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Structure of the Bacterial Cellulose Ribbon and Its Assembly-
Guiding Cytoskeleton by Electron Cryotomography
William J. Nicolas
,
a,b
Debnath Ghosal
,
a
*
Elitza I. Tocheva
,
a
*
Elliot M. Meyerowitz
,
a,b
Grant J. Jensen
a,b
a
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA
b
Howard Hughes Medical Institute, Pasadena, California, USA
ABSTRACT
Cellulose is a widespread component of bacterial bio
fi
lms, where its
properties of exceptional water retention, high tensile strength, and stiffness pre-
vent dehydration and mechanical disruption of the bio
fi
lm. Bacteria in the genus
Gluconacetobacter
secrete crystalline cellulose, with a structure very similar to that
found in plant cell walls. How this higher-order structure is produced is poorly
understood. We used cryo-electron tomography and focused-ion-beam milling of
native bacterial bio
fi
lms to image cellulose-synthesizing
Gluconacetobacter hanse-
nii
and
Gluconacetobacter xylinus
bacteria in a frozen-hydrated, near-native state.
We con
fi
rm previous results suggesting that cellulose crystallization occurs serially
following its secretion along one side of the cell, leading to a cellulose ribbon
that can reach several micrometers in length and combine with ribbons from
other cells to form a robust bio
fi
lmmatrix.Wewereabletotakedirectmeasure-
ments in a near-native state of the cellulose sheets. Our results also reveal a novel
cytoskeletal structure, which we have named the cortical belt, adjacent to the
inner membrane and underlying the sites where cellulose is seen emerging from
the cell. We found that this structure is not present in other cellulose-synthesizing
bacterial species,
Agrobacterium tumefaciens
and
Escherichia coli
1094, which do
not produce organized cellulose ribbons. We therefore propose that the cortical
belt holds the cellulose synthase complexes in a line to form higher-order cellu-
lose structures, such as sheets and ribbons.
IMPORTANCE
This work
s relevance for the microbiology community is twofold. It
delivers for the
fi
rst time high-resolution near-native snapshots of
Gluconacetobacter
spp. (previously
Komagataeibacter
spp.) in the process of cellulose ribbon synthesis,
in their native bio
fi
lm environment. It puts forward a noncharacterized cytoskeleton
element associated with the side of the cell where the cellulose synthesis occurs.
This represents a step forward in the understanding of the cell-guided process of
crystalline cellulose synthesis, studied speci
fi
cally in the
Gluconacetobacter
genus and
still not fully understood. Additionally, our successful attempt to use cryo-focused-
ion-beam milling through bio
fi
lms to image the cells in their native environment will
drive the community to use this tool for the morphological characterization of other
studied bio
fi
lms.
KEYWORDS
cellulose,
Gluconacetobacter
, electron cryotomography
H
umans rely on cellulose for building material, clothing, and fuel (1
3). Recently,
the polymer has sparked interest in the biotechnology
fi
eld as a potential source
of biofuel feedstock (4) and in the biomedical industry as a biologically neutral scaffold
to promote tissue regeneration (5, 6). Cellulose is a linear polymer of glucose molecules
connected with
b
-1,4 linkages by a glucosyltransferase. Individual linear glucan chains
can pack via hydrogen bonding and van der Waals interactions in various ways to form
different types of celluloses, with different properties (3, 7, 8). The most common way
Citation
Nicolas WJ, Ghosal D, Tocheva EI,
Meyerowitz EM, Jensen GJ. 2021. Structure of
the bacterial cellulose ribbon and its assembly-
guiding cytoskeleton by electron
cryotomography. J Bacteriol 203:e00371-20.
https://doi.org/10.1128/JB.00371-20.
Editor
Yves V. Brun, Université de Montréal
Copyright
© 2021 Nicolas et al. This is an
open-access article distributed under the terms
of the
Creative Commons Attribution 4.0
International license.
Address correspondence to Elliot M.
Meyerowitz, meyerow@caltech.edu, or Grant J.
Jensen, jensen@caltech.edu.
*
Present address: Debnath Ghosal, Division of
Medicine, Dentistry and Health Sciences,
University of Melbourne, Parkville, Victoria,
Australia; Elitza I. Tocheva, Department of
Microbiology and Immunology, University of
British Colombia, Vancouver, British Columbia,
Canada.
Received
29 June 2020
Accepted
26 October 2020
Accepted manuscript posted online
16
November 2020
Published
11 January 2021
February 2021 Volume 203 Issue 3 e00371-20
Journal of Bacteriology
jb.asm.org
1
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glucan chains organize in nature is to form hydrogen-bonded planes stacked into par-
allel layers via van der Waals interactions (9, 10). These stacked layers give rise to cellu-
lose I micro
fi
brils, or native cellulose, that can then coalesce to form larger arrays.
Because glucan chains pack in a regular lattice but cannot sustain this regular pattern
over their entire length, cellulose I is considered paracrystalline. Depending on how
the lattice is organized, cellulose I can be of the
a
form, bearing a triclinic unit cell, or
b
form, bearing a monoclinic unit cell (11, 12). Cellulose I
b
is mainly found in plants,
where it is a major structural element of the cell wall (13).
In the prokaryotic world, cellulose is an important component of bacterial bio
fi
lms
(14, 15), which increase cells
tolerance for a range of biotic and abiotic stresses and
enhance surface adhesion, cell cooperation, and resource capture (14). Cellulose-contain-
ing bio
fi
lms have also been shown to be involved in pathogenicity, enabling bacteria to
resist antibiotics and disinfection (16, 17). Most cellulose-synthesizing bacteria produce
amorphous (noncrystalline) cellulose, but a few genera, including
Gluconacetobacter
,can
produce cellulose I
a
micro
fi
brils. In
Gluconacetobacter
, these paracrystalline cellulose
micro
fi
brils can further aggregate into wide ribbon structures and larger arrays (18), giv-
ing rise to thick bio
fi
lms that are predominantly pure cellulose I.
Bacterial cellulose is synthesized by an envelope-spanning machinery called the
bacterial cellulose synthase (BCS) complex, encoded by the BCS gene cluster and
fi
rst
identi
fi
ed in
Gluconacetobacter
(15). While the components vary, most of the species
encode BcsA, a component in the inner membrane that, with BcsB, catalyzes transfer
of UDP-glucose to the nascent glucan chain (15, 19, 20). BcsD forms a periplasmic ring
thought to gather glucan chains from several BcsA/B units (21, 22). BcsA and BcsB are
essential for cellulose synthesis
in vivo
, and BcsD is essential for the crystallization of
nascent glucan chains (23). BcsC forms a pore in the outer membrane (OM), and very
recent work has solved its crystallographic structure (24). Consistent with previous
data relying on sequence homology with the exopolysaccharide secretin components
AlgE and AlgK from
Pseudomonas aeruginosa
, BcsC is found to form an outer-mem-
brane
b
-barrel pore at its C-terminal end, secreting the nascent elementary cellulose
fi
brils into the environment (23
27). It is hypothesized that the elementary cellulose
fi
brils can aggregate with neighboring elementary
fi
brils upon secretion to form
micro
fi
brils (28, 29). Genes
fl
anking the operon,
cmcAx
(endo-
b
-1,4-glucanase),
ccpAx
(unknown function), and
bglxA
(
b
-glucosidase), are essential for cellulose
crystallization, and despite knowledge of their enzymatic functions, how they take
part in this process is unclear (29
32).
In this report, the terms used to describe the cellulose assembly process are
adapted from the ones de
fi
ned in reference 29, elaborating on the cell-directed hier-
archical model for cellulose crystallization (7, 10). Glucan chains are linear polymers of
b
-1,4-linked glucose residues synthesized by a single catalytic site of a cellulose syn-
thase. An elementary
fi
bril (also termed a minicrystal in previous work [10, 33, 34]) is
the product of the periplasmic aggregation of multiple glucan chains which is then
extruded through a single BcsC subunit into the environment. Micro
fi
brils result from
the aggregation of several elementary
fi
brils, at least three according to earlier work
(34), outside the cell. These micro
fi
brils can then crystallize into sheets that stack on
each other to form ribbons. The latter terminology differs somewhat from previous
usage in that our de
fi
nition of a sheet is equivalent to the
bundles of micro
fi
brils,
the
polymerization step prior to the ribbon, described in reference 29.
Much work has already been done to understand the synthesis of paracrystalline
cellulose (18, 20, 21, 23, 30
33, 35
41). In particular, freeze fracture/freeze-etching elec-
tron microscopy (EM) studies have found that the
Gluconacetobacter hansenii
BCS com-
plexes are arrayed linearly along the side of the cell (18, 33, 38, 39), and this arrange-
ment seems to determine the extracellular organization of cellulose I into ribbons (18,
33, 39). How this linear arrangement is achieved is not known.
Here, we used cryo-electron tomography (cryo-ET) of isolated cells and cryo-
focused-ion-beam (cryo-FIB) milling of bio
fi
lms to visualize native cellulose production
Nicolas et al.
Journal of Bacteriology
February 2021 Volume 203 Issue 3 e00371-20
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