Trapped translocation intermediates establish the route for export of capsular polysaccharides across Escherichia coli outer membranes.
The outer membrane (OM) of Gram-negative bacteria is designed to exclude potentially harmful molecules. This property presents a challenge for bacteria that must secrete proteins and large glycoconjugates to grow, divide, and persist. Proteins involved in trafficking such molecules have been identified, but their precise roles are often unresolved due to the difficulty in capturing "snapshots" during the export pathway. Wza is the prototype for the large family of OM polysaccharide export proteins. In Escherichia coli, Wza is essential for the assembly of a capsule, a protective surface coat composed of long-chain polysaccharides. Wza creates an octameric α-helical channel spanning the OM, but the bulk of the protein exists as a large periplasmic structure enclosing an extensive lumen. Residues within the lumen of Wza were targeted for site-specific incorporation of the UV photo-cross-linkable unnatural amino acid p-benzoyl-L-phenylalanine. Using this in vivo photo-cross-linking strategy, we were able to trap polysaccharide translocation intermediates within the lumen of Wza, providing the first unequivocal evidence to our knowledge that nascent capsular polysaccharide chains exit the cell through the Wza portal.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1; and.
Nicholas N
NN
Nickerson
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1; and.
Iain L
IL
Mainprize
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1; and.
Lauren
L
Hampton
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1; and.
Michelle L
ML
Jones
Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews KY16 9ST, Scotland.
James H
JH
Naismith
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1; and cwhitfie@uoguelph.ca.
Chris
C
Whitfield
D000595:Amino Acid Sequence; D016667:Bacterial Capsules / Q000737:chemistry / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011135:Polysaccharides, Bacterial / Q000737:chemistry / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
D001425:Bacterial Outer Membrane Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D011135:Polysaccharides, Bacterial; C404158:Wza protein, E coli