title: Functional role of pyruvate kinase from Lactobacillus bulgaricus in acid tolerance and identification of its transcription factor by bacterial one-hybrid.
abstract: Lactobacillus delbrueckii subsp. bulgaricus develops acid tolerance response when subjected to acid stress conditions, such as the induction of enzymes associated with carbohydrate metabolism. In this study, pyk gene encoding pyruvate kinase was over-expressed in heterologous host Lactococcus lactis NZ9000, and SDS-PAGE analysis revealed the successful expression of this gene in NZ9000. The survival rate of Pyk-overproducing strain was 45-fold higher than the control under acid stress condition (pH 4.0). In order to determine the transcription factor (TF) which regulates the expression of pyk by bacterial one-hybrid, we constructed a TF library including 65 TFs of L. bulgaricus. Western blotting indicated that TFs in this library could be successfully expressed in host strains. Subsequently, the promoter of pfk-pyk operon in L. bulgaricus was identified by 5'-RACE PCR. The bait plasmid pH3U3-p01 carrying the deletion fragment of pfk-pyk promoter captured catabolite control protein A (CcpA) which could regulate the expression of pyk by binding to a putative catabolite-responsive element (5'-TGTAAGCCCTAACA-3') upstream the -35 region. Real-time qPCR analysis revealed the transcription of pyk was positively regulated by CcpA. This is the first report about identifying the TF of pyk in L. bulgaricus, which will provide new insight into the regulatory network.
authors.affiliation: Key Laboratory of Functional Dairy, Co-constructed by Ministry of Education and Beijing Municipality, College of Food Science &Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Zhengyuan
authors.initials: Z
authors.lastname: Zhai
authors.affiliation: Key Laboratory of Functional Dairy, Co-constructed by Ministry of Education and Beijing Municipality, College of Food Science &Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Haoran
authors.initials: H
authors.lastname: An
authors.affiliation: Key Laboratory of Functional Dairy, Co-constructed by Ministry of Education and Beijing Municipality, College of Food Science &Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Guohong
authors.initials: G
authors.lastname: Wang
authors.affiliation: Key Laboratory of Functional Dairy, Co-constructed by Ministry of Education and Beijing Municipality, College of Food Science &Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Yunbo
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Key Laboratory of Functional Dairy, Co-constructed by Ministry of Education and Beijing Municipality, College of Food Science &Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Hao
mesh_terms: D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial; D019426:Integrases / Q000378:metabolism; D019344:Lactic Acid / Q000494:pharmacology*; D007778:Lactobacillus / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D016133:Polymerase Chain Reaction; D011770:Pyruvate Kinase / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D024363:Transcription Initiation Site; D020798:Two-Hybrid System Techniques
chemical_list: D014157:Transcription Factors; D019344:Lactic Acid; D011770:Pyruvate Kinase; C045073:Cre recombinase; D019426:Integrases