Escherichia coil K-12 with a High-Copy Plasmid Encoding the Green Fluorescent Protein Reduces Growth: Implications for Predictive Microbiologyt

The green fluorescent protein (GFP) of the jellyfish Ac'qiiorea victoria has been widely used as a hiomarker 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 gf,i under the control of a tetracycline-inducible promoter (pTGP) was constructed. The plasniid was first introduced into a tetracyclineresistant strain of E.rclierichia coli K-12 to propagate it for subsequent transformation of tetracycline-resistant strains of Salmonella. In contrast to transformed E. co/i K-12, which only fluoresced in response to tetracycline. transformed Salmonella fluoresced maximally without tetracycline induction of gfi. Although pTGP did not function as intended in Salmonella, growth of parent and GFP E. coil K-12 was compared to test the hypothesis that induction of (IFP production reduced growth. Although GFP production was not induced during growth on sterile chicken in the absence of tetracycline, maximum specific growth rate (p) of GFP E. co/i K-12 was reduced 40 to 509t (P K t).05) at 10, 25. and 40°C compared with the parent strain. When growth of parent and GFP strains of E. coil K-12 was compared in sterile broth at 40°C. and maximum population density of the GFP strain were reduced (P K 0.05) to the same extent (50 to 60i/r) in the absence and presence of tetracycline. These results indicated that transformation reduced growth of E. co/i K-12 independent of gfp induction. Thus. use of a low-copy plasmid or insertion of gf into the chromosome may he required to construct valid strains for development of predictive models for growth of pathogens on naturall y contaminated food. The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has been widely used as a hiomarker in eukaryotic and prokar yotic cells (8, 20. 21, 31, 36). In prokaryotes, GFP has been used to follow growth, death, and dissemination of bacteria in water (9. 24). food (2, 7, 11, 15, 16), and environmental systems (13. 19, 34, 38). Many reports indicate that GFP production does not alter the biochemical, morphological, or behavioral characteristics of the transformed microbial cells (4. 12, 14. 15, 19, 28, 37). Transformation methods include use of high-copy plasmids (1, 15) or low-copy plasmids (17, 37) and insertion of gfr into the chromosome (16, 18, 22, 35). Production of GFP has been constitutive (14. 25, 26) or inducible (27, 29, 30), depending on the application of the marker organisms. A few reports have recently appeared indicating that GFP reduces growth of transformed bacteria. Rang et al. (29) found that doubling time of enteric bacteria increased in proportion to the amount of GFP produced, which was altered with an inducible promoter. Oscar (26) reported that constitutive expression of ,fp reduced growth of Sn/mone/la and that the magnitude of the effect depended on the strain and incubation temperature. * Author for correspondence. Tel: 410-651-6062; Fax: 410-651-8498: E-mail: toscar@ umes.edu . Mention of trade names or commercial products in this publication is solely for providing specific information and does not imply recoinmendation or endorsement by the U.S. Department of Agriculture. In a recent exposure assessment for food pathogens, the lack of models that predict behavior of patho gens in naturally contaminated food was noted (10). Pathogens transformed with gf, have potential as marker organisms for developing predictive models in food (26, 37). However, before a GFP-transfornied pathogen can he used to develop a model in food with competitive microflora. the behavior of the GFP pathogen must he confirmed as the same as that of its parent strain (26, 37). In predictive microbiology applications, production of GFP is not required during growth of a pathogen on food. Rather, GFP production is only required during pathogen enumeration. In the current study, a tetracycline-resistant strain of Escheric/,ia co/i K-12 and four tetracycliric-resistant strains of Salmonella were transformed with a highcopy plasmid encoding under the control of a tetracycline-inducible promoter (pTGP). This promoter was selected because tetracycline is not normally found in food. E. co/i K-12 was selected to create a strain for propagation of pTGP for transformation of Salmonella. The objective was to determine whether induction of GFP production altered microbial growth by comparing growth of parent and GFP strains of E. coli K-12. Growth of parent and GFP Salmonella was not compared because transformed cells were maximally fluorescent in the absence of tetracycline. Thus, effects of induction of GFP production on growth of Salmonella could not he directly assessed. J. lOOd Prot.. Vol. 69, No. 2 REDUCED GROWTH IN TRANSFORMED L. COLI K12 277 MATERIALS AND METHODS Plasmid construction. Two different high-copy plasmids. pEGFP (3.4 kb) and pPROtet.E (2.2 kb) (Clontech Company. Palo Alto. Calif.), were used to construct pTGP (2.9 kb). pPROiei.E has a chloramphenicol-resistant gene and a tetracycline-inducible promoteL and pEGFP has the gene for enhanced GFP. Plasmids were digested overnight at 37°C with the restriction enzymes Non and Ba,ulll (New England Biolabs. Beverly. Mass.) in a buffer composed of 150 mM NaCl. 10 uM Tris-HCI. 10 mM MgCl2. I mM dithioth re i tot, and 100 Vg/ml bovine serum albumin (all from Sigma Chemical Co.. St. Louis, MO.). Digested plasmids were separated in a 1% low-melting-point agarose gel, and hands with desired genes. pPROtet.E (2.2 kb) and gtp (0.7 kh), were excised from the gel and ligated using T4DNA ligase (Nev, England Biolabs) to generate pTGP Transformation. Competent cells of a tetracycline-resistant strain of E. co/i K12 (Carolina Biological Supply Company, Burlington. NC.) and tetracycline-resistant strains of Salmonella Hadar. Salmonella Kentucky. and Salmonella Tvphimurium (n -2) were transformed with pTGP (32). The ligation mixture containing pTGP was mixed with competent cells in a microcentrifugc tube and incubated on ice for 30 nun. The tube was then incubated at 37C for 2 inin in a water bath and chilled on ice for 5 miii. 951 p.1 of Luria-Bertani (1,B) broth (Becton Dickinson. Sparks. Md.) was added, and the translormant mixture was incubated at 37°C for 60 inin and then streaked onto LB agar that contained tetracycline (100 n o/ml: Sigma) and chloramphenicol (25 p.c/mI: Sigma). After overnight incubation at 37°C. the streak plate was examined for fluorescence with a hand-held 366-mm UV light. Stock cultures. A fluorescent colony from the streak plate of the transformant mixture was used to inoculate 5 ml of brain heart infusion (BHI) broth (Becton Dickinson) that contained 25 p.g/ml chloramphenicol in a 25-ml Erlenmeyer flask sealed with a foarn plug. The cultures were incubated at 30°C and ISO orbits per mm (opm) for 23 h. Aliquots (100 p.l) of the 23-hculture were transferred to freezer storage vials that contained 900 p.1 of BHI broth supplemented with 153 vol/vol) glycerol. Vials of the GFP strain and vials of the parent strain, which were prepared in a similar manner but with BHI broth as the growth medium. were stored at 70°C until they were used in the growth experiments. Sterile chicken. Ten-gram portions of ground chicken breast meat were formed into uniform patties that were cooked in an autoclave at 121'C for IS nun to kill all microorganisms and to inactivate residual tetracycline that could be present from treatment of live birds with the antibiotic. After cooling, a sterile 1.5cm-diameter cork borer was used to cut plugs from the center of the patties. The plugs were cut in half and trimmed with a sterile scalpel to yield I­ samples of sterile chicken, which were transferred to individual wells of a sterile 12-well tissue culture dish for subsequent inoculation. Inoculum culture. Thawed and resuspended stock culture (5 p.!) was added to 5 ml of BHI broth for the parent strain or 5 ml of BHI broth plus chloramphenicol (25 p.g/mh for the GFP strain in a 25-nil Erlenmever flask sealed with a foam plug. Inoculum cultures for the Pffeiit and GFP strains were incubated in air at 30°C and 150 opm for 23 h before serial dilution in buffered peptone water (BPW: Becton Dickinson). Growth experiments. Diluted inoculum cultume (5 p.l) was pipetted onto the surface of sterile chicken portions for an initial density of 10 15 CFtJ/g. Inoculated chicken samples were incubated in air at 10. 25. or 40°C. At selected incubation times, a 1g portion of sterile chicken was homogenized (model 80 stomacher blender. Seward. London. UK) for I min at normal speed in 9 ml of BPW in it Whirl-Pak hag (9.4 by 17.5 cm) containing a filter screen (Nasco, Fort Atkinson, Wis.). Samples of stomachate (50 p.1). either tmndiluted or serially diluted in BPW. were spiral plated (Whitle y Automatic Spiral Flatci. Microbiology International, Frederick. Md.) onto BHI agar (Becton Dickinson) for the parent strain and onto BI-Il agar plus chloramphenicol (25 p.g/ml ) and tetracycline (100 ng/ml) for the (HP strain. Tetracycline was included in the BHI agar for the OFF strain to induce production of GFP and to simulate how the OFF strain would he used in a predictive microbiology experiment with naturally contaminated food. Spiral plates were incubated oi air at 30°C for 24 Ii. and then colonies were counted with an automated counter (ProtoCol. Microbiology International). Three replicate trials of the growth experiment were conducted per temperattire. each with a different batch of chicken portions. Growth modeling. Colony counts (lo(, CFU per gram) were graphed as a function of time and fit to a three-phase linear model (6) using version 4.0 of Prism (GraphPad Software. Inc.. San Di-