Open AccessEscherichia coli O127 group 4 capsule proteins assemble at the outer membrane
Author(s) -
Matthew R. Larson,
Kassia Biddle,
Adam D. Gorman,
Sarah Boutom,
Ilan Rosenshine,
Mark A. Saper
Publication year - 2021
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0259900
Subject(s) - bacterial outer membrane , operon , escherichia coli , bacterial capsule , biology , secretion , biochemistry , chemistry , microbiology and biotechnology , biophysics , gene , virulence
Enteropathogenic Escherichia coli O127 is encapsulated by a protective layer of polysaccharide made of the same strain specific O-antigen as the serotype lipopolysaccharide. Seven genes encoding capsule export functions comprise the group 4 capsule ( gfc ) operon. Genes gfcE , etk and etp encode homologs of the group 1 capsule secretion system but the upstream gfcABCD genes encode unknown functions specific to group 4 capsule export. We have developed an expression system for the large-scale production of the outer membrane protein GfcD. Contrary to annotations, we find that GfcD is a non-acylated integral membrane protein. Circular dichroism spectroscopy, light-scattering data, and the HHomp server suggested that GfcD is a monomeric β-barrel with 26 β-strands and an internal globular domain. We identified a set of novel protein-protein interactions between GfcB, GfcC, and GfcD, both in vivo and in vitro , and quantified the binding properties with isothermal calorimetry and biolayer interferometry. GfcC and GfcB form a high-affinity heterodimer with a K D near 100 nM. This heterodimer binds to GfcD ( K D = 28 μM) significantly better than either GfcB or GfcC alone. These gfc proteins may form a complex at the outer membrane for group 4 capsule secretion or for a yet unknown function.