Quantitative detection of Streptococcus pneumoniae cells harbouring single or multiple copies of the gene encoding the green fluorescent protein

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Abstract
A modified gfp gene from Aequorea victoria, encoding a variant of the green fluorescent protein (GFP), was subcloned into the mobilizable plasmid pMV158. gfp was placed under the control of the inducible P M promoter of the Streptococcus pneumoniae gene malM, cloned in plasmid pLS70. The P M promoter is regulated by the product of the pneumococcal malR gene, which is inactivated by growing the cells in maltose-containing media. By homologous recombination, the P M–gfp construction was integrated into the host chromosome in a single copy. In both conditions (single and multiple copies), the pneumococcal cells were able to express GFP in an inducible or constitutive form, depending on whether the S. pneumoniae strain harboured a wild-type or a mutant malR gene. Quantification of the levels of GFP expressed by cultures supplemented with sucrose or maltose as carbon sources was feasible by fluorescence spectroscopy. Phase-contrast and fluorescence microscopy allowed pneumococcal cells expressing GFP in mixed cultures to be distinguished from those not carrying the gfp gene.