Dataset Information

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.

SUBMITTER: Zhai Z

PROVIDER: S-EPMC4652205 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Functional role of pyruvate kinase from Lactobacillus bulgaricus in acid tolerance and identification of its transcription factor by bacterial one-hybrid.

Zhai Zhengyuan Z An Haoran H Wang Guohong G Luo Yunbo Y Hao Yanling Y

Scientific reports 20151119

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 ...[more]

PMID: 26581248

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Project description:We examine the use of high-throughput sequencing on binding sites recovered using a bacterial one-hybrid (B1H) system and find that improved models of transcription factor (TF) binding specificity can be obtained compared to standard methods of sequencing a small subset of the selected clones. We can obtain even more accurate binding models using a modified version of B1H selection method with constrained variation (CV-B1H). However, achieving these improved models using CV-B1H data required the development of a new method of analysis - GRaMS (Growth Rate Modeling of Specificity) - that estimates bacterial growth rates as a function of the quality of the recognition sequence. We benchmark these different methods of motif discovery using Zif268, a well characterized C2H2 zinc finger transcription factor on both a 28bp randomized library for the standard B1H method and on 6bp randomized library for the CV-B1H method for which forty-five different experimental conditions were tested: 5 time points and three different IPTG and 3-AT concentrations. We find that GRaMS analysis is robust to the different experimental parameters whereas other analysis methods give widely varying results depending on the conditions of the experiment. Finally, we demonstrate that the CV-B1H assay can be performed in liquid media, which produces recognition models that are similar in quality to sequences recovered from selection on solid media. All selections were performed using the bait transcription factor Zif268. 45 different B1H selections were performed on plates and the selected sites were multiplexed and sequenced in a single lane. A single selection was performed in liquid media and sequenced along with other multiplexed sets of sites in a separate lane. For the experiments performed on plates, the of the selections assays was varied (4, 8, 12, 18, 24 hours). The stringency of the selections was also varied by changing the concentration of IPTG (0, 10, 50 uM) and 3-AT (0.5, 1, 2 mM). The initial randomized library used for all selections was also sequenced in a single lane. The randomized portion of each transcription factor binding site is 6bp long and is immediately 5' of the constant sequence, GCGG, which is the consensus binding site for finger 1 of Zif268. Upstream of the randomized binding site is a 4bp randomized region referred to as the key region. The purpose of the key region is to detect possible PCR 'jackpotting.' The single liquid media experiment was run for 4 hours using 50uM IPTG and 5mM 3-AT. Four selections were performed using a prey library with a 28bp long randomized region and not fixed consensus site region. These selections were performed at either 36 or 48 hours using 10uM IPTG and 0, 2, or 5 mM 3-AT.

Dataset Information

Functional role of pyruvate kinase from Lactobacillus bulgaricus in acid tolerance and identification of its transcription factor by bacterial one-hybrid.

Publications

Functional role of pyruvate kinase from Lactobacillus bulgaricus in acid tolerance and identification of its transcription factor by bacterial one-hybrid.

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