Insights into phosphoethanolamine cellulose synthesis and secretion across the Gram-negative cell envelope

Abstract

Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the catalytically active BcsA enzyme, a hexameric semicircle of the periplasmic BcsB protein, and the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a periplasmic cellulase of unknown biological function. While cellulose synthesis and translocation by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three BcsG copies near the nascent cellulose polymer. Further, the semicircle's terminal BcsB subunit tethers the N-terminus of a single BcsC protein in a trans-envelope secretion system. BcsC's TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of translocation incompetent cellulose. Lastly, protein engineering introduces cellulose pEtN modification in orthogonal cellulose biosynthetic systems. These findings advance our understanding of pEtN cellulose modification and secretion.

Fig. 1. BcsA coordinates a BcsG trimer.

a Schematic representation of the pEtN transfer reaction catalyzed by BcsG. PE lipid Phosphatidylethanolamine, Ser Ser278 as catalytic nucleophile, pEtN Phosphoethanolamine. b Cartoon illustration of the Ec cellulose synthase complex. The putative cellulose secretion path is shown as a dashed line. c Low-resolution cryo-EM map of the Ec inner membrane-associated cellulose synthase complex (IMC, EMD-23267). d AlphaFold2-predicted structure of Ec BcsG (AF-P37659-F1) with close-up showing the acidic cavity extending from the putative membrane surface. e Cryo-EM composite map of the IMC after focused refinements of the periplasmic BcsB hexamer and the BcsG trimer associated with BcsA, respectively. BcsB subunits are colored from light gray to pink, BcsA and trimeric BcsG are colored in shades of blue and yellow respectively, and the associated BcsB transmembrane (TM) helix is colored gray. Contour level: 5σ. f AlphaFold2-predicted complex of BcsA, BcsB’s TM anchor, and the trimeric BcsG colored as in (e). g Co-purification of Ec BcsG (His-tagged) with the N-terminal domain of Ec BcsA (NTD, Strep-tagged) by immobilized metal (IMAC) and Strep-Tactin affinity chromatography followed by size exclusion chromatography (SEC). Source data are provided as a Source Data File.

Fig. 2. Engineering pEtN cellulose biosynthesis.

a Illustration of the Rs-Ec (R/E) BcsA chimera shown as a light and dark gray surface for BcsA and BcsB, respectively (PDB: 4P00). The introduced Ec N- and C-terminal domains are shown as cartoons colored blue and red, respectively. b Size exclusion chromatography of the chimeric BcsAB-BcsG complex. Inset: Coomassie-stained SDS-PAGE of the peak fraction. c In vitro catalytic activity of the purified chimeric BcsAB-BcsG complex. DPM disintegrations per minute. d Representative 2D class averages of the chimeric BcsAB-BcsG complex. e Low-resolution cryo-EM map (semitransparent surface, contoured at 4.8σ) of the chimeric BcsAB-BcsG complex overlaid with the refined map. Insets show a carved map of the chimeric BcsA NTD-BcsG complex as well as a focused refinement of BcsAB, respectively. Ec BcsA NTD is colored blue and trimeric BcsG is colored in shades of yellow. f Solid state NMR spectrum of pEtN cellulose produced by the chimeric BcsAB-BcsG complex in vivo (R/E-FG; black line), overlaid with a reference spectrum of pure pEtN cellulose from Ec (dashed red line). Peaks labeled C1-8 correspond to the chemical shifts expected for the indicated carbon atoms (see inset). The inset represents the chemical structure of a pEtN modified cellobiose unit with carbon atoms numbered. g Western blots of IMVs containing the wild type (WT) Rs BcsAB complex alone or the WT or chimeric BcsAB complex (R/E) co-expressed with BcsF and BcsG (FG). BcsA and BcsG are His- and Flag-tagged, respectively. * Indicates an N-terminal degradation product of the chimeric BcsA. h Polysaccharide analysis by carbohydrate gel electrophoresis (PACE) of in vitro synthesized pEtN cellulose. IMVs shown in (g) were used for in vitro synthesis reactions. Cellulase (BcsZ) released and Alexa Fluor 647-labeled cello-oligosaccharides are resolved by PACE. Cello-oligosaccharide standards are ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid) labeled and range from mono (Glc) to hexasaccharides (CE2-6). This experiment has been repeated twice with similar results. i Quantification of cellulose biosynthesis by the IMVs shown in (g) containing the indicated BcsA variants and based on incorporation of ³H-glucose into the water-insoluble polymer. Error bars in (c) and (i) represent standard deviations from the means of three replicas. Source data are provided as a Source Data File.

Fig. 3. Interactions of BcsC with BcsB.

a Low-resolution map of the Bcs complex in the presence of BcsC’s periplasmic domain. The terminal BcsB subunit is colored light pink and BcsC is colored green and light blue for TPR#1 and #2, respectively. TPR tetratricopeptide repeat. The map is contoured at 1.2σ. Inset: High-resolution map of the BcsB-BcsC complex obtained after fusing BcsC’s TPRs#1–4 to the N-terminus of BcsB and focused refinement of the BcsB subunit bound to BcsC (contoured at 4.8σ). Density likely representing BcsG’s periplasmic domain (PPD) is encircled. b Detailed interactions of BcsB and BcsC. FD flavodoxin-like domain and CBD carbohydrate-binding domain.

Fig. 4. Interactions of BcsC with cellulose.

a Low-resolution cryo-EM maps of BcsC. Top panel: BcsC in the absence of cellotetraose with the full-length AlphaFold2-BcsC model (AF-P37650-F1) docked into the density. TPR#1 and #2 are colored green and lightblue, respectively, TPR#15–19 are colored as in (b) and (c) (TPR tetratricopeptide repeat). Bottom panel: BcsC in the presence of cellotetratose. The model of the refined C-terminal BcsC fragment is docked into the density with the putative ligand colored blue. b Close-up views of the putative cellulose binding sites of the refined maps in the absence (apo) and the presence of cellotetraose (shown as sticks colored cyan and red). Both maps are contoured at 10.5σ. c Surface representation of BcsC bound to the putative cellotetraose ligand.

Fig. 5. Cellulase activity is necessary for cellulose secretion.

a Congo red (CR) fluorescence image of Ec macrocolonies expressing the indicating components as part of the Bcs complex. ‘All Bcs’ express the inner membrane complex (IMC) together with BcsZ and BcsC. ΔBcsZ: no BcsZ. b Carboxymethylcellulose digestion on agar plates using periplasmic Ec extracts. Cel9M and CMCax: Periplasmic extracts of cells expressing all Bcs components with BcsZ replaced by the indicated enzyme. BSA and A. niger: Controls with purified BSA or Aspergillus niger cellulase spotted on the agar plates. Cellulose digestion was imaged after CR staining, resulting in the observed plaques. c Evaluation of CR fluorescence exhibited by Ec macrocolonies expressing the indicated components as part of the Bcs complex. d Representative 2D class averages of BcsZ tetramers. e Model of the BcsZ tetramer shown as a semitransparent surface and cartoon, overlaid with the cellopentaose-bound crystal structure (PDB: 3QXQ, only cellopentaose is shown as ball-and-sticks in cyan and red). Two-fold symmetry axes are indicated by black ellipses. f Detailed views of the boxed regions in (e). CR and cellulase plate assays were repeated at least three times with similar results. Source data are provided as a Source Data File.

Fig. 6. Model of cellulose pEtN modification and secretion.

BcsA recruits three copies of BcsG to the cellulose biosynthesis site via its N-terminal cytosolic domain. The catalytic domain of BcsG either faces the lipid bilayer to receive a pEtN group or contacts the nascent cellulose chain for modification. BcsC interacts with the terminal BcsB subunit of the semicircle to establish an envelope-spanning complex. Cellulose is guided towards the OM through interactions with the TPR solenoid. BcsZ may degrade cellulose to prevent stalling of the biosynthetic machinery or mislocalization of cellulose to the periplasm. The cytosolic BcsE and BcsQR as well as BcsF components are omitted for clarity. PE phosphatidylethanolamine lipid, DAG diacylglycerol, pEtN phosphoethanolamine, IM, and OM inner and outer membrane, respectively.