High-level fluorescence labeling of gram-positive pathogens

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Fluorescence labeling is crucial for observing bacterial pathogens within host cells.
• Existing methods may have limitations in broad applicability across different bacterial genera.

Purpose of the Study:

• To construct and validate a novel, versatile EGFP reporter plasmid for high-efficiency fluorescence labeling of Gram-positive pathogens.
• To assess the broad applicability of the developed plasmid across multiple bacterial species.

Main Methods:

• Construction of a novel shuttle vector, pBSU101, based on the E. coli streptococcal shuttle plasmid pAT28.
• Incorporation of a promoterless enhanced green fluorescent protein (EGFP) gene under the control of the Streptococcus agalactiae CAMP-factor gene (cfb) promoter.
• Transformation of various Gram-positive bacterial species and assessment of EGFP expression via fluorescence microscopy and FACS analysis.

Main Results:

• The pBSU101 plasmid successfully facilitated distinct and detectable fluorescence in transformed bacteria.
• FACS analysis showed 10-50 fold increased fluorescence compared to controls.
• Successful labeling was achieved across multiple species including Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, and Staphylococcus aureus.
• High-level EGFP expression was observed, which could be further enhanced under high oxygen conditions.

Conclusions:

• The pBSU101 plasmid is a highly effective tool for fluorescence labeling of a wide range of Gram-positive bacterial pathogens.
• This novel reporter system offers significant improvements in bacterial detection and observation for research applications.
• The ability to enhance expression under specific conditions provides flexibility for experimental design.