Construction of a new reporter system to study the NaCl-dependent dnaK promoter activity of Lactobacillus sanfranciscensis

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 alpha-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.

Electrotransformation and Expression of Cellulase Genes in Wild-Type <b><i>Lactobacillus reuteri</i></b>

“…The fragment was then digested with Sma I and Bam HI and ligated into pLEM415 [Serror et al, 2002], which was digested with the same restriction enzymes. To build the pLEM4152 plasmid, the dna K promoter was digested from plasmid pSH85351 [Hormann et al, 2006] and ligated into the pLEM4151, which was digested with Spe I and Xba I. To build plasmids pLEM4153 and pLEM4154, the two cellulase genes were cloned from B. subtilis and ligated into the pLEM4152, which was digested with Sac II and Sac I, respectively ( fig.…”