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Quantitative detection of Streptococcus pneumoniae cells harbouring single or multiple copies of the gene encoding
Paloma Acebo1, Concha Nieto1, Marı A Angeles Corrales1
1Centro de Investigaciones Biológicas, CSIC, Velázquez, 144, E-28006 Madrid, Spain1.
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.
Insights
Researchers developed a novel method to track Streptococcus pneumoniae using a modified green fluorescent protein (GFP). This system allows for inducible or constitutive expression, enabling visualization and quantification of bacterial cells.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Green fluorescent protein (GFP) is a widely used reporter protein.
- Streptococcus pneumoniae is a significant human pathogen.
- Gene expression in S. pneumoniae can be regulated by specific promoters.
Purpose of the Study:
- To develop a system for visualizing and quantifying Streptococcus pneumoniae.
- To engineer a modified green fluorescent protein (GFP) expression system in S. pneumoniae.
- To control GFP expression using an inducible promoter.
Main Methods:
- Subcloning a modified gfp gene into a mobilizable plasmid.
- Placing gfp under the control of the inducible P(M) promoter from Streptococcus pneumoniae.
- Integrating the P(M)-gfp construct into the host chromosome via homologous recombination.
- Culturing cells in media with different carbon sources (sucrose or maltose) to regulate promoter activity.
- Utilizing fluorescence spectroscopy and microscopy for quantification and visualization.
Main Results:
- Successful expression of GFP in Streptococcus pneumoniae, either inducibly or constitutively.
- Expression levels of GFP were quantifiable using fluorescence spectroscopy.
- The P(M) promoter's activity was modulated by the malR gene and carbon source availability.
- Fluorescence microscopy enabled differentiation of GFP-expressing cells in mixed cultures.
Conclusions:
- A versatile GFP-based reporter system was established for Streptococcus pneumoniae.
- This system allows for monitoring bacterial presence and expression levels.
- The developed system has potential applications in studying pneumococcal pathogenesis and population dynamics.