Constitutive and inducible green fluorescent protein expression in Bartonella henselae

Abstract

The green fluorescent protein (GFP) gene was expressed on a plasmid in B. henselae, and GFP-expressing bacteria were visualized by fluorescence microscopy. HEp-2 cells infected with GFP-expressing bacteria were separated from uninfected cells with a fluorescence activated cell sorter. Promoter fusions of B. henselae chromosomal DNA to gfp were examined by flow cytometry, and a B. henselae groEL promoter fusion which induced expression at 37 degreesC was isolated.

FIG. 1

(a) B. henselae strain 882-ANT4 expresses GFP. Bacteria were visualized with an epifluorescence microscope with a fluorescein isothiocyanate (FITC) filter. (b) JK13-ANT5 has about a 475-fold-higher mean fluorescence intensity (x) than non-GFP-expressing bacteria. Fluorescence levels were examined with a FACScalibur scanner (Becton Dickinson). Cell Quest software (Becton Dickinson) was used for the analysis and quantitation of fluorescence. (c) Intracellular bacteria can be distinguished from extracellular bacteria by antibody labelling. All bacteria express GFP. However, only extracellular bacteria are labelled red. Colocalization of GFP and TRITC markers renders extracellular bacteria yellow, while intracellular bacteria remain green. Cells were visualized with an epifluorescence microscope with FITC and TRITC filters to visualize green and red fluorescence, respectively. Images were superimposed with Adobe Photoshop 3.0.5 software. (d) HEp-2 cells infected with JK13-ANT5 can be distinguished from uninfected cells with the FACStar cytometer (Becton Dickinson). Cells within the gated region have cell-associated, GFP-expressing B. henselae and were separated from uninfected cells.

FIG. 2

Native B. henselae promoters drove GFP expression at varying levels of fluorescence intensity. x, mean fluorescence intensity for the given clonal population of bacteria, Bhcon-1, Bhcon-2, or Bhcon-3.

FIG. 3

GFP could be expressed differentially when the B. henselae groEL promoter was fused to gfpmut3. At 26°C, little GFP was expressed, but when shifted to 37°C, the groEL gene was turned on, and GFP expression was induced fivefold. x, mean fluorescence intensity.