CaFbFP fluorescence in yeast transformants. (A) Fluorescence emission spectra of crude extracts of S. cerevisiae transformants (0.5 mg protein/ml) carrying plasmid pIE3 (CaFbFP) or control vector p426-Gal1. Fluorescence was measured with a Perkin Elmer model LS50B luminescence spectrometer (excitation wavelength, 450 nm); fluorescence intensity is expressed in arbitrary units (A.U.). (B) Fluorescence of yeast transformants synthesizing GFP or CaFbFP following normoxic or anoxic growth. S. cerevisiae strain YKMI expressing a fusion of GFP to the GAL1 promoter or strain THY.AP4 carrying a GAL1p fusion to CaFbFP (pIE3) was grown on SGR agar (2% galactose-1% raffinose) at 30°C for 48 h under normoxia or for 72 h in a jar generating anoxic conditions (Oxoid AnaeroGen AN0025A). Grown colonies and streaks of transformants were photographed directly or examined for fluorescence by using a Zeiss Axioplan 2 microscope (filter set, Zeiss FS38; excitation BP, 470/40; emission, 525/50; magnification, 100-fold). (C) Fluorescence of CaFbFP in single cells of S. cerevisiae transformants carrying pIE3. Cells were examined by phase microscopy or by fluorescence microscopy using a Zeiss Axioplan 2 microscope equipped with an HBO100 lamp (filter set, Zeiss FS38; excitation BP, 470/40; emission, 525/50; magnification, 1,000-fold). Transformants carrying empty vector p426-Gal1 were used as controls.

Expression of FMN-binding fluorescent protein (CaFbFP) in fungi. (A) The codon usage of EcFbFP (1) was adapted to C. albicans, and the resulting CaFbFP gene was placed under the transcriptional control of the GAL1 promoter for S. cerevisiae expression or under the control of the ACT1 or THD3 promoter for C. albicans expression. A tandem CaFbFP fusion was constructed using the indicated asymmetrical AvaI sites. (B) Detection of CaFbFP in transformants of C. albicans strain CAI4 carrying plasmids pIE1.1, pIE1.2, pIE5.1, and pIE5.2 and in S. cerevisiae strain THY.AP4 containing plasmid pIE3. Crude extracts (50 μg protein) were analyzed by immunoblotting using anti-EcFbFP antiserum; extracts of transformants carrying empty vectors pDS1044 and p426 were tested as controls (c). The migration of standard proteins was as indicated (kDa). (C) Detection of CaFbFP in crude extracts (500 μg protein) of S. cerevisiae strain THY.AP4 containing plasmid pIE3 by Coomassie staining of a two-dimensional polyacrylamide gel electrophoresis gel (left) and subsequent immunoblotting using anti-EcFbFP antiserum (right). Arrows indicate CaFbFP proteins.

(A) Fluorescence of C. albicans CAI4 transformants carrying plasmid pIE1.2 or pIE5.2 synthesizing tandem CaFbFP following growth on SD agar (2% glucose) at 30°C for 24 h under normoxia or for 72 h in a jar generating anoxic conditions (Oxoid AnaeroGen AN0025A). Grown colonies were photographed directly or examined for fluorescence by using a Zeiss Axioplan 2 microscope (filter set, Zeiss FS38; excitation BP, 470/40; emission, 525/50; magnification, 100-fold). (B) Fluorescence of tandem CaFbFP in single cells of anoxically grown C. albicans CAI4 transformants carrying pIE5.2.