title: Use of immunofluorescence to visualize cell-specific gene expression during sporulation in Bacillus subtilis.
abstract: We have adapted immunofluorescence microscopy for use in Bacillus subtilis and have employed this procedure for visualizing cell-specific gene expression at early to intermediate stages of sporulation. Sporangia were doubly stained with propidium iodide to visualize the forespore and mother cell nucleoids and with fluorescein-conjugated antibodies to visualize the location of beta-galactosidase produced under the control of the sporulation RNA polymerase sigma factors sigma E and sigma F. In confirmation and extension of earlier reports, we found that expression of a lacZ fusion under the control of sigma E was confined to the mother cell compartment of sporangia at the septation (II) and engulfment (III) stages of morphogenesis. Conversely, sigma F-directed gene expression was confined to the forespore compartment of sporangia at postseptation stages of development. Little indication was found for sigma E- or sigma F-directed gene expression prior to septation or in both compartments of postseptation sporangia. Gene expression under the control of the forespore sigma factor sigma G also exhibited a high level of compartmentalization. A high proportion of sporangia exhibited fluorescence in our immunostaining protocol, which should be suitable for the subcellular localization of sporulation proteins for which specific antibodies are available.
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
authors.forename: E J
authors.initials: EJ
authors.lastname: Harry
authors.forename: K
authors.initials: K
authors.lastname: Pogliano
authors.forename: R
authors.initials: R
authors.lastname: Losick
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D008858:Microscopy, Phase-Contrast; D012808:Sigma Factor / Q000502:physiology; D013171:Spores, Bacterial / Q000378:metabolism; D001616:beta-Galactosidase / Q000096:biosynthesis
chemical_list: D012808:Sigma Factor; D001616:beta-Galactosidase