Development of an electroporation procedure for gene disruption in Lactobacillus helveticus CNRZ-32

An electroporation-mediated transformation method was developed and optimized for Lactobacillus helveticus CNRZ 32. The effects of electroporation buffers, growth conditions, cell-wall-weakening agents and field strength on transformation frequency were examined. Optimal conditions yielded a frequency of 2 x lo4 transformants (pgpGK12)-'. Using this procedure, an integration plasmid was introduced into L. helveticus CNRZ 32 to inactivate the chromosomal X-prolyl dipeptidyl aminopeptidase gene (pepXP). The integration plasmid, designated pSUW200, consisted of pUC19, the pE194 erythromycin resistance gene and a 1.6 kb internal fragment of the pepXP gene. The erythromycin resistance transformants obtained were X-prolyl dipeptidyl aminopeptidase (X-PDAP) negative. Southern hybridization results indicated that pSUW200 had integrated into the L. helveticus CNRZ 32 chromosome via Campbell-type integration. After growth of the pSUW200-derived transformants for 112 generations under non-selective conditions, erythromycin-sensitive X-PDAP+ isolates were obtained. Southern hybridization results indicated that precise excision of pSUW200 had occurred via recombination between the 1-6 kb pepXP-derived nontandem repeats