ycjX和ycjF基因在布鲁氏菌热应激中的作用

Roles of ycjX and ycjF in the response of Brucella to heat stress

摘要:

【背景】细菌应对环境压力的能力对其存活和增殖及引起感染具有重要意义。充分揭示布鲁氏菌应激机制，可为防控布鲁氏菌病提供理论依据。研究发现，ycjX基因在细菌热应激时高表达且可能受σ³²调节，ycjF基因在细菌败血症期间表达显著上升，二者在革兰阴性菌中可能构成操纵子。【目的】研究ycjX和ycjF基因在布鲁氏菌热应激中的作用。【方法】对布鲁氏菌(Brucella)及其ycjX和ycjF双基因缺失株(△ycjXF)和回补株(C△ycjXF)进行热应激试验，计算3株菌的存活率。利用RT-PCR和β-半乳糖苷酶活性检测试验鉴定ycjX和ycjF的操纵子模式和启动子区域活性。利用ChIP试验分析σ³²与ycjX和ycjF的靶向调节关系。表达并纯化pGEX-4T-1-σ³²重组蛋白，凝胶电泳迁移试验分析σ³²与ycjX和ycjF之间的结合关系。【结果】热刺激后△ycjXF存活显著低于Brucella suis S2和C△ycjXF。明确布鲁氏菌ycjX和ycjF为同一转录本，确定其启动子区域具有活性。ChIP试验表明，σ³²可靶向富集在ycjXF启动子区，凝胶电泳迁移试验确定σ³²可与ycjXF体外直接结合。【结论】在σ³²的调节下，ycjX和ycjF在布鲁氏菌热应激中发挥正向作用。

Abstract:

[Background] The response to environmental stimuli is important for the survival, proliferation, and infection of bacteria. Fully revealing the molecular mechanism of Brucella in coping with environmental stimuli can provide a theoretical basis for the prevention and control of brucellosis. The available studies have demonstrated that ycjX is highly expressed in the bacteria under heat stress and may be regulated by σ³² and the expression of ycjF rises significantly during bacterial sepsis. The two genes may constitute an operon in Gram-negative bacteria. [Objective] To explore the roles of ycjX and ycjF in the response of Brucella to heat stress. [Methods] Heat stress test was performed on Brucella suis S2, △ycjXF, and C△ycjXF and the survival rates of the three strains were calculated. The operon modes of ycjX and ycjF were identified via RT-PCR method. The activity of β-galactosidase was determined to measure the promoter region activity. The chromatin immunoprecipitation (ChIP) assay was employed to explore the endogenous regulation of ycjX and ycjF by σ³². The pGEX-4T-1-σ³² recombinant protein was expressed and purified. The electrophoretic mobility shift assay (EMSA) was employed to analyze the binding relationship of σ³² with ycjX and ycjF. [Results] The growth rate of △ycjXF slowed down compared with B.suis S2 and C△ycjXF under heat stress. The co-transcriptional analysis showed that ycjX and ycjF were in the one transcript of Brucella. The promoter region of the two genes was active. The ChIP assay showed that σ³² can be enriched in the promoter region of ycjXF, and the EMSA determined that σ³² can directly bind to ycjXF in vitro. [Conclusion] Under the regulation of σ³², ycjX and ycjF play a role in the response of Brucella to heat stress.