Construction of a new reporter system to study the NaCl-dependentdnaKpromoter activity ofLactobacillus sanfranciscensis

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Abstract

A reporter system was developed to study gene expression in Lactobacillus sanfranciscensis. It was based on the Escherichia coli/Lactobacillus shuttle vector pLP3537 and the melA gene encoding α-galactosidase originating from Lactobacillus plantarum. melA was functionally expressed in E. coli and L. sanfranciscensis, and activity was easily monitored in vivo as well as in vitro by applying an optimized enzyme assay. The reporter system was validated by demonstrating the induction of the dnaK operon of L. sanfranciscensis by NaCl stress. The complete operon, which was composed of hrcA, grpE, dnaK, and dnaJ, was sequenced. A 299-bp sequence upstream of this operon, including a putative sigmaA-type promoter and a single conserved Controlling Inverted Repeat of Chaperone Expression element, was amplified. This amplicon was cloned directly upstream of melA. Both reporter enzyme activity and Northern hybridization analyses of dnaK and melA revealed a transcriptional induction, reaching its maximum when the culture was exposed to 0.75 M NaCl.

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