains unknown. This study aims to reveal the role of gene B739_RS03695 in the strain resistance to oxidative stress and antibiotics. [Methods] We constructed the B739_RS03695-deleted strain and studied its response to oxidative stress by establishing the growth curve and conducting the oxidative stress assay. The role of the gene in antibiotic resistance was measured by the minimum inhibitory concentration (MIC) and time-dependent killing experiment. [Results] Compared with the parent strain, RA CH-1ΔB739_RS03695 presented unaffected growth in the GCB medium, inhibited growth in the iron-restricted medium, increased sensitivity to H₂O₂, and enhanced resistance to gentamicin. The real-time PCR results showed that compared with the control group, the transcription level of B739_RS03695 was significantly up-regulated under iron restriction and hydrogen peroxide stress conditions. [Conclusion] The deletion of B739_RS03695 impaired the growth of RA CH-1 in iron-restricted medium. B739_RS03695 was required for the resistance of RA CH-1 to H₂O₂ and the killing effect of gentamicin on RA CH-1.

Key words:Riemerella anatipestifer;iron-sulfide-binding protein;oxidative stress;antibiotic resistance