Activation of sigma G during formation of spores by Bacillus subtilis depends on completion of engulfment, which follows translocation of the chromosome into the prespore

Abstract

The process of sporulation in Bacillus subtilis serves as a paradigm for study of sporulation in all Bacillus and Clostridium species. During the process of sporulation there is a sequential and cell type specific activation of RNA polymerase sigma factors. The asymmetric sporulation septum forms, which divides the cell into the smaller prespore and the larger mother cell. Then, óF becomes active in the prespore and óE becomes active in the mother cell. Following completion of engulfment of the prespore by the mother cell, óG becomes active in the prespore and finally óK becomes active in the mother cell. At the time the sporulation septum forms only the 30% origin proximal portion of the chromosome is within the prespore. It is the responsibility of the SpoIIIE translocase to pump the remaining chromosome destined for the prespore into the prespore. We hypothesize that activation of óG does not occur until the complete chromosome has been translocated into the prespore, and the prespore has been engulfed by the mother cell. Our first method of investigation was to increase the time required to complete translocation of the chromosome into the prespore. Strains in which large inserts of foreign DNA have considerably increased the genome size were obtained and characterized. The strains have shown a delay in the completion of engulfment, which still occurred before the activation of óG. Activation was identified by visualization of GFP from a óG-directed promoter indicating translation of a product transcribed by óG. We have also shown that the terminus region of the chromosome entered the prespore shortly before the completion of engulfment. It was determined that the increased genome size did not result in a delay of the transcription of the structural gene for óG,spoIIIG. Using a strain in which the activity of the SpoIIIE translocase was reduced 2.5 fold we used a óG-directed GFP reporter to study óG activation under both time course and time lapse conditions. We again found that óG only became active following the completion of engulfment. Our second avenue of investigation was to change the site of replication termination. A strain was used in which several terminus associated genes have been relocated from their usual location near the terminus (172°) resulted in termination of replication occurring at 145° on the chromosome. In this strain, it has been found that, under both time course and time lapse microscopy conditions, óG activation still occurred only following completion of engulfment. The translocation of the chromosome in this strain was studied by time lapse microscopy using a óF- directed reporter and it was determined that the origin proximal region, the site of replication termination, and the traditional terminus region, were all translocated into the prespore prior to the completion of engulfment. The results support the hypothesis that there is a strong link requiring complete translocation of the chromosome, followed by engulfment of the prespore, before óG becomes active.