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

【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σ^(32)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(ΔycjXF)和回补株(CΔycjXF)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σ^(32)与ycjX和ycjF的靶向调节关系。表达并纯化pGEX-4T-1-σ^(32)重组蛋白,凝胶电泳迁移试验分析σ^(32)与ycjX和ycjF之间的结合关系。【结果】热刺激后ΔycjXF存活显著低于Brucella suis S2和CΔycjXF。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,σ^(32)可靶向富集在ycjXF启动子区,凝胶电泳迁移试验确定σ^(32)可与ycjXF体外直接结合。【结论】在σ^(32)的调节下,ycjX和ycjF在布鲁氏菌热应激中发挥正向作用。

[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 byσ^(32) 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,ΔycjXF,and CΔycjXF 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 ofβ-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 byσ^(32).The pGEX-4T-1-σ^(32) recombinant protein was expressed and purified.The electrophoretic mobility shift assay(EMSA)was employed to analyze the binding relationship ofσ^(32) with ycjX and ycjF.[Results]The growth rate ofΔycjXF slowed down compared with B.suis S2 and CΔycjXF 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 thatσ^(32) can be enriched in the promoter region of ycjXF,and the EMSA determined thatσ^(32) can directly bind to ycjXF in vitro.[Conclusion]Under the regulation ofσ^(32),ycjX and ycjF play a role in the response of Brucella to heat stress.