Stable Expression of Modified Green Fluorescent Protein in Group B Streptococci To Enable Visualization in Experimental Systems

Group B Streptococcus (GBS) is a gram-positive bacterium associated with various diseases in humans and animals. Many studies have examined GBS physiology, virulence and microbe-host interactions using diverse imaging approaches, including fluorescence microscopy. Strategies to label and visualize GBS using fluorescence biomarkers have been limited to antibody-based methods or non-specific stains that bind DNA or protein; an effective plasmid-based system to label GBS with a fluorescence biomarker would represent a useful visualization tool. In this study, we developed and validated a green fluorescence protein (GFP)-variant-expressing plasmid, pGU2664 that can be applied as a marker to visualize GBS in experimental studies. The synthetic, constitutively active CP25 promoter drives strong and stable expression of GFPmut3 in GBS carrying pGU2664. GBS maintains GFPmut3 activity at different phases of growth. Application of fluorescence polarization enables easy discrimination of GBS GFPmut3 activity from the auto-fluorescence of culture media commonly used to grow GBS. Differential interference contrast microscopy, in combination with epifluorescence microscopy to detect GFPmut3 in GBS, enabled visualization of bacterial attachment to live human epithelial cells in real time. Plasmid pGU2664 was also used to visualize phenotypic differences in the adherence of wild-type GBS and an isogenic gene-deficient mutant strain lacking the control of global virulence regulator CovR in adhesion assays. The system for GFPmut3 expression in GBS described in this study provides a new tool for the visualization of this organism in diverse research applications. We discuss the advantages and consider the limitations of this fluorescent biomarker system developed for GBS. Importance Section Group B Streptococcus (GBS) is a bacterium associated with various diseases in humans and animals. This study describes the development of a strategy to label and visualize GBS using a fluorescence biomarker, termed green fluorescence protein (GFP)mut3. We show that this biomarker can be successfully applied to track the growth of the bacteria in liquid medium, and enables the detailed visualization of GBS in the context of live human cells in real time microscopic analysis. The system for GFPmut3 expression in GBS described in this study provides a new tool for the visualization of this organism in diverse research applications.