Protein localization and peptidoglycan hydrolysis during engulfment in Bacillus subtilis.

摘要

In response to unfavorable environmental conditions such as nutrient starvation, the gram positive soil bacterium Bacillus subtilis enters into a developmental pathway known as sporulation. Shortly following the commitment to sporulation, B. subtilis divides asymmetrically, dividing the cell into two compartments, a smaller forespore (the future spore) and a larger mother cell. Subsequently, in a process that resembles eukaryotic phagocytosis, known as engulfment, the mother cell membranes migrate up and around the forespore. The completion of engulfment is marked by the mother cell membranes fusing at the forespore pole, releasing the immature spore into the mother cell cytoplasm where further spore maturation takes place. Three mother cell expressed proteins, SpoIID, SpoIIM, and SpoIIP have been shown to mediate both the early step of engulfment, septal thinning and subsequent membrane migration. Research presented in this dissertation addresses how SpoIID, SpoIIM and SpoIIP are targeted to the sporulation septum, and in particular, the role of SpoIID during engulfment.; My studies of the engulfment proteins led to the discovery of two pathways that are involved in targeting SpoIID, SpoIIM, and SpoIIP (DMP) to the septum. Normally, SpoIID, SpoIIM, and SpoIIP localize to the septum in a SpoIIB-dependent manner. However, in the absence of SpoIIB, cells were able to complete engulfment, albeit at a slower rate than wild type. This suggested the presence of a secondary, SpoIIB-independent pathway for targeting the DMP complex to the septum. In fact, my studies showed that a secondary, compensatory targeting pathway does exist, and is mediated by the SpoIIQ-SpoIIIAH (Q-AH) zipper via the mother cell expressed proteins SpoIVFA and SpoIVFB. Thus, the Q-AH zipper not only provides a compensatory mechanism for membrane migration during engulfment when DMP activity is reduced, but also indirectly mediates a compensatory septal localization pathway for DMP when its primary targeting pathway is disrupted.; My research also demonstrated that SpoIID functions as a peptidoglycan hydrolase. Site directed mutagenesis of conserved amino acids within SpoIID has led to the identification of residues important for the proteins enzymatic activity as well as its function in vivo. I have thereby demonstrated that the peptidoglycan hydrolase activity of SpoIID is required during engulfment, and have found mutations which suggest that SpoIID also functions later in sporulation, as they block spore formation but not membrane migration. In collaboration with the Popham lab at Virginia Tech, we have determined that SpoIID acts as an endopeptidase capable of cleaving the peptide cross-bridges that link adjoining glycan strands. These studies, in total, have added to our mechanistic knowledge of engulfment by further characterizing the biochemical activity of SpoIID and correlating its in vitro activities with its function in vivo.