Development of gfp vectors for expression in Listeria monocytogenes and other low G+C gram positive bacteria

The gfp (green fluorescent protein) gene has previously been used to constr uct a variety of reporter plasmids for Cram-positive bacteria for bacterial localization and gene expression studies. When a native red-shifted gfp va riant (gfp3) was cloned into an expression vector using the P-xyn promoter and used to transform the soil-borne pathogen Listeria monocytogenes, only a small proportion of the population was seen to fluoresce when examined by epifluorescence microscopy. When the P-xyn promoter was replaced with the P-xyn promoter, with accompanying modification of the translation initiatio n region of the gfp3 gene, a homogeneously fluorescent population of cells was obtained. When expressed in other Gram-positive organisms, such as Stap hylococcus aureus and Bacillus subtilis, the translationally enhanced gene also resulted in high-level and homogeneous GFP production within the bacte rial population. High-level expression of these reporter constructs in L. m onocytogenes was evaluated to determine if it had any detrimental biologica l effect during intracellular infection of eukaryotic cell lines. The gfp(3 +)Listeria were found to invade equally as well as the wild-type cells; sho wing that these expression systems can be used to monitor the bacterium in natural environments. Based on these results, similar translationally enhan ced vectors were also developed using unstable GFP3 variants, which retain their short-half life characteristics in L. monocytogenes and therefore can be used as a sensitive monitor of gene expression.