A four-dimensional view of assembly of a morphogenetic protein during sporulation in Bacillus subtilis

We report the use of a fusion to the green fluorescent protein to visualize the assembly of the morphogenetic protein SpoIVA around the developing forespore during the process of sporulation in the bacterium Bacillus subtilis. Using a deconvolution algorithm to process digitally-collected optical sections, we show that SpoIVA, which is synthesized in the mother cell chamber of the sporangium, assembled into a spherical shell around the outer surface of the forespore. Time-lapse fluorescence microscopy showed that this assembly process commenced at the time of polar division and seemed to continue after engulfment of the forespore was complete. SpoIVA remained present throughout the late stages of morphogenesis and was present as a component of the fully mature spore. Evidence indicates that assembly of SpoIVA depended on the extreme C-terminal region of the protein and an additional region that directly or indirectly facilitated interaction among SpoIVA molecules. The N- and C-terminal regions of SpoIVA, including the extreme C terminus, are highly similar to the corresponding regions of the homologous protein from the distantly related endospore-forming bacterium Clostridium acetobutylicum, attesting to their importance in the function of the protein. Finally, we show that proper localization of SpoIVA required the expression of one or more genes which, like spoIVA, are under the control of the mother cell transcription factor sigmaE. One such gene was spoVM, whose product was required for efficient targeting of SpoIVA to the outer surface of the forespore.