Impact of Membrane Fusion and Proteolysis on SpoIIQ Dynamics and Interaction with SpoIIIAH

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 G , which produces high levels of the secreted SpoIVB protease that is required for activation of the late mother cell transcription factor K . Engulfment also triggers the proteolytic cleavage of SpoIIQ, an event that depends on the SpoIVB protease but not on G 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 G 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.Endospore formation in Bacillus subtilis and its relatives depends on engulfment, a phagocytosis-like process that mediates a dramatic rearrangement of the sporangium from two adjacent daughter cells, to an endospore in which the forespore lies within the cytoplasm of the larger mother cell (see Fig. 1; reviewed by Refs. 1 and 2). Engulfment is critical for sporulation, because it allows spore assembly to occur in a protected environment. It also serves as a morphological checkpoint for activation of the late forespore and mother cell transcription factors G and K , respectively (reviewed by Refs. 1-4). The endospore-forming bacteria must therefore have some mechanism to sense the completion of engulfment and to couple this morphological event to the onset of late gene expression.The forespore transcription factor G is the first to become active after engulfment, but it remains unclear how G is held inactive during engulfment or activated after engulfment. More is known about the regulation of the second late transcription factor, K , which becomes active in the mother cell (summarized in Fig. 1B and reviewed by Refs. 1-4). The K factor is initially synthesized as an inactive pro-protein containing a hydrophobic leader sequence, which functions as a covalently attached anti-sigma factor (5). This leader sequence is removed by the intramembrane protease SpoIVFB, which cleaves pro-K within the membrane ...

A Membrane-Embedded Amino Acid Couples the SpoIIQ Channel Protein to Anti-Sigma Factor Transcriptional Repression during Bacillus subtilis Sporulation

“…The first is that Q may undergo a change in topology such that Tyr-28 gains access to the cytosol. Intriguingly, Q is subject to proteolysis by the secreted protease SpoIVB at a site not far downstream of the TMD (after Val-72), resulting in an N-terminal fragment that becomes cytosolic (45,46). However, we have found that csfB expression is not misregulated in a strain in which spoIVB is deleted (our unpublished results), arguing against a model in which a SpoIVBgenerated fragment of Q is responsible for csfB regulation.…”

A channel connecting the mother cell and forespore during bacterial endospore formation

“…The interaction with SpoIIQ is necessary for the septal localization and stability of SpoIIIAH (4,5,30). This suggests that SpoIIQ should be necessary for the forespore accessibility and stability of SpoIIIAH-BAP.…”

Holin triggering in real time

“…FRAP experiments were conducted as previously described (35,36). Briefly, photobleaching experiments to assess the mobility of S105-GFP (or derivatives) used cells grown in liquid culture (0.25× LB, 0.5 μg/mL of FM 4-64); 10 μL of cells were removed at the indicated times and applied to coverslips.…”