Introduction of Silent Mutations in a Proteinase Gene of Lactococcus lactis as a Useful Marker for Monitoring Studies

A 1.3 kb BamHI-EcoRI fragment of the cloned proteinase PrtP gene (pNZ511) of Lactococcus lactis subsp. cremoris SK11 was subcloned in the vector pBluescript and Escherichia coli. The nucleotides at the third positions of three adjacent codons were changed by in vitro mutagenesis. The predicted amino acid sequence was not altered and the average codon usage maintained. The 1.3 kb BamHI-EcoRI fragment of pNZ511 was replaced by the modified fragment. Lactococcus lactis subps. lactis was electrotransformed with the modified plasmid pLMP1. Specific hybridization probes were designed for the modified and the corresponding unmodified gene regions. These probes in combination with species specific hybridization probes were used to analyze presence and distribution of the gene variants in pure and mixed cultures. This is the first example of introducing silent mutations in a gene from an industrially used microorganism with the purpose of following the fate of the genetically modified microorganism.