SpoIID-Mediated Peptidoglycan Degradation Is Required throughout Engulfment during Bacillus subtilis Sporulation

Abstract

SpoIID is a membrane-anchored enzyme that degrades peptidoglycan and is essential for engulfment and sporulation in Bacillus subtilis. SpoIID is targeted to the sporulation septum, where it interacts with two other proteins required for engulfment: SpoIIP and SpoIIM. We changed conserved amino acids in SpoIID to alanine to determine whether there was a correlation between the effect of each substitution on the in vivo and in vitro activities of SpoIID. We identified one amino acid substitution, E88A, that eliminated peptidoglycan degradation activity and one, D210A, that reduced it, as well as two substitutions that destabilized the protein in B. subtilis (R106A and K203A). Using these mutants, we show that the peptidoglycan degradation activity of SpoIID is required for the first step of engulfment (septal thinning), as well as throughout membrane migration, and we show that SpoIID levels are substantially above the minimum required for engulfment. The inactive mutant E88A shows increased septal localization compared to the wild type, suggesting that the degradation cycle of the SpoIID/SpoIIP complex is accompanied by the activity-dependent release of SpoIID from the complex and subsequent rebinding. This mutant is also capable of moving SpoIIP across the sporulation septum, suggesting that SpoIID binding, but not peptidoglycan degradation activity, is needed for relocalization of SpoIIP. Finally, the mutant with reduced activity (D210A) causes uneven engulfment and time-lapse microscopy indicates that the fastest-moving membrane arm has greater concentrations of SpoIIP than the slower-moving arm, demonstrating a correlation between SpoIIP protein levels and the rate of membrane migration.Endospore formation is an evolutionarily conserved process that allows Bacillus subtilis and related Gram-positive bacteria to adapt to changes in the environment, such as nutrient depletion. Many dramatic morphological changes occur during sporulation, each requiring a multitude of specialized proteins (reviewed in references 13 and 17). First, a sporulation septum is formed near one of the cell poles, forming two separate compartments of unequal sizes and with differing fates (Fig. 1A). The smaller of the two, the forespore, will eventually become the spore, while the larger, the mother cell, will ultimately lyse. Next, the mother cell membranes move up and around the forespore in the poorly understood process of engulfment. Although this process is superficially similar to eukaryotic engulfment, it is complicated by the thick cell wall that surrounds and separates the two compartments. After engulfment, the migrating membranes pinch off from the mother cell membrane, thereby releasing the forespore into the cytoplasm of the mother cell, where it can be enveloped with protective coat proteins and eventually released into the environment as a mature spore. Sporulation provides an ideal, nonessential system for understanding how bacterial cells are capable of undergoing dramatic morphological changes.Engulfment involves dynam...

Crystal Structures of the SpoIID Lytic Transglycosylases Essential for Bacterial Sporulation

“…The Glu-101 to Ala substitution inactivated the enzyme. This result, complemented by the structural data and by the fact that the corresponding Glu-88 in the BsSpoIID has been previously proposed to be the catalytic residue (1,4), confirms that Glu-101 of CdSpoIID is the essential catalytic residue for the reaction. Interestingly, the single mutations of His-145, Cys-146, or Cys-140, three of the residues involved in the zinc coordination, also inactivated the enzyme, highlighting the importance of this structural motif for CdSpoIID activity.…”

“…First, we confirmed that both CdSpoIID and BaSpoIID require peptide-free glycan chains as substrate for their hydrolase activities (1,4). In fact, no blue release was detected in the supernatant for reactions containing BaSpoIID and CdSpoIID when incubated with RBB-labeled PGN.…”

“…Sporulation is characterized by an asymmetric cell division, followed by migration of the mother cells' membrane around the nascent spore in a phagocyte-like process (2,3). Engulfment of what will become the spore requires PGN 2 degradation by the SpoIIM/SpoIIP/ SpoIID molecular machine (4,5). In this complex, SpoIIM serves as a scaffold upon which the SpoIID and SpoIIP enzymes assemble (1,4,6).…”

“…Engulfment of what will become the spore requires PGN 2 degradation by the SpoIIM/SpoIIP/ SpoIID molecular machine (4,5). In this complex, SpoIIM serves as a scaffold upon which the SpoIID and SpoIIP enzymes assemble (1,4,6). SpoIIP is single-pass transmembrane protein with a large extracellular portion that has both amidase and endopeptidase activities.…”

“…The requirement for a peptide-free glycan strand defines an ordered and sequential set of events during the engulfment process. SpoIID also functions to stimulate SpoIIP activity (4).…”