Development of an efficient gene transfer system for Lactobacillus helveticus WSU-19
Principal Investigators: Gulhan U. Yuksel, University of Idaho
Co-Investigators: Joe R. Powers, Washington State University
Develop/optimize an electrotransformation prcedure for Lb. Helveticus WSU-19; It has been reported that lactobacilli can be transfromed with vectors by electrtransformation of Lb. acidophilus, Lb. bavaricus, Lb. casei, Lb. curvatus, Lb. fermenum, Lb. halveticus, Lb. reuteri, Lb. sake and others. Although electrotransformation has been successfully applied in many Lactobacillus strains, transformation efficiencies and optimal electrotransformation parameters vary greatly from strain to strain, especially in the case of Lb. helveticus. In the proposed study, the electrotransformation of Lb helveticus WSU-19 will be carried out using selected procedures established for other Lactobacillus strains. The efficiency of transformation will be determined in each case. The vectors of interest will include pSA3 and pGK12. PSA3 is a temperature-sensitive integration vector that we plan to use for the construction of isogenic Lb. helveticus WSU-19 strains that are deficient in selected peptidase enzymatic acitvities. PGK12 is a vector that is commonly used to develop/optimize eletrotransformtation procedures for Lactobacillus. The most efficient electrotransformation procedure will be subject to an optimization process in which the effects of several parameters on transformation frequency will be studied. These parameters will include electroportation buffers (type, pH, ionic strencth), growth media (MRS, APT), cell-wall weakening concentration, and field strength (4000-12,500 V/cm). Our goal is to achieve an electrotransformation efficiency of 10 to the 4th- 10 to the 5th / _g pGK12.
Confirm the effectiveness of the electrotransformation procedure by construction a peptidase-deficient mutant of Lb. helveticus WSU-19: To confirm the effectiveness of the newly established procedure, a pSA3-based integration plasmid that contains a deleted version of the Aminopeptidase N gene (pepN) will be constructed and introduced into Lb. helveticus WSU-19. An efficient method for gene replacement using the temperature-sensitve vector pSA3 will be applied to replace the wild-type chromosomal pepN gene with the deleted version of the gene. The PepN-deficient derivative of Lb. helveticus WSU-19 will be confirmed genotypically and phenotypically. To accomplish our long-range goal, future research will deal with Cheddar-type cheese trials using the PepN-deficient strain and other peptidase-deficient strains as adjuncts. A Lactococcus lactis strain that is known to cause bitter Cheddar cheese will be used as the starter culture in these trials.