VISUALIZATION OF THE SUBCELLULAR LOCATION OF SPORULATION PROTEINS IN BACILLUS-SUBTILIS USING IMMUNOFLUORESCENCE MICROSCOPY

We describe the application of immunofluorescence microscopy to visual ization of the subcellular localization of proteins involved in coat m orphogenesis and chromosome packaging during the process of sporulatio n in Bacillus subtilis. In confirmation and extension of previous find ings, we show that SpolVA, which is responsible for guiding coat forma tion to the surface of the outer membrane that surrounds the developin g spore, assembles into a shell that is located close to or on the sur face of this enveloping membrane. CotE, which is responsible for the f ormation of the outer layer of the coat, assembles into a second shell of apparently larger diameter, Assembly of SpolVA could be detected a s early as the morphological stage of polar septation and closely foll owed the enveloping membrane of the mother cell during the stage of en gulfment, thereby providing a sensitive and diagnostic marker for this phagocytic-like process. Surprisingly, the chromosome of the developi ng spore and the small, acid-soluble proteins, known as alpha/beta-typ e SASPs, that are known to coat the spore chromosome, were found to co -localize to a doughnut-like ring of approximately 1 mu m in diameter. The use of a double mutant lacking the alpha/beta-type SASP demonstra ted that these high abundance, DNA-binding proteins are responsible fo r packaging the chromosome of the developing spore into this unusual s tructure, We conclude that sporulation in B. subtilis is a fertile sys tem for addressing cell biological problems in a bacterium and that im munofluorescence microscopy provides a sensitive method for visualizin g protein subcellular localization at high resolution.