Merck

High-level fluorescence labeling of gram-positive pathogens.

PloS one (2011-07-07)
Simone Aymanns, Stefanie Mauerer, Ger van Zandbergen, Christiane Wolz, Barbara Spellerberg
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.

MATERIALS
Product Number
Brand
Product Description

SAFC
RPMI-1640 Medium, HEPES Modification, with L-glutamine and 25mM HEPES, without sodium bicarbonate, powder, suitable for cell culture
Sigma-Aldrich
RPMI-1640 Medium, Modified, with sodium bicarbonate, without L-glutamine and phenol red, liquid, sterile-filtered, suitable for cell culture
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RPMI-1640 Medium, Modified, with L-glutamine, without phenol red and sodium bicarbonate, powder, suitable for cell culture
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RPMI-1640 Medium, With sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
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RPMI-1640 Medium, With L-glutamine, without glucose and sodium bicarbonate, powder, suitable for cell culture
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RPMI-1640 Medium, With L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
SAFC
RPMI-1640 Medium, Hybri-Max, Modified, with L-glutamine, 4500 mg/L glucose and 15mM HEPES, without sodium bicarbonate, powder, suitable for hybridoma
SAFC
RPMI-1640 Medium, 10 ×, Without L-glutamine, folic acid and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
SAFC
RPMI-1640 Medium, AutoMod, without L-glutamine and sodium bicarbonate, powder, suitable for cell culture
SAFC
RPMI-1640 Medium, HEPES Modification, With 25 mM HEPES, without L-glutamine., liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
RPMI-1640 Medium, Dutch Modification, with sodium bicarbonate and 20mM HEPES, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
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RPMI-1640 Medium, Modified, with 20 mM HEPES and L-glutamine, without sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
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RPMI-1640 Medium, Modified, with sodium bicarbonate, without methionine, cystine and L-glutamine, liquid, sterile-filtered, suitable for cell culture
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RPMI-1640 Medium, With L-glutamine, without sodium bicarbonate, powder, suitable for cell culture
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Phorbol 12-myristate 13-acetate, synthetic, ≥98.0% (TLC)
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RPMI-1640 Medium, With L-glutamine and sodium bicarbonate. Without arginine, leucine, lysine, and phenol red, liquid, sterile-filtered, suitable for cell culture, designed for isotope labeling for cell culture applications
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PMA, for use in molecular biology applications, ≥99% (HPLC)
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Phorbol 12-myristate 13-acetate, ≥99% (TLC), film or powder
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StableCell RPMI-1640, With stable glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture