Proteomic Characterization Of The Acid Tolerance Response In Lactobacillus Delbrueckii Subsp. Bulgaricus CAUH1and Functional Identification Of Acid Stress-related Genes Ldb0677and Pyk

Yoghurt has been considered beneficial to the human health, but post-acidification has been a serious problem which restricts the development of yogurt industry. Previous studies indicate that controlling the growth of Lactobacilus delbrueckii subsp. bulgaricus is the key to solve the post-acidification. In this study, proteomics approach complemented by transcriptional analysis was employed to separate and identify the proteins which were differentially expressed in response to acid stress, and further characterize their function during acid adaptation. This study will get more useful information for comprehensively understanding the mechanism of acid tolerance response in L. bulgaricus, meanwhile the results will also provide new insights and ideas for resolving the post-acidification problem in the yoghurt production and storage. The research contents and results in this dissertation were as followed.（1） Identification and transcriptional analysis of the proteins which were differentially expressed in L. bulgaricus CAUH1under acid stress. Twenty seven proteins were separated by2-DE approach and identified by MALDI-TOF/TOF-MS/MS. The changes of these proteins in L. bulgaricus CAUH1at transcription level were detected using real-time reverse transcription PCR. These results also demonstrated that all the changes in protein levels were accompanied by concordant changes in the expression of corresponding mRNAs, which further revealed that the expression of some key genes were regulated mainly at transcriptional level in L. bulgaricus under acid stress condition.（2） Bioinformatics analysis of the mechanism of acid tolerance response in L. bulgaricus. The metabolic pathways participated by the differently expressing genes and their functions were studied using bioinformatics analysis, revealing that when grown under acid stress condition, L. bulgaricus CAUH1enhanced the expression of glycolysis-associated enzymes to improve the carbohydrate catabolism and energy supply; rerouted the pyruvate metabolism to fatty acid biosynthesis to increase the ratio of saturated fatty acids in the cell membrane and enhance the rigidity and impermeability of the membrane; modulated the abundance of elongation factors （EF-Tu, EF-G） and ribosomal protein S1（RpsA） to reduce the rate of protein synthesis and enhance the translational accuracy.（3） Functional identification of acid stress-related genes by heterologous over-expression. Heterologous over-expression of six acid stress-related genes were carried out using the L. lactis NICE system. The results indicated that Pyk-overproducing strain showed45-fold increase in survival under acid stress condition and Ldb0677-overproducing strain showed200-fold increase in survival. SDS-PAGE indicated that Pyk and Ldb0677were successfully expressed, thus these two protein were considered to play important roles in acid tolerance response. In addtion, over-expression of Ldb0677also enhanced the bile resistance of the host strain. Over-expression of Pyk enhanced the bile and cold resistance of the host strain.（4） Characterization of a novel acid stress-related transcription factor Ldb0677. The DNA-binding motif （SSTAGACR） of Ldb0677was determined using bacterial one-hybrid. Twenty two target genes were predicted according to the DNA-binding sequence feature. The Ldb0677with a His-tag was expressed using L. lactis NICE system, and purified through Nickel-affinity chromatography column. And the DNA-binding specificity of Ldb0677to target genes LBU₁764and Ldb0486were further assessed by electrophoretic mobility shift assay （EMSA）, respectively. These results indicated that Ldb0677was a novel transcription factor and might function as a major regulator in acid stress response in L. bulgaricus.（5） Identification of the transcription factor which regulates the acid stress-related gene pyk. Sixty five transcription factor genes were amplified by PCR from the genomic DNA of L. bulgaricus CAUH1and inserted into pB1H2w2to generate the L. bulgaricus transcription factor library. Western blot results confirmed that each TF had expressed as a carboxy-terminal fusion to the omega-subunit. The "Bait" vectors with no self-activation were constructed by deletion analysis method. The transcription factor which regulated the expression of pyk gene was identified to be the catabolite control protein A （CcpA） using the transcription factor library complemented by bacterial one-hybrid assay. Moreover, the binding site of CcpA was predicted to be upstream the-35region, and the sequence was TGTAAGCCCTAACA.