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

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.

Key words:Brucella;ycjF;ycjX;strain with double gene deletion;σ³²factor;heat stress