title: High-level fluorescence labeling of gram-positive pathogens.
abstract: Fluorescence labeling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbors a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene (cfb) promoter of Streptococcus agalactiae and was designated pBSU101. Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10-50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labeling in different gram-positive pathogens, numerous species were transformed. We successfully labeled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high-level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration.
authors: Institute of Medical Microbiology and Hygiene, University of Ulm, Ulm, Germany.
authors: Simone
authors: S
authors: Aymanns
authors: Stefanie
authors: S
authors: Mauerer
authors: Ger
authors: G
authors: van Zandbergen
authors: Christiane
authors: C
authors: Wolz
authors: Barbara
authors: B
authors: Spellerberg
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D005057:Eukaryotic Cells / Q000382:microbiology; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006094:Gram-Positive Bacteria / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D013291:Streptococcus / Q000166:cytology / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins