Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system

Abstract

Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that are synthesized by several types of bacterial cellulose secretion (Bcs) systems, which include conserved cyclic diguanylate (c-di-GMP)-dependent cellulose synthase modules together with diverse accessory subunits. In E. coli, the biogenesis of phosphoethanolamine (pEtN)-modified cellulose relies on the BcsRQABEFG macrocomplex, encompassing inner-membrane and cytosolic subunits, and an outer membrane porin, BcsC. Here, we use cryogenic electron microscopy to shed light on the molecular mechanisms of BcsA-dependent recruitment and stabilization of a trimeric BcsG pEtN-transferase for polymer modification, and a dimeric BcsF-dependent recruitment of an otherwise cytosolic BcsE₂R₂Q₂ regulatory complex. We further demonstrate that BcsE, a secondary c-di-GMP sensor, can remain dinucleotide-bound and retain the essential-for-secretion BcsRQ partners onto the synthase even in the absence of direct c-di-GMP-synthase complexation, likely lowering the threshold for c-di-GMP-dependent synthase activation. Such activation-by-proxy mechanism could allow Bcs secretion system activity even in the absence of substantial intracellular c-di-GMP increase, and is reminiscent of other widespread synthase-dependent polysaccharide secretion systems where dinucleotide sensing and/or synthase stabilization are carried out by key co-polymerase subunits.

Fig. 1. State-of-the-art and here-in presented structures of the Bcs macrocomplex from the E. coli Type II cellulose secretion system.

a Left, E. coli bcs operon organization, BcsA domain architecture and thumbnail representation of the secretion system topology in the E. coli envelope. Middle and right, current structural insights into complex assembly from X-ray crystallographic and electron microscopy structures^,–. Adapted with modifications from Krasteva 2024 under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/legalcode). NTD N-terminal domain (green), TMD transmembrane domain (wheat), GT glycosyl transferase domain (light green), PilZ c-di-GMP-sensing PilZ domain (dark red), CT C-terminal tail with amphipathic helices (orange), OM outer membrane, PG peptidoglycan, IM inner membrane, ATP adenosine triphosphate, c-di-GMP cyclic diguanylate, pEtN phosphoethanolamine, NTPase* (light gray) catalytically incompetent nucleoside triphosphatase domain, REC* (orange) phosphorylation-incompetent receiver domain, GGDEF* (dark red) degenerate diguanylate cyclase domain. Multidomain BcsB hexamerizes to form a periplasmic crown shown in two different views. The carbohydrate-binding domains are shown in shades of light purple, the flavodoxin-like domains in blue and pink, and the C-terminal tail-anchor (TA) in dark purple. Densities for BcsA^NTD (green), BcsG (light blue), BcsE (tricolor) and BcsF (dark blue) have remained practically unresolved in the macrocomplex and are represented as thumbnails, whereas crystallographic snapshots have captured two different conformations of BcsE, shown on the right^,. The relative REC* domain displacement and rotation are indicated (45 Å and 144 degrees, respectively). The formation of a composite c-di-GMP binding site by RxxD (arginine-two residues-aspartate) motifs from both the degenerate REC* and GGDEF* domains increases the affinity for dimeric c-di-GMP from the low micromolar to nanomolar range (bottom right). b Cartoon representations of the here-in-resolved cryo-EM structure of the assembled, c-di-GMP-saturated Bcs macrocomplex in five different views. c Cartoon representations of the here-in-resolved cryo-EM structure of the assembled Bcs macrocomplex featuring a c-di-GMP-free BcsA.

Fig. 2. Cryo-EM structure of the synthase:pEtN-transferase complex.

a Different views of a locally refined cryo-EM structure of the c-di-GMP-free BcsA-BcsB^TA-BcsG₃ assembly (BcsAG₃ for simplicity) with corresponding electron densities (left) and cartoon representation (right). b, c Zoom-ins on the specific protein-protein interfaces with key residues shown as sticks and the electron density as a mesh. d Composite predicted structure of full-length BcsG (catalytic domain: X-ray structure of the E. coli BcsG^CTD; NTD and linker, AlphaFold (AF)) and crystal structure of the lipid A pEtN-transferase from Neisseria meningitidis EptA. The flexible interdomain linkers are colored in purple. e Model for independent function of the three BcsG copies for substrate-extraction and cellulose modification. IM inner membrane, PE phosphatidyl-ethanolamine.

Fig. 3. BcsF-dependent BcsE recruitment and regulatory complex conformation in the c-di-GMP-saturated state.

a Locally refined cryo-EM structure of the BcsE₂F₂ assembly from the c-di-GMP-saturated synthase macrocomplex shown as electron density and in cartoon. IM, inner membrane. b BcsF dimerization shown as Coulombic electrostatic potential-colored surface (left, default −10 to 10 range) and in cartoon and sticks (right). c BcsE-BcsF interactions. Left, BcsE^NTD is shown as a lipophilicity-colored surface (default −20 to 20 range), BcsF residues—including the hydrophobic plug residues V⁴⁶ and L⁵²—are shown as sticks. Right, recombinant expression and purification of the Bcs macrocomplex with various BcsF variants (Bcs^HisRQA^HA-FLAGB + Bcs^strepEF*G). Protein-specific bands are identified as previously^,. BcsE and BcsA-specific signals are further detected by western blotting with epitope tag-specific antibodies in the bottom (representative data from three independent experiments). d The BcsE^NTD dimerization interface. e The BcsE^REC* dimerization interface. f The c-di-GMP-binding dual I-site pocket in closed BcsE. All interface parameters were calculated with PISA.

Fig. 4. The c-di-GMP-bound synthase macrocomplex.

a Locally refined cryo-EM map and fitted structure of the crownless c-di-GMP-saturated synthase macrocomplex in two different views. IM inner membrane. b Cartoon representation of the same assembly, summary of the BcsA interactions with the cytosolic vestibule partners and stimulatory effects of BcsR overexpression as detected by binding and UV-fluorescence of E. coli macrocolonies grown on Congo Red-supplemented plates. c A zoom-in on the BcsA-BcsR interface with key residues shown as sticks. The R-D-R triad is indicated with a yellow arrowhead. d c-di-GMP coordination, together with its corresponding electron density, and overall core synthase fold showing unstructured gating loop and an accessible active site. e Effects on cellulose secretion upon BcsR^NTD mutagenesis using plasmid-based complementation with various BcsR mutants. KDDA D²¹K-L²⁵D-F²⁹D-L³¹D, ADDDA D²¹A-L²⁵D-F²⁹D-L³¹D-Y³⁶A. CR Congo Red, CF calcofluor. Data representative of three independent experiments with two biological replicates each. f Consensus ColabFold structural models of Type II BcsA-BcsR (based on multiple BcsA homologs encoded by bcsR- and bcsEF-positive enterobacterial bcs clusters), Type III BcsA (derived from bcsK-positive bcs clusters) and Type I and hybrid BcsA-BcsP^NTD (derived from bcsPDQ-positive bcs clusters). BcsA is shown as Coulombic electrostatic potential-colored surface, and BcsR (magenta) and BcsP^NTD (cyan) are shown in cartoon. The stabilizing pairs of hydrophobic residues in BcsR and BcsP^NTD are shown as sticks.

Fig. 5. The synthase macrocomplex in limiting c-di-GMP.

a Locally refined cryo-EM map and fitted structure of the crownless synthase macrocomplex featuring a c-di-GMP-free synthase in two different views. IM inner membrane. b Cartoon representation of the same assembly. c The cryo-EM map and model of a locally refined BcsRQEF assembly. d A zoom-in on c-di-GMP binding by a composite, dual I-site pocket in closed BcsE. e REC* domain dimerization interface in the non-saturated macrocomplex. f A zoom-in on the BcsA:BcsR interface and summary of the synthase’s interactions with the cytosolic vestibule partners. g Overall core synthase fold showing unstructured gating loop and an accessible active site.

Fig. 6. Synthase activation and polymer modifications in β- and γ-Proteobacteria.

In addition to direct c-di-GMP complexation at micromolar dinucleotide concentrations, BcsA can be activated or stabilized in a catalytically competent conformation by a high-affinity c-di-GMP-sensing BcsRQEF cytosolic vestibule complex or by macromolecular intracellular scaffolds. In the periplasm, the polymer can undergo chemical modifications by the pEtN-transferase BcsG or by a multicomponent Wss cellulose acetylation complex. Finally, the polymer can undergo limited hydrolysis by the periplasmic endoglucanase BcsZ. OM outer membrane, PG peptidoglycan, IM inner membrane.