The Role Of GlnR In The Cross Adaptation Of Lactobacillus Rhamnosus To Heat Stress And Oxidative Stress

Lactobacillus rhamnosus is one of the most studied and applied probiotics.Previous studies have shown that nitrogen metabolism regulation is a common pathway for heat stressoxidative stress cross adaptation of L.rhamnosus hsryfm 1301.GlnR is the central element of nitrogen metabolism regulation in Lactobacillus,which may be an important node of heat stress-oxidative stress cross adaptation.In this paper,the effect of GlnR on the stress tolerance of hsryfm 1301 and its mechanism were investigated by knocking out the glnR gene.The main research findings are as follows:(1)The plasmid vector pUC19e-glnRUD for gene knockout was constructed and introduced into the genome of L.rhamnosus hsryfm 1301.The recombinant strain without erythromycin resistance was screened by one-by-one detection method,and the glnR gene knockout strain L.rhamhosus hsryfm 1301ΔglnR,namely ΔglnR strain,was successfully obtained.(2)The growth curves of L.rhamnosus hsryfm 1301 and L.rhamnosus hsryfm 1301ΔglnR under different nitrogen source medium were compared.The results showed that the deletion of glnR gene did not affect the growth of L.rhamnosus.There was no significant difference in growth trend and growth between L.rhamnosus hsryfm 1301 and L.rhamnosus hsryfm 1301ΔglnR.(3)To explore the effect of GlnR on different stress tolerance of L.rhamnosus hsryfm 1301.The results showed that the survival rate of L.rhamnosus hsryfm 1301 under acid stress was 6 times higher than that of L.rhamnosus hsryfm 1301ΔglnR,while there was no significant difference in bile salt stress and cold stress tolerance between them.This indicates that GlnR is related to the resistance of L.rhamnosus to acid stress,and may regulate the expression of acid stress stress proteins.(4)The effects of GlnR on the cross-adaptation of L.rhamnosus hsryfm 1301 to heatoxidative stress at different stages were investigated.The results showed that the regulatory role of GlnR in the cross-adaptation of heat stress and oxidative stress of L.rhamnosus was complex and affected by the growth period of the strain.In the early logarithmic phase,the survival rate of ΔglnR strain was 47%lower than that of wild strain after heat pretreatment and heat stress treatment.After heat pretreatment and oxidative stress treatment,the survival rate of ΔglnR strains was 70%lower than that of wild strains,indicating that GlnR played an important role in improving the heat resistance or oxidation resistance of strains under heat pretreatment.In the late logarithmic phase,GlnR not only plays an important role in the heat stress perception of the strain,but also plays a role in the cross adaptation of heat stress and oxidative stress.In the stationary phase,the deletion of glnR gene only reduced the oxidation resistance of the strain after oxidative pretreatment,and had no significant effect on other groups.In general,compared with oxidative stress,GlnR plays a more significant role in the heat stress perception of L.rhamnosus.(5)The effect of GlnR on the survival rate of L.rhamnosus hsryfm 1301 spray drying.The results showed that after glnR gene deletion,the spray drying survival rate of L.rhamnosus hsryfm 1301 heat pretreatment group and oxidation pretreatment group decreased significantly.It indicated that GlnR was a key component to help L.rhamnosus resist heat stress and oxidative stress during spray drying.In addition,the results of acid and bile salt resistance test showed that GlnR positively regulated the acid resistance of spray-dried powder under thermal pretreatment,while negatively regulated the acid resistance of spray-dried powder under oxidation pretreatment.In addition,compared with the control group and the oxidative pretreatment group,the bile salt tolerance of the wild strain powder in the heat pretreatment group was significantly stronger than that of the ΔglnR strain powder,indicating that the glnR gene played a more significant role in enhancing the bile salt tolerance of L.rhamnosus powder under heat stress.(6)The transcriptome sequencing results of L.rhamnosus hsryfm 1301ΔglnR after heat stress and oxidative stress in the early logarithmic phase were compared with those of wild strains under the same conditions.The results showed that after heat stress treatment,the DEGs related to amino acid metabolism in the ΔglnR strain were not down-regulated(the downregulation of DEGs related to amino acid metabolism in the wild-type strain made it have stronger heat resistance and oxidation resistance),indicating that the glnR gene plays an important role in the down-regulation of genes related to amino acid metabolism.After oxidative stress treatment,the DEGs of ascorbic acid and aldarate metabolism related to antioxidation were up-regulated,indicating that ascorbic acid and aldarate metabolism may serve as an alternative pathway to help ΔglnR strains resist oxidative stress.