Effects of Glutamate Dehydrogenase gdhA Gene Deficiency on Antioxidant Stress in Listeria monocytogenes
[Objective]The aim of this study was to elucidate the effect of glutamate dehydrogenase gdhA gene on the antioxidant stress ability of Listeria monocytogenes,and explore the regulatory effect of glnR gene on gdhA gene.[Method]The gdhA gene deletion strain of Listeria monocytogenes was constructed by homologous recombination technique.The growth ability of parental strain 10403S,deletion strain 10403S-ΔgdhA and complementation strain 10403S-CΔgdhA were measured by growth curves.The antioxidant stress survival test was used to detect the effect of gdhA gene deletion on the antioxidant stress ability of Listeria monocytogenes.The rabbit polyclonal antibody was prepared by inducing the expression and purification of GdhA protein,and the specificity of the antibody was detected by Western blotting.GFP reporter plasmid carrying gdhA gene promoter region was constructed to determine the effect of glnR gene deletion on gdhA gene transcription level under oxidative stress.Western blotting was used to detect the effect of glnR gene deletion on the expression level of GdhA protein under oxidative stress,and to detect the effect of glnR gene deletion on the survival ability of strains.[Result]The results of growth curve showed that the deletion of gdhA gene did not affect the growth ability of Listeria monocytogenes under normal conditions.The survival ability of deletion strain 10403S-ΔgdhA was extremely significantly higher than that of the parental strain 10403S under the condition of oxidative stress with 20 mmol/L H2O2(P＜0.01).Western blotting detection indicated that the rabbit polyclonal antibody to GdhA was successfully produced.GFP fluorescence analysis showed that the transcription level of the gdhA gene was extremely significantly increased after the deletion of glnR gene(P＜0.01).Under oxidative stress,the expression level of GdhA protein was extremely significantly increased after glnR gene deletion(P＜0.01).glnR gene deletion significantly decreased the viability of strains(P＜0.05).[Conclusion]The deletion of gdhA gene,which encoded for glutamate dehydrogenase,did not affect the normal growth of Listeria monocytogenes parental strain 10403S,but significantly increased the survival ability under the oxidative stress.The transcription level of gdhA gene and the expression level of GdhA protein were negatively regulated by glnR gene.
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