| In traditional fermentation and food preservation processes, lactic acid bacteria(LAB) are often challenged by high concentrations of salt. High osmotic pressurecaused by high salt concentration triggers intracellular water outflow, cytoplasmicseparation, causing cells to stop growing or even death. The cellular adaptation toosmotic stress is an important factor that determines growth and survival of LAB inthe natural ecosystems and industrial production. For maintenance of osmoticequilibrium, microorganisms have developed several strategies to adapt to osmoticcondition, including absorption of K+, effluxion of Na+, and synthesize and/oraccumulate compatible solutes. But the molecular basis of Lactobacillus plantarumST-III to salt stress was unclear. In this study, the ability of L. plantarum ST-III to salttolerance and the ability of absption of compatable solutes were investigated.RNA-seq was used to investigate the changes in gene transcription of L. plantarumST-III cultured in chemically defined medium (CDM) with or without salt and glycinebetiane (GB). Furthermore, the ability of salt stressed cell to the function of ProU andthe results of transcriptomic analysis were studied. The main results are described asfollows：(1) L. plantarum ST-III can growth in CDM with8%NaCl, and the growth of L.plantarum ST-III was affected by addition of salt. As the salt concentration increased,the lag phase was prolonging. The addition of compatable solute alone did not affectthe L. plantarum ST-III cell growth. Except choline, GB and carnitine improved thegrowth of L. plantarum ST-III in CDM with salt. GB and carnitine protected L.plantarum ST-III cells by moving through into the cell and was not physicallyadsorbed on the cell surface.(2) RNA-seq was used to investigate the response of L. plantarum ST-III to salttolerance. The results suggested that salt have a strong effect on physiology of L.plantarum ST-III, including ion balance; carbon metabolism, cell envelopebiosynthesis, and DNA repair. When L. plantarum ST-III was cultured in6%NaCl,the expression level of transcription, replication, recombination and repair wereup-regulated more than in2%NaCl. Moreover, a total of146ncRNAs were predictedin the three samples (control,2%NaCl, and6%NaCl), and33,54, and10ncRNAwere only expressed in the control,2%NaCl, and6%NaCl samples, respectively.Thirty-seven of these ncRNA predicted their mRNA target. (3) RNA-seq was used to investigate the role of GB in resistance of L. plantarumST-III to salt stress. The transcriptom analyses showed that the existence of GBchanged the expression levels of genes associated with carbohydrate transport andmetabolism, translation, ribosomal structure and biogenesis, cell wall/membranebiogenesis, and inorganic ion transport and metabolism. Whileas, the expressionlevels of gene encoding compatible solute transporter was not significant changed. Atotal of180ncRNAs were predicted in the three samples (1mmol/L GB,2%NaCland1mmol/L GB,6%NaCl and1mmol/L GB), and82,3, and1ncRNA were onlyexpressed in the1mmol/L GB,2%NaCl and1mmol/L GB, and6%NaCl and1mmol/L GB samples, respectively. Twenty-eight of these ncRNA predicted theirmRNA target.(4) Through phylogenetic analysis, the similarity of the proU to thecorresponding gene of the L. peutosus IG1is100%. The proU, and its three openreading frames proX, proW, and proV were cloned into pNZ8148, resulting in theconstruction of Lactococcus lactis expression systems. Introduction of expressionvectors in osmotic sensitive strain Lactococcus lactis NZ9000were achieved byelectrotransformation. To determine the ability of salt tolerance, recombinant strainswere cultured in CDM with3%NaCl and1mmol/L betaine. The results showed that,the gene proU, proX, proW, proV in recombinant strains were expressed, and theexpression level were at least106times higher than control strain. Thesalt tolerance ability of recombinant strains were more exceed than control strain. Theresults proved that ProU was directly related to salt tolerant ability of L. plantarumST-Ⅲ.(5) The transcriptom analyses showed that the expression levels of genes relatedto acid resistance, bile resistance and adherence increased in salt concentrations.Hence, the ability of L. plantarum ST-III to resistance acid, bile salt, and the ability toadhere to intestinal epithelial cells were studies in this part. L. plantarum have a goodability to resistant acid. The cell membrane was almost no damage after incubatedwith pH2.0HCl for60min. Cultured in salt enhance the ability of L. plantarum ST-IIIto resistance bile salt and adhere to intestinal epithelial. And those capabilities wereproportional to the slat concentration. The effects of GB on bile resistance and on theadhesion of L. plantarum ST-III to HT-29cells were also studied. The resultssuggested that GB does not protect L. plantarum ST-III against bile salt stress, anddoes not enhance the ability of L. plantarum ST-III to adhere to intestinal epithelialcells. The cps2A gene was directly related to the ability of L. plantarum ST-III to adhere to intestinal epithelial cells. |
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