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Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose

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Science  19 Jan 2018:
Vol. 359, Issue 6373, pp. 334-338
DOI: 10.1126/science.aao4096
  • Fig. 1 E. coli produces phosphoethanolamine cellulose.

    (A) Representation of the chemical structure of glucose and pEtN glucose units in pEtN cellulose. (B) 13C CPMAS solid-state NMR spectra of the pure modified cellulose with two additional carbon contributions, C-7 (63 ppm) and C-8 (41 ppm). The C-6 carbon appears at 62 ppm for the unmodified glucose units and at 66 ppm for the modified glucose units (figs. S1 and S4). (C) 13C CPMAS spectra of the modified cellulose compared with that of the modified cellulose isolated from cells grown in the presence of CR. The pure CR spectrum is provided as an overlay (dashed red line). The comparison demonstrates that purification with CR does not influence the polysaccharide composition. δC, carbon chemical shift. (D) The C-6ʹ and C-7 carbon chemical-shift region exhibited the strongest dephasing in the 1-ms C{P} REDOR NMR measurement, followed by that of the C-5′ and C-8 carbons, suggesting the full structural assignment as pEtN cellulose, further confirmed by solution-state NMR and mass spectrometry (figs. S4 to S8). S0, REDOR full-echo spectrum; ΔS, REDOR difference spectrum. (E) The 13C CPMAS spectrum of the cellulosic material isolated from the bcsG derivative lacked modification carbons and contained only the 13C chemical shifts expected for standard amorphous cellulose.

  • Fig. 2 BcsG directly interacts with cellulose synthase and communicates with the c-di-GMP–binding BcsE via the transmembrane peptide BcsF.

    (A) Interactions of the indicated proteins were tested using a bacterial two-hybrid (2H) system based on the reconstitution of adenylate cyclase (AC) (18), which allows the utilization of maltose by W3110Δcya, resulting in red color on MacConkey agar plate. The 2H vector plasmids allow the attachment of the respective AC domain tags (18, 25), either at the N terminus (pKT25, pUT18c) or the C terminus (pKNT25, pUT18) of a protein. For BcsA, BcsB, BcsE, and BcsF, the tags were located at the C terminus; for BcsG, the tags were located at the N terminus. Zip-zip, leucine zipper domain of the yeast GCN4 protein, used as a positive control. (B) Transmembrane orientation of BcsF and BcsG was determined by assaying enzymatic activities of hybrid proteins between N-terminal parts from BcsF and BcsG fused to LacZ and PhoA expressed from low–copy number plasmids in strains W3110∆lac(I-A) and W3110∆phoA. Fusion joints were after codon 1 (all combinations), codon 24 (of bcsF fused to both reporter genes), codon 162 (bcsG::lacZ), or codon 158 (bcsG::phoA). (C) A schematic model of the directly interacting modules for cellulose synthesis (BcsAB) and modification (BcsEFG) summarizes the protein-protein interactions (double-headed arrows) detected in (A), the transmembrane orientation of BcsG and BcsF as tested in (B), and dual control by the second messenger c-di-GMP, which binds to both BcsA (6) and BcsE (17). NTD, N-terminal domain; CTD, C-terminal domain; UDP-Glc, uridine diphosphate–glucose.

  • Fig. 3 Phosphoethanolamine cellulose production is detected in curli-integrated E. coli biofilm matrices with isotopic serine labeling and is also produced by Salmonella enterica.

    (A) Isotopic labeling with l-[3-13C]Ser–supplemented YESCA nutrient medium resulted in enrichment of the pEtN cellulose C-7 carbon in an isolated pEtN sample, consistent with routing through a possible substrate such as phosphatidylethanolamine. (B) Isotopic labeling with l-[15N]Ser was evaluated by 15N CPMAS NMR on extracellular matrix samples containing both curli and cellulosic material. The 15N-amide signals correspond to curli amides. The loss of the 15N-amine signal in the bcsG derivative (right) confirmed the amine nitrogen assignment as that from pEtN cellulose. Loss of the modification was accompanied by loss of the wrinkled macrocolony morphology (inset photographs). (C) The 13C CPMAS spectrum of the cellulosic material isolated from Salmonella enterica serovar Typhimurium strain IR715ΔcsgBA matched that of pEtN cellulose from AR3110ΔcsgBA.

  • Fig. 4 Eliminating BcsG changes macroscopic morphology and microscopic matrix architecture of E. coli macrocolony biofilms.

    (A) Macrocolonies of strain AR3110, which produces both cellulose and curli fibers, and the indicated mutant derivatives (i to v) were grown for three days on salt-free LB agar plates containing either Congo red or the green-fluorescent thioflavin S, which stain cellulose and curli without affecting the overall matrix architecture and colony morphotype. The microscopic architecture of thioflavin S–stained matrix was visualized in thin cross sections of macrocolonies, with color-coded boxed areas being further enlarged adjacently. (B) The surface of macrocolonies was visualized at high resolution by scanning electron microscopy. The classical E. coli K-12 lab strain W3110 is isogenic to AR3110, except for a bcsQstop mutation, which eliminates the ability to produce cellulose (12). Because of polarity, the ΔcsgB mutation also eliminates the expression of CsgA (from the csgBA operon), i.e., both curli subunits encoded by csgBA are not produced.

Supplementary Materials

  • Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose

    Wiriya Thongsomboon, Diego O. Serra, Alexandra Possling, Chris Hadjineophytou, Regine Hengge, Lynette Cegelski

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S16
    • Table S1
    • References

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