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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.

scientific reports

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