Biosynthesis, assembly and secretion of cell surface polysaccharides
EMSL Project ID
51084
Abstract
All forms of life produce extracellular complex carbohydrate to form cell walls, protective coats, or to define an extracellular matrix aiding in cell-to-cell communication and adhesion. Cellulose, a linear polymer of glucose molecules, is the most abundant biopolymer on earth produced primarily by vascular plants but also by biofilm-forming bacteria and tunicates. Cellulose synthase is an amazing enzyme that synthesizes and secretes cellulose using UDP-activated glucose as substrate. In Gram-negative bacteria, this reaction is catalyzed by the BcsA-BcsB complex. Transport across the periplasm and the outer membrane most likely involves the BcsC subunit that partners with the BcsA-BcsB complex in the inner membrane. During cellulose biosynthesis, BcsA forms a transmembrane channel to shuttle the nascent polymer across the inner membrane. In the absent of a polymer, however, this channel must close to maintain the permeability barrier of the plasma membrane. To delineate this gating mechanism, we seek to determine structures of the cellulose-bound and –free BcsA-BcsB complex by single particle cryo electron microscopy (Aim 1). Enterobacteria, such as E. coli, modify cellulose in the periplasm with phosphoethanolamine to stabilize its interaction with curli fibers on the cell surface. This reaction requires the association of the BscA-BcsB core complex with up to 5 other subunits, thereby forming a membrane-embedded supramolecular complex. We seek to determine the structure of this complex to establish the molecular basis for cellulose modification in enterobacteria (Aim 2). Cellulose microfibrils (CMFs) form the load-bearing component of plant cell walls. CMF formation requires the assembly of multiple cellulose synthases into CMF-forming complexes (CSCs) that are structurally uncharacterized to date. Freeze-fracture electron microscopy analysis of native tissues from various plants revealed that CSCs are pseudo six-fold symmetric particles containing 3-4 cellulose synthases per repeating unit. In contrast to the bacterial enzymes, plant cellulose synthases contain additional domains, including an N-terminal RING-like domain assumed to mediate oligomerization. We established the expression, functional reconstitution, and in vitro assembly of plant cellulose synthase and seek to determine the cryo EM structures of these oligomers prior to and during cellulose biosynthesis.
Project Details
Start Date
2019-10-15
End Date
2021-03-17
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members