Use of green fluorescent protein for detection of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis.
Wild-type and mutant forms of the gene encoding green fluorescent protein (GFP) from Aequorea victoria have been introduced into Bacillus subtilis as translational fusions to the prespore-specific and mother-cell-specific genes dacF and spoIVA. In both cases, the protein was readily detected by fluorescence microscopy, and its synthesis was correctly localized. The S65T substitution, which improves the quantum yield and rate of development of fluorescence, also produced a spectral shift that allowed the protein to be colocalized with DNA, after staining with 4',6-diamidino-2-phenylindole. Three different translational fusions to the N-terminal region of GFP all produced active protein. Moreover, a full-length SpoIVA-GFP fusion showed proper targeting to the surface of the spore, albeit at low temperature and in the presence of wild-type SpoIVA protein. A mutation in the gfp gene which changes the light emitted by the protein from green to blue was found not to be useful because of the intrinsic autofluorescence of B. subtilis in the blue part of the spectrum.
Lewis
Peter J
PJ
Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK.
Errington
Jeffery
J
Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK.
D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D037401:Scyphozoa / Q000235:genetics; D012808:Sigma Factor; D013171:Spores, Bacterial / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014157:Transcription Factors
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D005819:Genetic Markers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C092468:spoIIR protein, Bacillus subtilis; C030069:spore-specific proteins, Bacillus; D049452:Green Fluorescent Proteins