Plasmid transformation by electroporation of Leuconostoc paramesenteroides and its use in molecular cloning.
In this report, we demonstrate the utility of electroporation as an efficient method for genetic transformation of Leuconostoc paramesenteroides. We optimized several factors which determine the transformation frequency, resulting in transformation efficiencies of up to 4 x 10(3) transformants per micrograms of pNZ12 DNA, which contains the promiscuous Lactococcus lactis pSH71 replicon. Slightly lower efficiencies were obtained with a deletion derivative of the broad-host-range plasmid pAM beta 1. These plasmids could be stably maintained in L. paramesenteroides NZ6009 for more than 100 generations, even in the absence of selective pressure. In order to show the use of the developed host-vector system, we cloned the Lactococcus lactis gene encoding phospho-beta-galactosidase in L. paramesenteroides. Expression of this heterologous gene in L. paramesenteroides under control of Lactococcus lactis expression signals was evident from the presence, in transformants, of phospho-beta-galactosidase activity and a specific phospho-beta-galactosidase protein band on Western blots (immunoblots). In addition, we transformed a lactose-deficient derivative of L. paramesenteroides with a plasmid carrying a Lactococcus lactis-Escherichia coli lacZ gene fusion. The resulting transformants synthesized high levels of beta-galactosidase, indicating the efficiency of heterologous gene expression signals in L. paramesenteroides.
David
S
S
Department of Biophysical Chemistry, Netherlands Institute for Dairy Research (NIZO), BA EDE.
Simons
G
G
De Vos
W M
WM
D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007778:Lactobacillus / Q000235:genetics; D007934:Leuconostoc / Q000235:genetics*; D010957:Plasmids; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D004269:DNA, Bacterial; D001616:beta-Galactosidase