Role of the anti-sigma factor SpoIIAB in regulation of sigmaG during Bacillus subtilis sporulation

Autor: Mónica Serrano, Charles P. Moran, Alexandre Neves, Adriano O. Henriques, Cláudio M. Soares
Rok vydání: 2004
Předmět:
Zdroj: Journal of bacteriology. 186(12)
ISSN: 0021-9193
Popis: Gene expression in the prespore and mother cell chambers of sporulating Bacillus subtilis is controlled by RNA polymerase sigma subunits whose activity is restricted to a specific cell type (22, 31, 37, 46). The activation of the sporulation-specific sigma factors is tightly coupled to the completion of key morphological intermediates in the process and also relies on signaling pathways that operate between the two cell types and that keep the prespore and mother cell lines of gene expression in close register (22, 31, 37, 46). Soon after the asymmetric division of the sporangial cell, an event that creates the prespore and the much larger mother cell, the first compartment-specific sigma factor σF becomes active in the prespore (22, 31, 37, 46). σF triggers the activation of σE in the mother cell, which together with σF drive the migration of the septal membranes around the prespore. This process is termed engulfment and results in the formation of a protoplast isolated from the external medium, fully encircled by the mother cell cytoplasm (22, 31, 37, 46). After engulfment, σF is replaced by σG, which controls late stages of development in this compartment and which also triggers the activation of the late mother cell-specific regulator σK (22, 31, 37, 46). The activities of both σG and σK are required for the assembly of the protective layers that encase the mature spore (22, 31, 37, 46). Synthesis of σF occurs in the predivisional cell, but its activation is restricted to the prespore by the action of three regulatory proteins, SpoIIAA, SpoIIAB, and SpoIIE. SpoIIAB is an anti-sigma factor that binds to σF as a dimer, preventing its association with RNA polymerase, whereas SpoIIAA is an anti-anti-sigma factor that in an unphosphorylated state interacts with SpoIIAB and releases σF from the SpoIIAB-σF complex (1, 2; reviewed in references 31 and 37). SpoIIE is a septum-bound phosphatase that is also produced in the predivisional cell that promotes the preferential dephosphorylation of SpoIIAA-P in the prespore (reviewed in references 31 and 37). The transcriptional activity of σF can be divided into an early phase and a late phase. Transcription of the spoIIIG gene (encoding σG) is induced as part of the late phase, towards the end of the engulfment process (29). After synthesis, σG does not become active until the engulfment process is complete (29). Once activated and since σG efficiently recognizes its own promoter, its cellular levels increase rapidly, allowing for the deployment of the σG regulon (17, 47). Because of this autoregulation, both the late transcription of spoIIIG and the negative regulation of σG appear to ensure that its transcriptional activity is effectively coupled to completion of the engulfment process and does not occur prematurely or ectopically (31, 37, 45). The tight coupling of σG activation to the conclusion of the engulfment sequence may serve to ensure that biogenesis of the spore integuments is not initiated during movement of the engulfment membranes (31, 37, 45, 46). Conclusion of the engulfment process is not sufficient for the activation of σG, which further requires expression of several genes, including the eight cistrons of the spoIIIA operon and the spoIIIJ gene (6, 19, 34). σG accumulates in spoIIIA or spoIIIJ mutant cells but is unable to activate transcription from its target promoters (19, 41). The spoIIIA operon encodes several putative membrane proteins and is expressed in the mother cell under the direction of σE (15). The spoIIIJ gene is expressed during vegetative growth and encodes a membrane protein translocase of the YidC/Oxap1 family (6, 27, 48). Despite the fact that its product may accumulate in both the prespore and the mother cell (6, 27), expression of spoIIIJ in the prespore is sufficient for the activation of σG and sporulation (41). Two negative regulators of σG are known, the anti-sigma factor SpoIIAB and the LonA protease (3, 8, 19, 21, 35, 40). Expression of spoIIIG prior to the asymmetric division of the sporangial cell blocks sporulation, a phenotype that can be suppressed by a multicopy allele of spoIIAB (21), and certain point mutations in spoIIAB result in expression of σG-dependent genes under conditions that do not support efficient sporulation (8, 35). Moreover, SpoIIAB binds to σG in vitro under conditions that also promote binding of SpoIIAB to σF (19), and the structure of a dimer of Bacillus stearothermophilus SpoIIAB in complex with σF shows that most of the residues involved in the interaction are conserved in σG, but not in other sigma factors (2). While it seems clear that SpoIIAB can regulate σG under nonsporulation conditions or in the predivisional cell at the onset of sporulation, the evidence for a role in the control of σG in the prespore is less clear (3, 8, 19, 21, 35). On the one hand, SpoIIAB seems to disappear from the prespore coincidently with the first manifestations of σG activity, but it persists in the prespore of a spoIIIA mutant (21). In addition, production of a form of σG (σGE155K) that is not efficiently bound by SpoIIAB in vitro allows expression of the σG-controlled sspE gene in spoIIIA or spoIIIJ mutants, suggesting that the expression of both loci is required to antagonize the inhibitory action of SpoIIAB upon σG (19, 41). However, expression of sspE in spoIIIGE155K cells bearing mutations in either spoIIIA or spoIIIJ does not occur prematurely, suggesting that the activity of σGE155K is still regulated in the double mutants (19, 41). Also, there seems to be very little, if any, free SpoIIAB in the prespore (28), and the anti-sigma factor would have to be able to negatively regulate σG at a time when SpoIIAB itself is antagonized by the anti-anti-sigma SpoIIAA in order to release active σF (reviewed in references 31 and 37). Since the interaction of SpoIIAB with σG appears to be very similar to the interaction of SpoIIAB with σF (7), it seems unlikely that at least prior to completion of the engulfment process, SpoIIAB decisively contributes to the regulation of σG. Mutations in lonA, coding for the ATP-dependent LonA protease, also result in σG activity under nonsporulation conditions, and result in some expression of sspE-lacZ in cells of a spoIIIA mutant during sporulation (40). Here we have analyzed the role SpoIIAB plays in the regulation of σG in sporulating cells. We screened for mutations in spoIIIG that allowed expression of the σG-controlled sspE-lacZ fusion in a spoIIIA background and found a single mutation that converted a glutamate at position 156 of σG to a lysine. However, we found that expression of sspE-lacZ in a spoIIIGE156K spoIIIA double mutant was delayed relative to the completion of the engulfment process and was not confined to the prespore. Rather, β-galactosidase accumulated throughout the whole cell at late times of sporulation. We also forced the early expression of spoIIIGE156K in the prespore from the spoIIQ promoter and found no premature induction of sspE-lacZ expression. In contrast, expression of spoIIIGE156K in the mother cell readily results in sspE-lacZ expression. The results suggest that the activity of σG is regulated in the prespore compartment by a SpoIIAB-independent mechanism and that SpoIIAB is either redundant or plays only a minor role.
Databáze: OpenAIRE
Externí odkaz: