Abstract
Cellulose is frequently found in communities of sessile bacteria called biofilms. Escherichia coli and other enterobacteriaceae modify cellulose with phosphoethanolamine (pEtN) to promote host tissue adhesion. The E. coli pEtN cellulose biosynthesis machinery contains the catalytic BcsA–B complex that synthesizes and secretes cellulose, in addition to five other subunits. The membrane-anchored periplasmic BcsG subunit catalyzes pEtN modification. Here we present the structure of the roughly 1 MDa E. coli Bcs complex, consisting of one BcsA enzyme associated with six copies of BcsB, determined by single-particle cryo-electron microscopy. BcsB homo-oligomerizes primarily through interactions between its carbohydrate-binding domains as well as intermolecular beta-sheet formation. The BcsB hexamer creates a half spiral whose open side accommodates two BcsG subunits, directly adjacent to BcsA’s periplasmic channel exit. The cytosolic BcsE and BcsQ subunits associate with BcsA’s regulatory PilZ domain. The macrocomplex is a fascinating example of cellulose synthase specification.
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Data availability
Coordinates and EM maps have been deposited at the Protein Data Bank (PDB) under accession codes 7L2Z and 7LBY and the Electron Microscopy Data Bank (EMDB) under accession codes EMD-23146 and EMD-23267. Unaligned video frames are available at EMPIAR under accession codes 10627 and 10626. Source data are provided with this paper.
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Acknowledgements
We thank K. Dryden and M. Purdy from the University of Virginia Molecular Electron Microscopy Core (MEMC) facility for help with data collection. MEMC is funded by a National Institutes of Health (NIH) Recovery grant 1G20RR31199. The MEMC Titan Krios and Gatan K3/GIF detector were funded through NIH grant nos. SIG S10-RR025067 and U24-GM116790, respectively. We thank N. Sherman and the University of Virginia Biomolecular Analysis Center for mass spectrometry analysis and G. Skiniotis for discussions. J.F.A. was supported in part by NIH grant no. 1F32GM126647-01. R.H. was supported by the Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Basic Energy Sciences (award no. DESC0001090). J.Z. is supported by NIH grant no. 5R01GM101001. L.C. acknowledges support from the National Science Foundation (NSF) award no. 2001189. N.F.G. is a recipient of the NSF Graduate Research Fellowship.
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J.F.A. and J.Z. designed the experiments. J.F.A. cloned, expressed and purified the IMC. R.H. and J.F.A. screened and optimized cryo grid preparation. R.H. collected the data. J.F.A. processed all data. J.Z. and J.F.A. built the atomic models. N.F.G. and L.C. performed the ssNMR analysis. All authors edited the manuscript.
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Peer review information Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended data
Extended Data Fig. 1 Cartoon representations of Bcs subunits.
Predicted TM topologies and secondary structures of the E. coli cellulose synthase subunits.
Extended Data Fig. 2 Expression, purification and characterization of the Ec IMC.
a, Clumping cells upon expression of the recombinant Bcs genes with AdrA in liquid cultures. Cells without BcsC remain planktonic. b, Cultures in (a) were grown on Congo red agar plates and photographed on a Fisher Scientific UV lightbox. c, Overlay of the ssNMR spectrum shown in Fig. 1 with the pEtN standard. d, Gel filtration purification of Ec IMC, 256 and 280 nm absorbances are shown in red and blue, respectively. Inset: SDS-PAGE of the peak fractions. e, Catalytic activity determined by UDP-Glc[13H] incorporation as described24. The synthesized polymer is sensitive to cellulase (BcsZ) degradation. DPM: Disintegrations per minute. f, Western blot analysis of the purified Ec IMC. Right panel: Coomassie-stained SDS-PAGE of the purified complex. Percentages indicate peptide covered in MS-sequencing based on the identified unique peptides (also shown). R/NR: Reducing and non-reducing conditions; * denotes a BcsG proteolytic fragment. g, Identification of Myc-tagged BcsF by Western blotting in the purified complex (left Coomassie-stained SDS-PAGE; right: anti-Myc Western blot). h, i, Kinetic analysis of the purified detergent-solubilized samples. UDP generation was quantified in real time using an enzyme-coupled assay that monitors the oxidation of NADH, as described24. An uncropped image of panel (f) is available as source data.
Extended Data Fig. 3 Bcs catalytic activity and complex formation of MBP-BcsE and BcsQ-R.
a, Time course of cellulose biosynthesis from IMVs containing BcsA-B-F-G. b, The BcsQ-R complex, MBP-BcsE, and the BcsQ-R-MBP-BcsE complex were analyzed by S200 size exclusion chromatography. The samples elute at the volumes indicated above the main peaks. Inset: Coomassie-stained SDS-PAGE of all samples (1: BcsQ-R-MBP-BcsE, 2: MBP-BcsE, 3: BcsQ-R).
Extended Data Fig. 4 Examples of BcsB density.
a-d, Map quality is shown for each of the BcsB domains of Chain B including β-strands, loops, and α-helices. Map is contoured to σ=2.0, and carve=2.0. e, Disulfide bond between CBD-1 and CBD-2, σ=1.5 and carve=2.0. f, Oligomerization β-strands and loops between FD domains of chain A and chain B, σ=1.5 and carve=2.0. Maps were converted to CCP4 format with Phenix, and figures were created in Pymol.
Extended Data Fig. 5 Comparison of BcsB from R. sphaeroides and E. coli.
The OL region forms an amphipathic interface helix at the periplasmic water/lipid interface in R. sphaeroides BcsB (PDB: 4P00). In E. coli BcsB, the OL is helical (residues 622-629) followed by a poorly order stretch (residues 631-658) in chain A. This region forms the interface with neighboring subunits in all other chains. Conserved cysteines covalently linking the CBDs are shown as spheres. The BcsB TM anchor is indicated as a cylinder.
Extended Data Fig. 6 Comparison of BcsB sequences.
BcsB sequences from E. coli, K. xylinus, and R. sphaeroides were aligned in MUSCLE38 and displayed in Jalview39 colored based on sequence conservation. Secondary structure elements observed in the Ec and Rs BcsB structures are indicated as bars and arrows for helices and β-strands, respectively. The Kx BcsB secondary structure of the OL was predicted in Jpred40 (colored magenta). Residues mediating intermolecular contacts in the Ec BcsB hexamer are indicated by yellow circles.
Extended Data Fig. 7 The Ec BcsA poly-alanine model.
The Ec BcsA poly-Ala model was generated based on Rs BcsA (PDB: 4P00) and manually docked into the cryo-EM density. TM helices were adjusted individually by rigid body docking in Coot. The GT and PilZ domains were only corrected for missing or additional residues and not adjusted due to insufficient resolution. Side chains of residues used to guide the register assignment are included in the model. The map is contoured at 7.5σ.
Extended Data Fig. 8 Comparison of Rs and Ec BcsA.
a, Rs BcsA (PDB: 4P00) was superimposed with the Ec BcsA model by secondary structure matching. Regions not present in Rs BcsA are shown in dark blue. b, Comparison of the Rs and Ec BcsA-B complexes. BcsB is colored green.
Extended Data Fig. 9 Putative BcsG density.
Overall Bcs cryo-EM map contoured at the indicated levels. Additional density is visible for BcsG at low contour levels. Bottom right: The map contoured at 0.8σ was Gaussian filtered at a width of 2.6 in Chimera. Two copies of Ec BcsG (PDB: 6PCZ) were manually docked into the putative BcsG density and colored blue to red from the N- to the C-terminus. Helices representing the helical cluster next to BcsA are shown as cartoons in black and magenta. The first and last BcsB subunits are shown as spheres in green and steel-blue, respectively.
Extended Data Fig. 10 The cytosolic Bcs components.
a, 2D class averages of Bcs particles lacking BcsE and BcsQ-R and (b) corresponding 3D volume (blue surface) superimposed with the BcsE-Q-R-containing macrocomplex (mesh) revealing only cytosolic density corresponding to BcsA. c, Localization of MBP-fused BcsE in the IMC. Volumes are contoured at 0.7σ and Gaussian filtered at a width of 5.2 in Chimera. Densities assigned to BcsQ-R and BcsE are colored orange and magenta, respectively, for the MBP-BcsE containing complex. d, Available structures of Bcs components manually docked into their corresponding densities. e, Domain organization of BcsE with corresponding functions. Adapted from reference (18). f, SDS-PAGE and western blot analyses of the MBP-BcsE containing IMC. The gels were run under reducing conditions in which BcsB co-migrates with BcsA. (1: IMC, 2: MBP-BcsE control; a, b, c: MBP-BcsE, reduced BcsA/BcsB, BcsG, respectively). (*) indicates non-specific antibody binding to the strong BcsA/B band. An uncropped image of panel (f) is available as source data.
Supplementary information
Supplementary Information
Supplementary Methods, Figs. 1 and 2 and Table 1.
Source data
Source Data Fig 1
Unprocessed western blot and SDS–PAGE gel.
Source Data Fig 1
Catalytic activity measurements.
Source Data Fig 2
Unprocessed gel.
Source Data Extended Data Fig. 2
Unprocessed western blots.
Source Data Extended Data Fig. 10
Unprocessed western blots.
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Acheson, J.F., Ho, R., Goularte, N.F. et al. Molecular organization of the E. coli cellulose synthase macrocomplex. Nat Struct Mol Biol 28, 310–318 (2021). https://doi.org/10.1038/s41594-021-00569-7
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DOI: https://doi.org/10.1038/s41594-021-00569-7
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