He X Y, Li Y Q, Zhou X D
West China Hospital of Stomatology, Sichuan University & State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, Chengdu 610041, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2022 Jan 9;57(1):68-75. doi: 10.3760/cma.j.cn112144-20210520-00260.
To explore the effect of csn2 gene deficiency on starvation tolerance and extracellular polysaccharides (EPS) synthesis in an oligotrophic environment of (Sm). The csn2 gene deletion strains and complementary strains of Sm were cultivated and then an oligotrophic growth environment for Sm growth by setting different concentration gradient media were created. Cell growth in oligotrophic environment was detected by growth curve. Biofilm volume was measured by crystalline violet staining. Scanning electron microscopy (SEM) and laser confocal microscope were performed to observe the biofilm structure of Sm. The synthesis of EPS was measured by the anthrone-sulfuric acid method. The expression of genes related to EPS synthesis was evaluated by quantitative real-time PCR (qRT-PCR). The growth curve results showed that the deletion of csn2 gene inhibited the growth of Sm under starvation stress. Furthermore, the results of laser confocal microscope showed that the biofilm EPS/bacteria ratios produced by the wild-type strain, csn2 gene-deficient strain and complement strains under nutrient sufficient culture conditions were 0.44±0.07, 1.05±0.13 and 0.57±0.08 respectively, while the ratios of EPS/bacteria in an oligotrophic environment were 0.93±0.24, 3.05±0.21 and 1.32±0.46 respectively, indicating that the deletion of csn2 gene enhanced the ability of extracellular polysaccharide synthesis of Sm in the oligotrophic environment. The expression levels of EPS synthesis-related genes gtfB and gtfC were up-regulated by 2.5 fold and 1.8 fold respectively and the expression level of gtfD was down-regulated by two-thirds. The csn2 gene deficiency showed multiple effects on the physiological functions and virulence characteristics of Sm, including starvation tolerance and EPS synthesis. These changes might be related to the shift of the complex regulative network caused by csn2 gene deletion.
为探究csn2基因缺失对嗜麦芽窄食单胞菌(Sm)在贫营养环境下饥饿耐受性及胞外多糖(EPS)合成的影响。培养Sm的csn2基因缺失菌株和互补菌株,然后通过设置不同浓度梯度的培养基为Sm生长创造贫营养生长环境。通过生长曲线检测贫营养环境中的细胞生长情况。用结晶紫染色法测量生物膜体积。进行扫描电子显微镜(SEM)和激光共聚焦显微镜观察Sm的生物膜结构。用蒽酮 - 硫酸法测定EPS的合成。通过定量实时PCR(qRT-PCR)评估与EPS合成相关基因的表达。生长曲线结果表明,csn2基因缺失抑制了饥饿胁迫下Sm的生长。此外,激光共聚焦显微镜结果显示,在营养充足的培养条件下,野生型菌株、csn2基因缺失菌株和互补菌株产生的生物膜EPS/细菌比率分别为0.44±0.07、1.05±0.13和0.57±0.08,而在贫营养环境中EPS/细菌比率分别为0.93±0.24、3.05±0.21和1.32±0.46,表明csn2基因缺失增强了Sm在贫营养环境中胞外多糖的合成能力。EPS合成相关基因gtfB和gtfC的表达水平分别上调了2.5倍和1.8倍,而gtfD的表达水平下调了三分之二。csn2基因缺失对Sm的生理功能和毒力特性表现出多种影响,包括饥饿耐受性和EPS合成。这些变化可能与csn2基因缺失导致的复杂调控网络的转变有关。