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We thank D. Cazalla and E. Cao for critique of the manuscript
and P. J. Aruscavage for technical assistance. EM was
performed at University of Utah EM Core Laboratory, with
computational support from Utah Center for High Performance
Computing. RNA was synthesized by the DNA/Peptide
facility (Health Sciences Center Cores at University of Utah).
This work was supported by funding from the National
Institute of General Medical Sciences (R01GM121706) and
the H. A. and Edna Benning Presidential Endowed Chair (to
B.L.B.). The authors declare no competing financial interests.
The models and cryo-EM maps are available via the
following accession numbers: Protein Data Bank (PDB)
6BUA, Electron Microscopy Data Bank (EMDB) EMD-7291,
EMD-7292 (apo-dmDcr-2RIII); PDB 6BU9, EMD-7290
(dmDcr-2RIII•52 BLT dsRNA•ATP-gS complex).
Materials and Methods
Figs. S1 to S16
Tables S1 to S3
29 September 2017; accepted 11 December 2017
Published online 21 December 2017
A naturally produced chemically
Wiriya Thongsomboon,1 Diego O. Serra,2 Alexandra Possling,2 Chris Hadjineophytou,2,3*
Regine Hengge,2† Lynette Cegelski1†
Cellulose is a major contributor to the chemical and mechanical properties of plants and
assumes structural roles in bacterial communities termed biofilms. We find that Escherichia
coli produces chemically modified cellulose that is required for extracellular matrix assembly
and biofilm architecture. Solid-state nuclear magnetic resonance spectroscopy of the intact
and insoluble material elucidates the zwitterionic phosphoethanolamine modification that had
evaded detection by conventional methods. Installation of the phosphoethanolamine group
requires BcsG, a proposed phosphoethanolamine transferase, with biofilm-promoting cyclic
diguanylate monophosphate input through a BcsE-BcsF-BcsG transmembrane signaling
pathway. The bcsEFG operon is present in many bacteria, including Salmonella species, that
also produce the modified cellulose. The discovery of phosphoethanolamine cellulose and
the genetic and molecular basis for its production offers opportunities to modulate its
production in bacteria and inspires efforts to biosynthetically engineer alternatively modified
Cellulose is the most abundant biopolymer on Earth. Plants rely on the tensile strength and mechanical properties of cellulose to stand upright (1). Chemically, cellulose is a linear polysaccharide composed of b-1,4–
linked glucosyl residues. Individual strands par-
ticipate in strong hydrogen-bonding networks with
neighboring strands and contribute to the physical
and chemical integrity of plant cell walls and
cellulosic materials (2). Microorganisms are also
major producers of cellulose (3). The essential
genetic and protein machinery for cellulose pro-
duction in bacteria include the cellulose synthase
genes, termed bcsA and bcsB, which encode
cellulose synthase subunits BcsA and BcsB (4).
BcsA is an integral membrane protein containing
the catalytic active site. BcsB interacts with BcsA
at the periplasmic face of the inner membrane
in Gram-negative bacteria, with the two subunits
forming a channel for cosynthetic secretion of
cellulose. Cellulose biosynthesis requires activa-
tion by the ubiquitous bacterial second messenger
cyclic diguanylate monophosphate (c-di-GMP) (5),
which directly binds to BcsA (6). Intense curiosity
has emerged in understanding the diversity of
additional genes in cellulose biosynthesis operons
that are present in many microorganisms (3). Here
we report on the determination of the structure
of a modified cellulose, phosphoethanolamine
(pEtN) cellulose, produced naturally by Escherichia
coli and other Gram-negative bacteria. We provide
the genetic basis for its production and the
functional implications of gene-directed pEtN
E. coli and Salmonella are among the best-studied microorganisms reported to produce cellulose. These include human pathogens such as
uropathogenic and enterohemorrhagic E. coli.
Functionally, the exopolysaccharide cellulose is a
major component of the self-produced extracellular
matrix in biofilms, which represent physiologically heterogeneous and spatially structured bacterial communities (7, 8). Biofilm formation is of
high medical relevance, as it confers enhanced
resistance to antibiotics and host defenses during
infection (9). Within the biofilm matrix, cellulose
forms a nanocomposite with amyloid curli fibers
that encapsulates individual cells in supramolecular
basketlike structures, enmeshes the bacterial community, and confers cohesion and elasticity that
allow biofilms to fold and buckle up in a tissuelike
manner (10–12). Biochemical and solid-state nuclear
magnetic resonance (NMR) measurements with
the clinically important uropathogenic E. coli
strain UTI89 established that the matrix was
composed of curli fibers and cellulosic material
in a 6:1 ratio by mass. During this bottom-up
analysis involving 13C and 15N NMR analysis of
the purified components, we also discovered that
the cellulose portion appears to be modified in
some way with an aminoethyl functionality (11).
Solid-state NMR analysis of the intact cellulosic material, complemented by solution-state
NMR and mass spectrometry analysis of acid-digested material, has now enabled the determination of the chemical structure of the modified
334 19 JANUARY 2018 • VOL 359 ISSUE 6373 sciencemag.org SCIENCE
1Department of Chemistry, Stanford University, Stanford, CA
94305, USA. 2Institute of Biology, Microbiology, Humboldt–
Universität zu Berlin, 10115 Berlin, Germany. 3Department of
Chemistry and Molecular Biology, University of Gothenburg,
41296 Gothenburg, Sweden.
*Present address: Department of Biosciences, University of Oslo, 0371
†Corresponding author. Email: firstname.lastname@example.org (L.C.);