Discovery of an amidase-activator complex that controls Staphylococcus aureus cell growth and division
Access StatusFull text of the requested work is not available in DASH at this time ("dark deposit"). For more information on dark deposits, see our FAQ.
Gunther, Truc Do
MetadataShow full item record
CitationGunther, Truc Do. 2020. Discovery of an amidase-activator complex that controls Staphylococcus aureus cell growth and division. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractBacterial cell survival depends on the integrity of the peptidoglycan cell wall, which surrounds and protects the cell from osmotic lysis. Peptidoglycan synthases first polymerize a disaccharide-peptide precursor to make glycan chains and then crosslink the peptides on these chains to the existing cell wall matrix. In Staphylococcus aureus, these enzymes move during the cell cycle from the periphery, where they are active during growth, to the site of division where they build the partition between daughter cells. But how peptidoglycan synthesis is regulated throughout the cell cycle is poorly understood. Our lab has conducted transposon screens to identify possible regulators of peptidoglycan synthesis in S. aureus. One hit mapped to a gene called lytH, which encodes a putative cell wall amidase. Amidases are a class of hydrolases that cleave the stem peptides from peptidoglycan; they typically hydrolyze crosslinked peptidoglycan between daughter cells so the cells can separate.
Here I show that LytH is an amidase that instead regulates S. aureus cell growth and division. Reconstitution of LytH activity required a membrane protein partner, ActH, that forms a stable complex with the amidase. Using uncrosslinked and crosslinked substrates made in vitro, I found that the protein complex only removes uncrosslinked stem peptides; cellular experiments were consistent with this preference. In the absence of this complex, peptidoglycan synthesis becomes spatially dysregulated, causing cells to grow so large that cell division is defective. Genetics combined with biochemistry and microscopy showed that decreasing the activity of the major peptidoglycan synthase, PBP2, corrects the cell size and division defects due to lytH deletion. Mislocalization of PBP2 in the absence of LytH contributes to dysregulation of peptidoglycan synthesis. Taken together, our findings support a model wherein the LytH-ActH complex acts to remove free stem peptides from peptidoglycan in order to control the density of peptidoglycan assembly sites at a given subcellular location, and thereby to control cell expansion. My work uncovered two key biological insights. I (i) identified a novel role for amidases in controlling cell growth and (ii) reported perhaps the first example of an activator that directly regulates a Gram-positive peptidoglycan hydrolase.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37365129
- FAS Theses and Dissertations