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.

Cis and trans elements regulating expression of the varicella zoster virus gI gene.

We have identified cis- and trans-acting elements involved in the VZV IE62 protein-activated expression of the varicella zoster virus (VZV) gene which encodes the viral gI glycoprotein. The cis-acting elements include a non-canonical TATA box and a novel 19 base pair sequence located just upstream of the TATA element designated the "activating upstream sequence" or AUS. The AUS is a movable element and its presence results in IE62 activation of a chimeric promoter consisting of the VZV gC TATA box and the gI AUS. We have also determined that the VZV ORF 29 protein modulates the regulatory activity of the IE62 protein at the gI promoter. In combination with the IE62 transactivator, it yields a 10 to 15-fold increase in expression over the levels seen with the IE62 protein alone in T lymphocytes. The upmodulatory activity requires the presence of a 40 base pair sequence, designated the 29RE, which maps between positions -220 and -180 in the gI promoter. In this paper we review these and earlier findings from our laboratories concerning the regulation of the gI promoter.

The varicella-zoster virus (VZV) open-reading frame 29 protein acts as a modulator of a late VZV gene promoter.

Transient expression assays have shown that the varicella-zoster virus (VZV) open-reading frame (ORF) 29 gene product can act as a modulator of VZV gene expression. The ORF 29 protein alone does not appear to have any effect on transcription; however, in its presence, changes in the level of reporter gene activity mediated by the VZV immediate early (IE) 62 major transactivator are seen. Increased expression was observed in human fibroblasts, MeWo cells, HeLa cells, and T cells. In contrast, the presence of the ORF 29 protein results in a down-regulation of IE62 activation in PC-12 rat neuronal cells. Competition filter binding assays indicate that the ORF 29 protein binds specifically to the glycoprotein I promoter. Since transcripts for ORF 29 and ORF 62 have been detected in latently infected ganglia, the gene regulatory properties of the ORF 29 protein may be relevant to maintenance or establishment of VZV latency.