Effects Of Salt Stress Response Gene On Division And Proliferation On The L. Bulgaricus

Lactobacillus bulgaricus is the main strain of the lactic acid bacterium starter,which suffers from salt stress generated by the flowing-alkaline method for the inhibition of acid during high density culture.Previous research shows that exogenous glycine betaine can effectively relieve the stress pressure on the bacteria,but it can`t restore the bacteria to the state where no salt stress.The reason is considered to be due to the effects of salt stress on the division and proliferation of L.bulgaricus.Now,there are few studies on the influence of L.bulgaricus in response to salt stress on its own division and proliferation as the starting point.Therefore,this project is based on L.bulgaricus to analyze the differential expression level of salt stress-responsive genes and then construct mutant strains.A preliminary study was conducted on the role of related genes in the process of cell division and proliferation.In this study,we used quantitative PCR to analyze the expression levels of genes that might respond to salt stress.It was found that the relative expression levels of the six response regulators of L.bulgaricus 34.5 rr1-rr6 under salt stress were all increased,and the relative expression of these gene in response to salt stress tended to decline after glycine betaine added.By analyzing the expression levels of the five membrane transporter genes selected,L.bulgaricus 34.5 responded to salt stress mainly by regulating the expression of the primary transporter genes.Among them,the expression of membrane transporter gene feoA gene was upregulated by 3.59 times under salt stress.After glycine betaine was added,the expression level was increased by 2.65 times.In order to study the effects of feoA gene on the division and proliferation of Lactobacillus bulgaricus,based on the principle of homologous recombination,the tet gene was as a resistance selection marker and the feoA gene of L.bulgaricus 34.5 was knocked out,and then the growth curve,cell morphology and the differential expression level of division-related genes were analyzed.The relationship between feoA gene and the bacterial growth,proliferation was discussed.The study found that the loss of the gene feoA led to the prolongation of the lag phase of L.bulgaricus 34.5 by 1.5 h.After the logarithmic phase of growth,the biomass of the bacteria decreased by 15%.At the stationary phase,the biomass of the bacteria was basically the same as wild strains.Microscopic observations revealed that the mutant ?feoA strain showed an irregular curved or semicircular morphology in the logarithmic growth phase,and a typical short rod-like morphology of lactic acid bacteria in the stationary phase.By comparing the expression levels of division-related genes,it was found that L.bulgaricus 34.5 strain significantly increased the expression levels of the genes ftsZ,mbl,and mreB in the logarithmic phase to adapt to the high-speed division and proliferation state of the bacteria.The loss of the gene feoA caused the expression of the genes ftsZ and mreB to differ from that of the wild type,and the expression of the two genes decreased by 0.61 and 0.31 times,respectively,in the late logarithmic phase where the division tended to be slow.It was speculated that the loss of the gene feoA can affect the cell division and proliferation by affecting the expression of ftsZ and mreB.This effect was reflected in the decrease in the logarithmic growth of the mutant ? feoA,and the morphological change,but the stable cells were not affected by the loss of the gene feoA.