Visualization of the subcellular location of sporulation proteins in Bacillus subtilis using immunofluorescence microscopy

Abstract

We describe the application of immunofluorescence microscopy to visualization of the subcellular localization of proteins involved in coat morphogenesis and chromosome packaging during the process of sporulation in Bacillus subtilis. In confirmation and extension of previous findings, we show that SpolVA, which is responsible for guiding coat formation to the surface of the outer membrane that surrounds the developing spore, assembles into a shell that is located close to or on the surface of this enveloping membrane. CotE, which is responsible for the formation of the outer layer of the coat, assembles into a second shell of apparently larger diameter. Assembly of SpolVA could be detected as early as the morphological stage of polar septation and closely followed the enveloping membrane of the mother cell during the stage of engulfment, thereby providing a sensitive and diagnostic marker for this phagocytic-like process. Surprisingly, the chromosome of the developing spore and the small, acid-soluble proteins, known as alpha/beta-type SASPs, that are known to coat the spore chromosome, were found to co-localize to a doughnut-like ring of approximately 1 micrometer in diameter. The use of a double mutant lacking the alpha/beta-type SASP demonstrated that these high abundance, DNA-binding proteins are responsible for packaging the chromosome of the developing spore into this unusual structure. We conclude that sporulation in B. subtilis is a fertile system for addressing cell biological problems in a bacterium and that immunofluorescence microscopy provides a sensitive method for visualizing protein subcellular localization at high resolution.