Isolation,characterization And The Mechanism Of The H~+-ATPase-defective Mutants From Lactobacillus Plantarum

Lactobacillus plantarum is an important probiotic bacteria which has a significant impact on human health.L.plantarum has a high ability to produces lactic acid and survival under acidic pH conditions.While postacidification always occurs during the storage because the L.plantarum will keep produce lactic acid in fermentation process,which leads to food fermentation with high acidity,and affects the sensory,shelf life of the fermentative products.A large number of studies have shown that the H~+-ATPase is one of the key enzymes involved in energy metabolism of lactic acid bacteria.It has been reported that the primary function of the H~+-ATPase in bacteria is to maintain intracellur pH.But the mechanism of the H~+-ATPase to regulate the intracellur pH is still rarely reported.In this study,two mutant strains with reduced H~+-ATPase activity have been isolated as neomycin resistant-mutants.The acid yield,glucose metabolism rate,lactic acid production and H~+-ATPase activity of the wile-type ZUST,the mutants ZUST-1 and ZUST-2 were measured,respectively.Then the mutation sites of the mutant strains were identified,the levels of H~+-ATPase genes expression were measured.Furthermore,the key genes of H~+-ATPase in regulation of the intracellular pH in L.plantarum were found out,and the root cause of the reduced H~+-ATPase activity was explored.The main work and success of this paper are as follows:1.Two mutant strains ZUST-1 and ZUST-2 with reduced H~+-ATPase activity were successfully isolated as neomycin-resistant mutants.2.The acid production ability,glucose metabolism rate,lactic acid yield and H~+-ATPase activity of ZUST and mutants ZUST-1,ZUST-2 were measured,respectively.The results showed that the growth ability of mutant ZUST-1 was slightly lower than that of the wile-type ZUST,and it was similar after entering the stationary phase.The OD660 was 1.5 and the pH was 3.7 at 24 h.Glucose metabolism rate and lactic acid production was also similar to the wile-type ZUST.The glucose concentration was 3.6 g/L,the lactic acid concentration was 19.3 g/L,and the activity of H~+-ATPase was 10.1% lower than the wile-type ZUST in stationary phase.The growth ability of the mutant ZUST-2 was the weakest,the OD660 was 0.8 and pH was 4.0 at 24 h.Glucose concentration was 6.2 g/L,lactic acid concentration was 14.8 g/L.H~+-ATPase activity was 28.8% lower than that of wile-type ZUST.3.To find out the mutant sites of the mutants,the H~+-ATPase of the wile-type and mutants were cloned and sequenced.Results showed that atpA gene of the mutants ZUST-1 and ZUST-2 exists 22 mutations by alignment of the wild-type sequence,atpC gene of ZUST-2 exists 6 mutations.The analysis of the amino acid sequence and protein three-dimensional structure show that mutations in the atpA gene of the mutants ZUST-1 and ZUST-2 were synonymous mutations,and the encoded amino acid sequence didn't change.While the mutations in atpC gene of ZUST-2 leads the amino acid sequence change,and ultimately the atpC encoded protein structure was miss an ? helix.4.The real-time RT PCR was used to evaluate the relative quantification of the H~+-ATPase genes expression.The results showed that the mutants ZUST-1 and ZUST-2 atp A gene expression were 41.1% and 35.7% lower than that of the wild-type in exponential phase,43.6% and 14.2% in stationary phase,respectively.The atpC gene expression of the ZUST-1 was similar to that of the wild-type in exponential phase,and was 30% higher than that of the wild-type in stationary phase,and the ZUST-2 atpC gene was not expressed.The mutants with lower H~+-ATPase activity were found to up-regulate the expression of H~+-ATPase genes in stationary phase,except the ZUST-2 atpC gene was not expressed.The H~+-ATPase activity has an important connection with the difference in gene expression of atpA and atpC.5.The mechanism of H~+-ATPase was further explored by adding DCCD to inhibit the activity of H~+-ATPase,and the cause of the reduced H~+-ATPase activity was confirmed.The results showed that all the coding genes of ZUST and mutants ZUST-1,ZUST-2 were higher than those in the exponential phase,which indicated that the expression of all genes was up-regulate due to the stress response after added DCCD.No matter DCCD was added or not,we found that atp A gene expression level of mutant ZUST-1 was lower than that of wile-type ZUST and the ZUST-2 atpC gene was not expressed.It was confirmed that the atp A gene and atpC gene were the key genes to regulate the faction of the H~+-ATPase.In this study two mutants strains ZUST-1 and ZUST-2 with reduced H~+-ATPase activity were obtained,the atpA and atp C were the key genes in regulation of H~+-ATPase.However,the faction of each coding subunit and the synergistic mechanism between each other have to be further studied.