Contrasting Effects of σ^Eon Compartmentalization of σ^FActivity during Sporulation ofBacillus subtilis

Abstract

Spore formation by Bacillus subtilis is a primitive form of development. In response to nutrient starvation and high cell density, B. subtilis divides asymmetrically, resulting in two cells with different sizes and cell fates. Immediately after division, the transcription factor F becomes active in the smaller prespore, which is followed by the activation of E in the larger mother cell. In this report, we examine the role of the mother cell-specific transcription factor E in maintaining the compartmentalization of gene expression during development. We have studied a strain with a deletion of the spoIIIE gene, encoding a DNA translocase, that exhibits uncompartmentalized F activity. We have determined that the deletion of spoIIIE alone does not substantially impact compartmentalization, but in the spoIIIE mutant, the expression of putative peptidoglycan hydrolases under the control of E in the mother cell destroys the integrity of the septum. As a consequence, small proteins can cross the septum, thereby abolishing compartmentalization. In addition, we have found that in a mutant with partially impaired control of F , the activation of E in the mother cell is important to prevent the activation of F in this compartment. Therefore, the activity of E can either maintain or abolish the compartmentalization of F , depending upon the genetic makeup of the strain. We conclude that E activity must be carefully regulated in order to maintain compartmentalization of gene expression during development. Sporulation inBacillus subtilis has become a paradigm for studying prokaryotic development. In response to nutrient starvation and high cell density, a developmental program that results in the formation of environmentally resistant endospores is initiated. An early hallmark event in sporulation is an asymmetric division that divides the cell into two compartments, the smaller prespore and the larger mother cell. The prespore will eventually form a mature spore, whereas the mother cell will assist in the developmental process but will eventually lyse and die, releasing the spore into the environment (29). Gene expression is tightly compartmentalized in these two cells during sporulation, with the RNA polymerase factor F becoming active exclusively in the prespore and E becoming active only in the mother cell (32). The mechanisms by which this compartmentalization process occurs have been a major focus of study. However, how compartmentalization is maintained throughout development has not been as thoroughly examined. We have utilized two separate lines of investigation in order to explore this aspect of sporulation. At the time of asymmetric division, only the origin-proximal one-third of a chromosome is present in the prespore (44). The spoIIIE gene encodes a DNA translocase that pumps the rest of the chromosome destined for the prespore into this compartment after asymmetric division (5, 42). In addition to their defects in chromosome partitioning, spoIIIE mutants display two different phenotypes with regard to F activity...

Compartmentalization of Gene Expression duringBacillus subtilisSpore Formation

“…However, a separate study found that ␤-galactosidase could not diffuse across the septa of class II mutants and proposed that persistence of SpoIIE in the mother cell rather than a hole in the septum was responsible for the loss of compartmentalization (234). Our recent studies support the existence of a small hole permeable to factors or their regulators and to GFP (110).…”

“…Given that E -directed genes are required to cause septal wall autolysis during engulfment (1,37,57,85,228,232,246,271,272), it seemed plausible that similar activity might enlarge a breach in the septum resulting from loss of SpoIIIE. Indeed, E -directed transcription of spoIID and spoIIP is required to disrupt compartmentalization in spoIIIE class II mutants (110). Presumably, autolytic activity associated with SpoIID and SpoIIP enlarges the putative hole sufficiently to allow passage of F or its regulators and of GFP and possibly of E .…”

Use of the fluorescent probe LAURDAN to label and measure inner membrane fluidity of endospores of Clostridium spp.

“…Such protein fluorophores exhibit great utility in studying protein localization and gene expression during sporulation, specifically in Bacillus spp. (Asai et al, 2001;Hilbert et al, 2004;McBride et al, 2005). Green fluorescent protein and its analogues require oxygen for maturation and subsequent fluorescence (Reid and Flynn, 1997).…”

Separation of Chromosome Termini during Sporulation of Bacillus subtilis Depends on SpoIIIE

“…The SpoIIIE protein localizes to the middle of the sporulation division septum (38,48). It forms an effective seal around the DNA during translocation, preventing the diffusion of proteins between the prespore and mother cell (16,48). During engulfment, it migrates with the engulfing membrane to the cell pole, where it is required for completion of engulfment (38).…”