Abstract
The onset of engulfment-dependent gene expression during Bacillus subtilis sporulation requires the forespore membrane protein SpoIIQ, which recruits mother cell proteins involved in late gene expression to the outer forespore membrane. Engulfment activates the late forespore transcription factor sigmaG, which produces high levels of the secreted SpoIVB protease that is required for activation of the late mother cell transcription factor sigmaK. Engulfment also triggers the proteolytic cleavage of SpoIIQ, an event that depends on the SpoIVB protease but not on sigmaG activity. To determine if SpoIVB directly cleaves SpoIIQ and to determine if this event participates in the onset of late gene expression, we purified SpoIVB, SpoIIQ, and SpoIVFA (another SpoIVB substrate). SpoIVB directly cleaved SpoIIQ at the same site in vitro and in vivo and cleaved SpoIVFA in at least three different locations. SpoIIQ cleavage depends on membrane fusion, but not on sigmaG activity, suggesting that the ability of SpoIVB to cleave substrates is regulated by membrane fusion. We isolated SpoIVB-resistant SpoIIQ proteins by random mutagenesis of codons at the cleavage site and demonstrated that SpoIIQ processing is dispensable for spore formation and for activation of late forespore and mother cell gene expression. Fluorescence recovery after photobleaching analysis demonstrated that membrane fusion releases SpoIIQ from an immobile complex, an event that could allow SpoIVB to cleave SpoIIQ. We propose that this membrane fusion-dependent reorganization in the complex, rather than SpoIIQ proteolysis itself, is necessary for the onset of late transcription.
Highlights
Bacillus subtilis sporulation requires the forespore membrane protein SpoIIQ, which recruits mother cell proteins involved in late gene expression to the outer forespore membrane
Activation of K is governed by two checkpoints: the forespore one slightly larger than that of checkpoint (i) in which G activation allows the production of high levels of SpoIVB [11], which cleaves SpoIVFA native SpoIIQ (which reacts with to relieve SpoIVFB from inhibition by BofA, and the engulfment checkpoint (ii) in which K activity is coupled to His -specific antibodies) and one engulfment by a BofA-independent mechanism [17]
Valine at the predicted SpoIVB cleavage site and permissive To distinguish between these models, we compared the levels substitutions at positions corresponding to Gly-73 and Lys-74 of G and K activity (using sspB-lacZ and cotD-lacZ, respecof B. subtilis SpoIIQ in strains carrying mutations that block (V72M and iheyensis, which had a non-permissive substitution at position G73E) or allow (V72C and G73V) SpoIIQ proteolysis
Summary
In the case of SpoIVFB, the signal is the prior activation of the late forespore-specific transcription factor G [11], which produces higher levels of the SpoIVB protease [12] This protease disrupts an inhibitory complex between the SpoIVFB intramembrane protease and two other proteins, SpoIVFA, which is necessary for complex assembly and localization [13], and BofA, which inhibits SpoIVFB activity (Fig. 1B) [7, 11, 13]. SpoIIQ, SpoIIIAH, and SpoIVFB all localize to foci surrounding the JANUARY 26, 2007 VOLUME 282 NUMBER 4 forespore (Fig. 4B) [17, 19, 23] These foci might represent synapse-like sites for intracellular signal transduction, perhaps allowing transcription in both cells to be coordinately regulated by engulfment. This reorganization in the SpoIIQ complex might control both proteolysis and signal transduction
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