gltC基因缺失对单增李斯特菌10403S抗酸应激能力的影响

Effect of gltC Gene Deletion on Acid Stress Resistance of Listeria monocytogenes 10403S

【目的】构建单增李斯特菌gltC基因缺失株,并阐明gltC基因对单增李斯特菌抗酸应激能力的影响。【方法】通过对单增李斯特菌参考菌株10403S进行添加或不添加外源性谷氨酸在致死性酸性条件的抗酸应激试验,测定谷氨酸对菌株抗酸应激存活能力的影响;利用同源重组方法构建单增李斯特菌gltC基因缺失株;通过生长曲线测定gltC基因缺失对菌株生长能力的影响;通过酸应激存活试验测定gltC基因缺失对单增李斯特菌酸应激能力的影响;通过Western blotting测定gltC基因缺失对GadD2蛋白表达水平的影响;通过构建携带gltBD启动子区域gfp报告质粒,测定在酸性和中性条件下gltC基因缺失对gltBD启动子区域转录水平的影响。【结果】酸应激结果显示,添加外源谷氨酸可极显著提高菌株10403S在pH 2.5酸性条件下的存活能力(P<0.01)。PCR结果显示,缺失株10403SΔgltC构建成功。生长曲线结果显示,gltC基因缺失不影响单增李斯特菌的生长能力。酸应激结果显示,在pH 2.5致死性酸性环境下缺失株10403SΔgltC生长能力弱于亲本株10403S(P<0.01)。Western blotting结果显示,gltC基因缺失不影响GadD2蛋白表达水平(P>0.05)。PCR结果显示,携带gltBD启动子区域的gfp报告质粒构建成功。GFP荧光分析结果显示,gltC基因缺失后在酸性和中性条件下均会极显著降低gltBD启动子区域转录水平(P<0.01)。【结论】gltC基因缺失不影响单增李斯特菌正常生长,缺失株10403SΔgltC表现出抗酸应激能力下降,且会降低gltBD启动子区域转录水平,但不影响GadD2蛋白表达水平。

【Objective】The experiment was aimed to construct a strain of Listeria monocytogenes with gltC gene deletion and elucidate the effect of gltC gene on the acid stress resistance of Listeria monocytogenes.【Method】The effect of glutamic acid on the survival ability of the 10403S was determined by acid stress test with or without the addition of exogenous glutamic acid under lethal acidic conditions.The homologous recombination method was used to construct a strain with a deletion of gltC gene.The effect of gltC gene deletion on the growth capacity of the strain was determined by growth curves.The effect of gltC gene deletion on the acid stress capacity of Listeria monocytogenes was determined by the acid stress survival assay.The effect of the gltC gene deletion on the expression level of GadD2 protein was determined by Western blotting.By constructing the gfp reporter plasmid carrying the gltBD promoter region,the effect of gltC gene deletion on the transcription level of the gltBD promoter region was measured under acidic and neutral conditions.【Result】Acid stress results showed that adding exogenous glutamic acid could extremely significantly improve the survival ability of strain 10403S under acidic conditions at pH 2.5(P<0.01).PCR results showed that the deletion strain 10403SΔgltC was successfully constructed.The growth curve results showed that gltC gene deletion did not affect the growth ability of Listeria monocytogenes.The results of acid stress showed that the growth ability of the deletion strain 10403SΔgltC was weaker than that of the parental strain 10403S in a lethal acidic environment at pH 2.5(P<0.01).The results of the Western blotting showed that the deletion of gltC gene did not affect the expression level of GadD2 protein(P>0.05).PCR results showed that gfp with gltBD promoter region reported plasmid was successfully constructed.GFP fluorescence analysis showed that the deletion of gltC gene extremely significantly decreased the transcription level of the gltBD promoter region under both acidic and neutral conditions(P<0.01).【Conclusion】Deletion of gltC gene did not affect the normal growth of the Listeria monocytogenes 10403S,but the deletion strain 10403SΔgltC reduced resistance to acid stress and decreased the level of transcription in the gltBD promoter region,without affecting the level of GadD2 protein expression.