Genetic engineering techniques for lactic acid bacteria: construction of a stable shuttle vector and expression vector for β-glucuronidase

The shuttle vector, pUL6erm, was constructed by using a replicon from pL2, a multiple cloning site, colE1 ori, the ori of Gram-negative bacteria from vector pUC19, and the erythromycin resistance gene from pVA838 as a selection marker. pUL6erm could be transformed easily and maintained stably in Lactococcus lactis, Streptococcus thermophilus, Lactobacillus plantarum and Lactobacillus casei. Transformation assays of pUL6erm indicated that it had a narrow host range. beta-Glucuronidase was induced in the presence of 0.3 M NaCl and 50 mM glutamate and expressed at 2.4 U mg(-1) with the expression vector (pUL6erm-gadR-GUS) constructed based on pUL6erm carrying beta-glucuronidase gene wuth a chloride-inducible (gadR) expression cassette using Pgad as promoter. Therefore, pUL6erm and pUL6erm-gadR-GUS might be a safe and useful genetic tool for the improvement of lactic acid bacteria.