Transformation of Escherichia coli K-12 with a high-copy plasmid encoding the green fluorescent protein reduces growth: implications for predictive microbiology.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has been widely used as a biomarker and has potential for use in developing predictive models for growth of pathogens on naturally contaminated food. However, constitutive production of GFP can reduce growth of transformed strains. Consequently, a high-copy plasmid with gfp under the control of a tetracycline-inducible promoter (pTGP) was constructed. The plasmid was first introduced into a tetracycline-resistant strain of Escherichia coli K-12 to propagate it for subsequent transformation of tetracycline-resistant strains of Salmonella. In contrast to transformed E. coli K-12, which only fluoresced in response to tetracycline, transformed Salmonella fluoresced maximally without tetracycline induction of gfp. Although pTGP did not function as intended in Salmonella, growth of parent and GFP E. coli K-12 was compared to test the hypothesis that induction of GFP production reduced growth. Although GFP production was not induced during growth on sterile chicken in the absence of tetracycline, maximum specific growth rate (mumax) of GFP E. coli K-12 was reduced 40 to 50% (P < 0.05) at 10, 25, and 40 degrees C compared with the parent strain. When growth of parent and GFP strains of E. coli K-12 was compared in sterile broth at 40 degrees C, mumax and maximum population density of the GFP strain were reduced (P < 0.05) to the same extent (50 to 60%) in the absence and presence of tetracycline. These results indicated that transformation reduced growth of E. coli K-12 independent of gfp induction. Thus, use of a low-copy plasmid or insertion of gfp into the chromosome may be required to construct valid strains for development of predictive models for growth of pathogens on naturally contaminated food.