TWIN SPORE FORMATION WITHIN ONE MOTHER CELL BY BACILLUS SUBTILIS

Abstract

Formation of spores by Bacillus subtilis is a primitive system of differentiation that has become a paradigm for studying cell differentiation in prokaryotes. Differential gene expression commences soon after the single, asymmetric sporulation division through the activation of different RNA polymerase sigma factors, sigma F in the smaller prespore and sigma E in the larger mother cell. sigma E activation relies on an inter-cellular signaling emanating from sigma F-directed gene expression. Formation of the asymmetric division septum and compartmentalized activity of both sigma factors occur prior to chromosome partitioning. At the time of septation, only 30% of the chromosome destined to be in the prespore is actually present in that compartment and the remaining 70% is in the mother cell. Thus, both cell types contain unequal DNA content. This study focused on the effect of this genetic asymmetry on sigma F-directed gene expression, and exploited this effect in order to study aspects of sigma F to sigma E inter-compartmental signaling. Perturbed signaling resulted in the discovery of a novel twin-spore forming morphology, which was further characterized. A DNA translocase is required to translocate the remaining portion of the chromosome from the mother cell to the prespore. The replication terminus region of the chromosome was observed to be the last to enter the prespore and thus, sigma F-directed genes showed delayed and reduced expression when moved to a terminus-proximal location. The studies indicate that this positional regulation of sigma F-directed gene expression is attributed to both delayed entry and inhibition in sigma F activity at late stages of sporulation. Moreover, the next prespore-specific sigma factor, sigma G, could have a role in inhibiting sigma F. The link between sigma F and sigma E activation is the spoIIR locus, which is transcribed in the prespore from a sigma F-directed promoter soon after the formation of the asymmetric septum. Inactivation of the structural genes for sigma F or sigma E or SpoIIR results in the formation of a second septum at the opposite pole; development proceeds no further, resulting in an "abortively disporic" phenotype. The second septum is formed about 20 min after the first, and sigma E activity is required to prevent its formation. As a sigma F-directed gene, spoIIR is subject to `positional regulation': a delay in spoIIR expression caused by moving it from its origin proximal position to the chromosome terminus, is sufficient to delay sigma E activation and block spore formation, giving the abortively disporic phenotype. The effects of delaying and enhancing spoIIR expression were tested. The changes delayed sigma E activation, and many organisms formed a septum at both ends. However, both prespores in these organisms were able to develop into mature spores (twins). Extra rounds of chromosome replication occured during twin formation, so that each twin had a chromosome and the mother cell had either one or two chromosomes. This over-initiation of chromosome replication is a prerequisite for twin spore formation. Moreover, the studies showed that mother cells of twin forming organisms were longer than those containing single spores; image analysis showed that mother cell length correlates with chromosome content. In contrast to twin spore formation, during normal spore development, there is usually one copy of the chromosome in the prespore and one in the mother cell, with no growth of either compartment. Therefore, the system allowed investigating regulation of chromosome replication and growth of the mother cell. The studies showed that replication and growth are permitted because of the absence of active sigma E and of reduced levels of transcription directed by the master regulator for entrance to spore formation, Spo0A. The results indicate that the burst of Spo0A-directed expression along with activation of sigma E provide mechanisms to block replication and growth of the mother cell.