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# Bacterial synthase-dependent exopolysaccharide secretion: a focus on cellulose

Petya V Krasteva

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Petya V Krasteva. Bacterial synthase-dependent exopolysaccharide secretion: a focus on cellulose. Current Opinion in Microbiology, 2024, 79, pp.102476. 10.1016/j.mib.2024.102476. hal-04575649

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https://hal.science/hal-04575649

Submitted on 15 May 2024

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HAL
open science

# Bacterial synthase-dependent exopolysaccharide secretion: a focus on cellulose

Petya V Krasteva

► To cite this version:

Petya V Krasteva. Bacterial synthase-dependent exopolysaccharide secretion: a focus on cellulose. Current Opinion in Microbiology, 2024, 79, pp.102476. 10.1016/j.mib.2024.102476. hal-04575649

HAL Id: hal-04575649

https://hal.science/hal-04575649v1

Submitted on 15 May 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

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Distributed under a Creative Commons Attribution 4.0 International License
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ELSEVIER

Available online at www.sciencedirect.com

ScienceDirect

Current Opinion in
Microbiology

# Bacterial synthase-dependent exopolysaccharide secretion: a focus on cellulose

Petya V. Krasteva<fnref n="1" />,<fnref n="2" />

<figure><img src="image_4.png" /></figure>

Bacterial biofilms are a prevalent multicellular life form in which individual members can undergo significant functional differentiation and are typically embedded in a complex extracellular matrix of proteinaceous fimbriae, extracellular DNA, and exopolysaccharides (EPS). Bacteria have evolved at least four major mechanisms for EPS biosynthesis, of which the synthase-dependent systems for bacterial cellulose secretion (Bcs) represent not only key biofilm determinants in a wide array of environmental and host-associated microbes, but also an important model system for the studies of processive glycan polymerization, cyclic diguanylate (c-di-GMP)-dependent synthase regulation, and biotechnological polymer applications. The secreted cellulosic chains can be decorated with additional chemical groups or can pack with various degrees of crystallinity depending on dedicated enzymatic complexes and/or cytoskeletal scaffolds. Here, I review recent progress in our understanding of synthase-dependent EPS biogenesis with a focus on common and idiosyncratic molecular mechanisms across diverse cellulose secretion systems.

## Addresses

Corresponding author: Krasteva, Petya V.

(pv.krasteva@iecb.u-bordeaux.fr)

**Current Opinion in Microbiology 2024, 79:102476**

This review comes from a themed issue on **Special section on Bacterial Nanomachines**

Edited by **Martin Thanbichler** and **Susan Schlimpert**

Available online xxxx

https://doi.org/10.1016/j.mib.2024.102476

1369-5274/© 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

Whether in abiotic environments or associated with eukaryotic hosts, bacteria typically live in close quarters as mono- or mixed-species multicellular communities

called biofilms. A key feature of the latter is spatial differentiation, where subsets of members are designated to secrete extracellular matrix components — such as extracellular DNA (eDNA), proteinaceous fimbriae (e.g. cell-surface adhesins or amyloid curli), and/or exopolysaccharides (EPS) — whereas others take charge of cell proliferation and biofilm dispersal [1–3]. EPS often provide the structural scaffold of biofilm macrocolonies and partake in redox control, biofilm hydration, nutrient exchange, surface colonization, and immune escape within the host. Bacteria have evolved a remarkable variety of systems for EPS and glycoconjugate biogenesis, which can be viewed as variations of four major mechanisms [4–7]. Apart from surface-associated sucrases, which generate dextrans or levans from hydrolyzed extracellular sucrose, the three other pathways — ATP-binding cassette (ABC) transporter-, Wzx/Wzy-, and synthase-dependent — rely on intracellular precursor polymerization and multicomponent assemblies for the export and modifications of the polymeric products [4–7].

Both ABC transporter- and Wzx (flippase)/Wzy (polymerase)-dependent systems involve the synthesis of isoprenoid- or phospholipid-linked polysaccharides or oligosaccharidic modules that are exported across the inner membrane (IM) using the energy of coupled ATP hydrolysis cycles or the countermovement of protons, respectively. ABC transporter-dependent pathways involve the biosynthesis of some lipopolysaccharide (LPS) O-antigens, isoprenoid-linked glycans for protein N-glycosylation, phosphatidylglycerol-linked capsular polysaccharides (CPS) in Gram-negative mucosal pathogens, and cell wall teichoic acids in Gram-positive bacteria [4–7]. Many O-antigen LPS are alternatively synthesized by Wzx/Wzy-like flippase-dependent systems and similar pathways ensure the biogenesis of diverse capsular and biofilm polysaccharides in both Gram-positive and Gram-negative bacteria (e.g. *Streptococcus pneumoniae* CPS, *Xanthomonas campestris* xanthan and *Xylella fastidiosa* fastidian gums, *Pseudomonas aeruginosa* Psl and *Vibrio cholerae* Vps, enterobacterial colanic acids, etc.) [4–7].

In contrast to the above, most synthase-dependent systems do not require lipid-linked initial acceptors and use highly processive polymerase/copolymerase tandems to couple linear glycan polymerization with IM translocation [4,7,8]. Examples include the alginate, poly-N-acetylglucosamine (PNAG) and Pel EPS (e.g. in *P*.

<footnote n="1">¹Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, Pessac F-33600, France</footnote>

<footnote n="2">²'Structural Biology of Biofilms' Group, European Institute of Chemistry and Biology (IECB), Pessac F-33600, France</footnote>
<figure><img src="image_3.png" /></figure>

ELSEVIER

Available online at www.sciencedirect.com

ScienceDirect

Current Opinion in
Microbiology

# Bacterial synthase-dependent exopolysaccharide secretion: a focus on cellulose

Petya V. Krasteva<fnref n="1" />,<fnref n="2" />

<figure><img src="image_4.png" /></figure>

Bacterial biofilms are a prevalent multicellular life form in which individual members can undergo significant functional differentiation and are typically embedded in a complex extracellular matrix of proteinaceous fimbriae, extracellular DNA, and exopolysaccharides (EPS). Bacteria have evolved at least four major mechanisms for EPS biosynthesis, of which the synthase-dependent systems for bacterial cellulose secretion (Bcs) represent not only key biofilm determinants in a wide array of environmental and host-associated microbes, but also an important model system for the studies of processive glycan polymerization, cyclic diguanylate (c-di-GMP)-dependent synthase regulation, and biotechnological polymer applications. The secreted cellulosic chains can be decorated with additional chemical groups or can pack with various degrees of crystallinity depending on dedicated enzymatic complexes and/or cytoskeletal scaffolds. Here, I review recent progress in our understanding of synthase-dependent EPS biogenesis with a focus on common and idiosyncratic molecular mechanisms across diverse cellulose secretion systems.

## Addresses

Corresponding author: Krasteva, Petya V.

(pv.krasteva@iecb.u-bordeaux.fr)

**Current Opinion in Microbiology 2024, 79:102476**

This review comes from a themed issue on **Special section on Bacterial Nanomachines**

Edited by **Martin Thanbichler** and **Susan Schlimpert**

Available online xxxx

https://doi.org/10.1016/j.mib.2024.102476

1369-5274/© 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

Whether in abiotic environments or associated with eukaryotic hosts, bacteria typically live in close quarters as mono- or mixed-species multicellular communities

called biofilms. A key feature of the latter is spatial differentiation, where subsets of members are designated to secrete extracellular matrix components — such as extracellular DNA (eDNA), proteinaceous fimbriae (e.g. cell-surface adhesins or amyloid curli), and/or exopolysaccharides (EPS) — whereas others take charge of cell proliferation and biofilm dispersal [1–3]. EPS often provide the structural scaffold of biofilm macrocolonies and partake in redox control, biofilm hydration, nutrient exchange, surface colonization, and immune escape within the host. Bacteria have evolved a remarkable variety of systems for EPS and glycoconjugate biogenesis, which can be viewed as variations of four major mechanisms [4–7]. Apart from surface-associated sucrases, which generate dextrans or levans from hydrolyzed extracellular sucrose, the three other pathways — ATP-binding cassette (ABC) transporter-, Wzx/Wzy-, and synthase-dependent — rely on intracellular precursor polymerization and multicomponent assemblies for the export and modifications of the polymeric products [4–7].

Both ABC transporter- and Wzx (flippase)/Wzy (polymerase)-dependent systems involve the synthesis of isoprenoid- or phospholipid-linked polysaccharides or oligosaccharidic modules that are exported across the inner membrane (IM) using the energy of coupled ATP hydrolysis cycles or the countermovement of protons, respectively. ABC transporter-dependent pathways involve the biosynthesis of some lipopolysaccharide (LPS) O-antigens, isoprenoid-linked glycans for protein N-glycosylation, phosphatidylglycerol-linked capsular polysaccharides (CPS) in Gram-negative mucosal pathogens, and cell wall teichoic acids in Gram-positive bacteria [4–7]. Many O-antigen LPS are alternatively synthesized by Wzx/Wzy-like flippase-dependent systems and similar pathways ensure the biogenesis of diverse capsular and biofilm polysaccharides in both Gram-positive and Gram-negative bacteria (e.g. *Streptococcus pneumoniae* CPS, *Xanthomonas campestris* xanthan and *Xylella fastidiosa* fastidian gums, *Pseudomonas aeruginosa* Psl and *Vibrio cholerae* Vps, enterobacterial colanic acids, etc.) [4–7].

In contrast to the above, most synthase-dependent systems do not require lipid-linked initial acceptors and use highly processive polymerase/copolymerase tandems to couple linear glycan polymerization with IM translocation [4,7,8]. Examples include the alginate, poly-N-acetylglucosamine (PNAG) and Pel EPS (e.g. in *P*.

<footnote n="1">¹Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, Pessac F-33600, France</footnote>

<footnote n="2">²‘Structural Biology of Biofilms’ Group, European Institute of Chemistry and Biology (IECB), Pessac F-33600, France</footnote>
*aeruginosa*), bacterial hyaluronic acid (e.g. in *Streptococcus pyogenes*), and cellulose secretion systems, encoded by dedicated operons or gene clusters with high mosaïcity [4,7–10].

Over the last few years, an impressive anthology of re-
solved structures, mechanistic idiosyncrasies, and com-
monalities among synthase-dependent EPS secretion
systems has been reported in the literature, whereas
comparative genomics and modeling studies allow to
identify and predict functionalities of homologous sys-
tems in an ever-growing catalog of microbes of interest.
This mini-review highlights key aspects of these im-
pressive machineries with focus on the widespread and
intrinsically diverse bacterial cellulose secretion (Bcs)
systems.

**Cellulose synthases: activity and regulation**

Glycan polymerization and IM translocation in synthase-
dependent systems can be carried out by separate pro-
teins (e.g. PelF and PelG, respectively, in Pel systems),
or be incorporated in a single subunit as is the case with
BcsA, Alg8, and PgaC in cellulose, alginate, and PNAG
biogenesis [7,8,11]. Importantly, EPS synthases are
likely to function as parts of macromolecular trans-en-
velope complexes, where similar functionalities have
evolved across multiple different systems, often despite
a lack of sequence or fold conservation across the re-
spective subunits [7,8,11]. These include tight associa-
tion with an IM copolymerase necessary for the
activation and/or stability of the catalytic subunit (e.g.
PelE, BcsB, Alg44, and PgaD); c-di-GMP-sensing pro-
tein modules for system activation (e.g. PilZ domains on
BcsA and Alg44, I-site motifs on degenerate [*] GGDEF
domains from BcsE and PelD, and a composite binding

site at the PgaC and PgaD interface); tetratricopeptide
repeat (TPR)-rich periplasmic scaffolds (e.g. AlgK and
periplasmic modules of BcsC, PelB, and PgaA); polymer-
modification (e.g. alginate and cellulose acetylation
complexes) and hydrolase/lyase enzymes (e.g. AlgL,
BcsZ, PelA, and PgaB) in the periplasm; and β-barrel
export modules in the outer membrane (AlgE and porin
domains from PelB, BcsC, and PgaA) [7,8,11] (Figure 1).

Dependent on encoded accessory subunits discussed
below, Bcs systems have been grouped into several
major types: Type-I systems characterized by the pre-
sence of the BcsD scaffolding subunit; Type-II or *E.
coli*-like systems encoding the BcsE c-di-GMP-sensing
and BcsG phosphoethanolamine (pEtN)-transferase
subunits; Type-III systems, which lack all three of the
above and often feature BcsK instead of BcsC in the
periplasm; Wss systems for acetylated cellulose secre-
tion; and hybrid systems combining features from the
above [7,10] (Figure 1).

BcsA's core for EPS polymerization and export is conserved from bacteria to higher plants and consists of an α-helical transmembrane domain (TMD) interrupted by a D,D,D,Q(Q/R)xRW-type glycosyltransferase (GT) domain from the GT2 family of enzymes, and a so-called gating loop that caps the active site pocket [7,12,13]. The last tryptophan from the Q(Q/R)xRW motif coordinates the terminal glycosyl moiety of the nascent EPS at the active site and two of the conserved spaced aspartates (D<sup>246</sup>xD in *R. sphaeroides* BcsA) coordinate the substrate's diphosphate via a divalent metal ion. The third aspartate (D<sup>343</sup> in *R. sphaeroides* BcsA) serves as the catalytic base and lies at the tip of the so-called 'finger helix,' whose minute movements aid polymer

<figure><img src="image_5.png" /><figcaption>Figure 1</figcaption></figure>

Major types of synthase-dependent EPS secretion systems in Gram-negative bacteria. *, ATP binding and hydrolysis are likely involved in Bcs system assembly and not in glucose polymerization and cellulose extrusion.
Updated and expanded from reference [7] (http://creativecommons.org/licenses/by-nc/4.0/).
*aeruginosa*), bacterial hyaluronic acid (e.g. in *Streptococcus pyogenes*), and cellulose secretion systems, encoded by dedicated operons or gene clusters with high mosaïcity [4,7–10].

Over the last few years, an impressive anthology of re-
solved structures, mechanistic idiosyncrasies, and com-
monalities among synthase-dependent EPS secretion
systems has been reported in the literature, whereas
comparative genomics and modeling studies allow to
identify and predict functionalities of homologous sys-
tems in an ever-growing catalog of microbes of interest.
This mini-review highlights key aspects of these im-
pressive machineries with focus on the widespread and
intrinsically diverse bacterial cellulose secretion (Bcs)
systems.

**Cellulose synthases: activity and regulation**

Glycan polymerization and IM translocation in synthase-
dependent systems can be carried out by separate pro-
teins (e.g. PelF and PelG, respectively, in Pel systems),
or be incorporated in a single subunit as is the case with
BcsA, Alg8, and PgaC in cellulose, alginate, and PNAG
biogenesis [7,8,11]. Importantly, EPS synthases are
likely to function as parts of macromolecular trans-en-
velope complexes, where similar functionalities have
evolved across multiple different systems, often despite
a lack of sequence or fold conservation across the re-
spective subunits [7,8,11]. These include tight associa-
tion with an IM copolymerase necessary for the
activation and/or stability of the catalytic subunit (e.g.
PelE, BcsB, Alg44, and PgaD); c-di-GMP-sensing pro-
tein modules for system activation (e.g. PilZ domains on
BcsA and Alg44, I-site motifs on degenerate [*] GGDEF
domains from BcsE and PelD, and a composite binding

site at the PgaC and PgaD interface); tetratricopeptide
repeat (TPR)-rich periplasmic scaffolds (e.g. AlgK and
periplasmic modules of BcsC, PelB, and PgaA); polymer-
modification (e.g. alginate and cellulose acetylation
complexes) and hydrolase/lyase enzymes (e.g. AlgL,
BcsZ, PelA, and PgaB) in the periplasm; and β-barrel
export modules in the outer membrane (AlgE and porin
domains from PelB, BcsC, and PgaA) [7,8,11] (Figure 1).

Dependent on encoded accessory subunits discussed
below, Bcs systems have been grouped into several
major types: Type-I systems characterized by the pre-
sence of the BcsD scaffolding subunit; Type-II or *E.
coli*-like systems encoding the BcsE c-di-GMP-sensing
and BcsG phosphoethanolamine (pEtN)-transferase
subunits; Type-III systems, which lack all three of the
above and often feature BcsK instead of BcsC in the
periplasm; Wss systems for acetylated cellulose secre-
tion; and hybrid systems combining features from the
above [7,10] (Figure 1).

BcsA's core for EPS polymerization and export is con-
served from bacteria to higher plants and consists of an
α-helical transmembrane domain (TMD) interrupted by
a D,D,D,Q(Q/R)xRW-type glycosyltransferase (GT)
domain from the GT2 family of enzymes, and a so-called
gating loop that caps the active site pocket [7,12,13].
The last tryptophan from the Q(Q/R)xRW motif co-
ordinates the terminal glycosyl moiety of the nascent
EPS at the active site and two of the conserved spaced
aspartates (D<sup>246</sup>xD in *R. sphaeroides* BcsA) coordinate the
substrate's diphosphate via a divalent metal ion. The
third aspartate (D<sup>343</sup> in *R. sphaeroides* BcsA) serves as the
catalytic base and lies at the tip of the so-called 'finger
helix,' whose minute movements aid polymer

<figure><img src="image_5.png" /><figcaption>Figure 1</figcaption></figure>

Major types of synthase-dependent EPS secretion systems in Gram-negative bacteria. *, ATP binding and hydrolysis are likely involved in Bcs system assembly and not in glucose polymerization and cellulose extrusion.
Updated and expanded from reference [7] (http://creativecommons.org/licenses/by-nc/4.0/).
<figure><img src="image_6.png" /><figcaption>Figure 2</figcaption></figure>

Structure-function insights into the pEtN-cellulose secretion system. TMD, transmembrane domain; AF, alphafold2 model; NTPase*, degenerate, that is, catalytically incompetent, NTPase-like domain; REC*, degenerate, that is, phosphorylation-incompetent, receiver domain; GGDEF*, degenerate, that is, diguanylate cyclase-incompetent, GGDEF domain; I-site, an RxxD c-di-GMP-binding motif found on diguanylate cyclase domains as an inhibitory or diguanylate signal relay motif; D, domain. Structural data from references [18,20,31,33,40]. (a) a dimeric BcsF stoichiometry has not been directly visualized but is consistent with the protein's running behavior in denaturing gel electrophoresis, bacterial two-hybrid (BACTH) assays of protein interactions, and the BcsF-dependent recruitment of BcsE to the membrane (BcsE is dimeric both in the soluble Bcs-ERQ complex and the membrane-embedded Bcs macrocomplex). (b) BACTH experiments have shown that BcsG interacts with the *E. coli*-specific BcsA<sup>NTD</sup> domain, whereas tubular densities in the detergent micelle have been interpreted as two copies of BcsG<sup>NTD</sup> in a low-resolution cryo-EM study of the Bcs macrocomplex. (c) ATP hydrolysis is not required for synthase activity in vitro, however, BcsRQ mutation analyses indicate that both ATP binding and hydrolysis might be necessary for cellulose biogenesis in vivo. In particular, *bcsQ* mutations disrupting ATP complexation (e.g. BcsQ<sup>N152A-R156A-N171A</sup>) lead to loss of cellulose secretion, mutations stimulating BcsRQ ATPase activity (e.g. BcsQ<sup>R156E</sup>) lead to enhanced cellulose secretion, and mutations that lead to a stable, ATP-binding, but catalytically inactive BcsRQ complex (e.g. BcsQ<sup>T15K</sup>) lead again to loss of cellulose secretion, thus excluding a purely structural role for nucleotide complexation.

translocation [7,12,13]. In bacteria, the cellulose synthase contains a third, PilZ domain, whose core-adjacent linker carries the RxxxR motif for dimeric cyclic diguanylate (c-di-GMP) coordination [7,13,14]. Dynamic dinucleotide binding causes gating loop relaxation and together with the 'finger helix' regulates the reiterative processes of uridine diphosphate (UDP) product release, polymer translocation, UDP-glucose substrate entry, gating loop closure, and catalysis [7,13].

Whereas the catalytic BcsA cycle has been characterized primarily *in crystallo* in saturating concentrations of dinucleotide [14], product, and/or substrate-like ligands, in nature, multiple mechanisms have evolved for the local enrichment of activating c-di-GMP. *E. coli*, for example, resorts to one or more dedicated diguanylate cyclases such as synthase-interacting AdrA/DgcA in some strains or YedQ/DgcQ in others [15,16]. In addition, *E. coli* BcsA forms a stable, multicomponent biosynthetic macrocomplex encompassing most of the inner-membrane and cytosolic subunits (BcsR<sub>2</sub>Q<sub>2</sub>AB<sub>5-6</sub>E<sub>2</sub>F<sub>2</sub>) (Figure 2). In it, the essential-for-secretion BcsRQ tandem has been proposed to regulate macrocomplex assembly in an ATP-dependent manner and to contribute to BcsA maintenance and catalytic activity in the membrane [17,18]. The BcsE protein, on the other hand, features a

tripartite architecture of catalytically or phosphotransfer-
incompetent NTPase (NTPase*), receiver (REC*), and
diguanylate cyclase (GGDEF*) domains. Dimeric BcsE
is recruited by BcsF to the membrane, where each BcsE
copy presents a composite site for dimeric c-di-GMP
coordination formed by two RxxD motifs on its degen-
erate receiver and diguanylate cyclase domains
(BcsE<sup>REC*-GGDEF*</sup>) [19,20] and, together with the
BcsRQ subunits, BcsE forms a c-di-GMP-binding cyto-
solic vestibule around BcsA's PilZ domain [17,18]
(Figure 2). Changes in the contribution of either RxxD
motif have been shown to alter the BcsE's affinity for c-
di-GMP and its observed dynamic structure — evi-
denced in both crystallographic and cryo-EM experi-
ments — has been proposed to act as a dynamic partner
for dinucleotide recycling in processive synthase activa-
tion [7,18,20].

As mentioned above, BcsA functions in tandem with the
BcsB copolymerase [7]. The latter features a donut-
shaped periplasmic module that incorporates 2 carbo-
hydrate-binding and 2 flavodoxin-like domains alter-
nating along the polypeptide chain, and a C-proximal
membrane anchor that is required for BcsA's catalytic
function and is composed of a short amphipathic and a
transmembrane α-helices [12,18,21]. Crystal structures
<figure><img src="image_6.png" /><figcaption>Figure 2</figcaption></figure>

Structure-function insights into the pEtN-cellulose secretion system. TMD, transmembrane domain; AF, alphafold2 model; NTPase*, degenerate, that is, catalytically incompetent, NTPase-like domain; REC*, degenerate, that is, phosphorylation-incompetent, receiver domain; GGDEF*, degenerate, that is, diguanylate cyclase-incompetent, GGDEF domain; I-site, an RxxD c-di-GMP-binding motif found on diguanylate cyclase domains as an inhibitory or diguanylate signal relay motif; D, domain. Structural data from references [18,20,31,33,40]. (a) a dimeric BcsF stoichiometry has not been directly visualized but is consistent with the protein's running behavior in denaturing gel electrophoresis, bacterial two-hybrid (BACTH) assays of protein interactions, and the BcsF-dependent recruitment of BcsE to the membrane (BcsE is dimeric both in the soluble Bcs-ERQ complex and the membrane-embedded Bcs macrocomplex). (b) BACTH experiments have shown that BcsG interacts with the *E. coli*-specific BcsA<sup>NTD</sup> domain, whereas tubular densities in the detergent micelle have been interpreted as two copies of BcsG<sup>NTD</sup> in a low-resolution cryo-EM study of the Bcs macrocomplex. (c) ATP hydrolysis is not required for synthase activity in vitro, however, BcsRQ mutation analyses indicate that both ATP binding and hydrolysis might be necessary for cellulose biogenesis in vivo. In particular, *bcsQ* mutations disrupting ATP complexation (e.g. BcsQ<sup>N152A-R156A-N171A</sup>) lead to loss of cellulose secretion, mutations stimulating BcsRQ ATPase activity (e.g. BcsQ<sup>R156E</sup>) lead to enhanced cellulose secretion, and mutations that lead to a stable, ATP-binding, but catalytically inactive BcsRQ complex (e.g. BcsQ<sup>T15K</sup>) lead again to loss of cellulose secretion, thus excluding a purely structural role for nucleotide complexation.

translocation [7,12,13]. In bacteria, the cellulose synthase contains a third, PilZ domain, whose core-adjacent linker carries the RxxxR motif for dimeric cyclic diguanylate (c-di-GMP) coordination [7,13,14]. Dynamic dinucleotide binding causes gating loop relaxation and together with the 'finger helix' regulates the reiterative processes of uridine diphosphate (UDP) product release, polymer translocation, UDP-glucose substrate entry, gating loop closure, and catalysis [7,13].

Whereas the catalytic BcsA cycle has been characterized primarily *in crystallo* in saturating concentrations of dinucleotide [14], product, and/or substrate-like ligands, in nature, multiple mechanisms have evolved for the local enrichment of activating c-di-GMP. *E. coli*, for example, resorts to one or more dedicated diguanylate cyclases such as synthase-interacting AdrA/DgcA in some strains or YedQ/DgcQ in others [15,16]. In addition, *E. coli* BcsA forms a stable, multicomponent biosynthetic macrocomplex encompassing most of the inner-membrane and cytosolic subunits (BcsR<sub>2</sub>Q<sub>2</sub>AB<sub>5-6</sub>E<sub>2</sub>F<sub>2</sub>) (Figure 2). In it, the essential-for-secretion BcsRQ tandem has been proposed to regulate macrocomplex assembly in an ATP-dependent manner and to contribute to BcsA maintenance and catalytic activity in the membrane [17,18]. The BcsE protein, on the other hand, features a

tripartite architecture of catalytically or phosphotransfer-
incompetent NTPase (NTPase*), receiver (REC*), and
diguanylate cyclase (GGDEF*) domains. Dimeric BcsE
is recruited by BcsF to the membrane, where each BcsE
copy presents a composite site for dimeric c-di-GMP
coordination formed by two RxxD motifs on its degen-
erate receiver and diguanylate cyclase domains
(BcsE<sup>REC*-GGDEF*</sup>) [19,20] and, together with the
BcsRQ subunits, BcsE forms a c-di-GMP-binding cyto-
solic vestibule around BcsA's PilZ domain [17,18]
(Figure 2). Changes in the contribution of either RxxD
motif have been shown to alter the BcsE's affinity for c-
di-GMP and its observed dynamic structure — evi-
denced in both crystallographic and cryo-EM experi-
ments — has been proposed to act as a dynamic partner
for dinucleotide recycling in processive synthase activa-
tion [7,18,20].

As mentioned above, BcsA functions in tandem with the
BcsB copolymerase [7]. The latter features a donut-
shaped periplasmic module that incorporates 2 carbo-
hydrate-binding and 2 flavodoxin-like domains alter-
nating along the polypeptide chain, and a C-proximal
membrane anchor that is required for BcsA's catalytic
function and is composed of a short amphipathic and a
transmembrane α-helices [12,18,21]. Crystal structures
of the *Rhodobacter sphaeroides* BcsAB tandem have re-
vealed a 1:1 complex where the C-terminal BcsB anchor
helix completes BcsA's export domain [12] and a similar
1:1 architecture was observed in a low-resolution nega-
tive-stain electron microscopy reconstruction of a het-
erodimeric *G. hansenii* BcsAB tandem [22], itself encoded
by a fused *bcsAB* gene and further supporting equimolar
BcsA:BcsB assembly [7,10].

Surprisingly, the cryo-EM structure of the *E. coli* Bcs
macrocomplex, revealed a drastically different, non-
canonical BcsA:BcsB stoichiometry [18]. In it, a single
BcsA subunit assembles with up to 6 BcsB protomers
that use a β-sheet complementation mechanism to form
a superhelical ‘crown’ in the periplasm, with cellulose
proposed to glide outward along stacked luminal loops
from BcsB’s carbohydrate-binding domains [18] (Figure
2). Importantly, the superhelical assembly of the peri-
plasmic modules and the presence of the transmem-
brane BcsB tail anchors likely induce and favor
significant negative curvature in the membrane [18]
(Figure 2). Interestingly, visualization of the assembly
ATPase BcsQ in cells and of secreted cellulose in biofilm
cryosections is consistent with elongated cell shape and
polar pEtN-cellulose secretion in the younger biofilm
layers (i.e. Bcs targeting to membrane domains with
the highest negative curvature), whereas the oldest
surface layers present rounded, nondividing cells fully
enclosed in a mesh of pEtN-cellulose and curli [23,24].
Whether subcellular targeting of the Bcs system can be
achieved by covarying cell shape and BcsA:BcsB stoi-
chiometry remains to be experimentally examined.

**Cellulosic homo- and heteropolymers**

Although some bacterial EPS-producing synthases can
yield mixed-linkage glycans [25], most of these enzymes
typically use a specific nucleotide sugar donor to transfer
the glycosyl moiety via a unique glycosidic linkage onto
a processively extruded polymer. Nevertheless, this
more prevalent “one enzyme–one substrate–one
linkage” principle does not necessarily translate into the
secretion of homopolymers as bacteria have evolved a
number of mechanisms to decorate and/or heterogenize
their biofilm polysaccharides [7,11,26] (Figure 1). For
example, the Pel system of *P. aeruginosa* uses the cyto-
solic PelF GT to polymerize UDP-N-acet-
ylgalactosamine (UDP-GalNAc) into a homopolymer
extruded through the PelG translocation pore in the
periplasm, where the nascent chain is partially deacety-
lated by the PelA deacetylase-hydrolase enzyme to yield
a positively charged polymer [11,27]. The latter — once
funneled through the PelC–PelB OM export tandem
— would then cross-link negatively charged eDNA in
the stalks of the typical mushroom-shaped biofilm
structures [27]. Similarly, alginate-secreting mucoid *P.
aeruginosa* polymerizes GDP-D-mannuronate via the

bifunctional GT Alg8 that also translocates the polymer
across the IM. Once in the periplasm, the polymer is
subject to both partial D-mannuronate to L-guluronate
epimerization by the periplasmic AlgG subunit and to O-
acetylation by the AlgIFJX complex [11] (Figure 1).

Similarly, secreted cellulosic polymers come in a variety
of shapes and flavors [7] (Figure 1). Some bacteria, such
as members of the *Gluconacetobacter* lineage, feature
longitudinal arrays of synthase terminal complexes
(TCs) whose secreted, chemically pure cellulose chains
bundle into so-called cellulose ribbons that lead to the
formation of thick biofilm mats with a high cellulose
crystallinity index [28,29]. Many enterobacteria, such as
*E. coli*, *S. enterica* serovar Typhimurium, and others, se-
crete a chemically modified cellulose where up to half of
the glycosyl moieties feature pEtN residues at the C⁶
position [30]. The pEtN-transferase function is carried
out in the periplasm by BcsG, an inner-membrane-em-
bedded, Zn-dependent enzyme from the alkaline
phosphatase superfamily that likely partakes in direct
but more dynamic interactions with the biosynthetic
BcsAB platform [7,30–33]. Indeed, bacterial BcsG was
found to interact with an *E. coli*-specific N-terminal
domain (NTD) of the BcsA synthase [17] and a separate
cryo-EM study of the Bcs macrocomplex attributed
tubular densities within the detergent micelle to two
copies of BcsGNTD, whose poorer resolution and lack of
corresponding resolved densities for the C-terminal
catalytic domain indicate highly heterogeneous occu-
pancy [33]. Interestingly, whereas in some species and
strains BcsG is dispensable for secretion of non-modified
cellulose in vivo (e.g. in AR3110, UTI89, or the naturally
BcsG-deficient rUT12 [30,34]), in others, it appears re-
quired for cellulose biogenesis. For example, in *E. coli*
1094, a nonpolar *bcsG* deletion effectively abolishes cel-
lulose secretion [17]; similarly, in *S. enterica* serovar Ty-
phimurium not only does the transmembrane NTD of
BcsG appear to influence the integrity of the cellulose
synthase, but also BcsG point mutants carrying a cata-
lytically inactive BcsG C-terminal domain do not secrete
cellulose even in the presence of wild-type BcsA levels,
thus suggesting direct requirement for the pEtN mod-
ification itself [32]. The pEtN-derivatized cellulose has
been further shown to determine biofilm architecture
and tensile strength by directly interacting with and af-
fecting the polymerization dynamics of the other major
enterobacterial matrix component, amyloid curli [35,36].
Finally, some species such as *Pseudomonas fluorescens*
SBW25 and *Orella dioscoreae* feature wss gene clusters,
whose products are proposed to introduce a different
chemical modification — cellulose acetylation — that
can affect the biofilm strength, architecture, and/or the
overall ecological success of the species [26,37,38]. The
predicted Wss subunits share structural and functional
homology with the AlgIFJX components from the algi-
nate acetylation complex and include a membrane-
of the *Rhodobacter sphaeroides* BcsAB tandem have re-
vealed a 1:1 complex where the C-terminal BcsB anchor
helix completes BcsA's export domain [12] and a similar
1:1 architecture was observed in a low-resolution nega-
tive-stain electron microscopy reconstruction of a het-
erodimeric *G. hansenii* BcsAB tandem [22], itself encoded
by a fused *bcsAB* gene and further supporting equimolar
BcsA:BcsB assembly [7,10].

Surprisingly, the cryo-EM structure of the *E. coli* Bcs
macrocomplex, revealed a drastically different, non-
canonical BcsA:BcsB stoichiometry [18]. In it, a single
BcsA subunit assembles with up to 6 BcsB protomers
that use a β-sheet complementation mechanism to form
a superhelical ‘crown’ in the periplasm, with cellulose
proposed to glide outward along stacked luminal loops
from BcsB’s carbohydrate-binding domains [18] (Figure
2). Importantly, the superhelical assembly of the peri-
plasmic modules and the presence of the transmem-
brane BcsB tail anchors likely induce and favor
significant negative curvature in the membrane [18]
(Figure 2). Interestingly, visualization of the assembly
ATPase BcsQ in cells and of secreted cellulose in biofilm
cryosections is consistent with elongated cell shape and
polar pEtN-cellulose secretion in the younger biofilm
layers (i.e. Bcs targeting to membrane domains with
the highest negative curvature), whereas the oldest
surface layers present rounded, nondividing cells fully
enclosed in a mesh of pEtN-cellulose and curli [23,24].
Whether subcellular targeting of the Bcs system can be
achieved by covarying cell shape and BcsA:BcsB stoi-
chiometry remains to be experimentally examined.

Cellulosic homo- and heteropolymers

Although some bacterial EPS-producing synthases can
yield mixed-linkage glycans [25], most of these enzymes
typically use a specific nucleotide sugar donor to transfer
the glycosyl moiety via a unique glycosidic linkage onto
a processively extruded polymer. Nevertheless, this
more prevalent “one enzyme–one substrate–one
linkage” principle does not necessarily translate into the
secretion of homopolymers as bacteria have evolved a
number of mechanisms to decorate and/or heterogenize
their biofilm polysaccharides [7,11,26] (Figure 1). For
example, the Pel system of *P. aeruginosa* uses the cyto-
solic PelF GT to polymerize UDP-N-acet-
ylgalactosamine (UDP-GalNAc) into a homopolymer
extruded through the PelG translocation pore in the
periplasm, where the nascent chain is partially deacety-
lated by the PelA deacetylase-hydrolase enzyme to yield
a positively charged polymer [11,27]. The latter — once
funneled through the PelC–PelB OM export tandem
— would then cross-link negatively charged eDNA in
the stalks of the typical mushroom-shaped biofilm
structures [27]. Similarly, alginate-secreting mucoid *P.
aeruginosa* polymerizes GDP-D-mannuronate via the

bifunctional GT Alg8 that also translocates the polymer
across the IM. Once in the periplasm, the polymer is
subject to both partial D-mannuronate to L-guluronate
epimerization by the periplasmic AlgG subunit and to O-
acetylation by the AlgIFJX complex [11] (Figure 1).

Similarly, secreted cellulosic polymers come in a variety
of shapes and flavors [7] (Figure 1). Some bacteria, such
as members of the *Gluconacetobacter* lineage, feature
longitudinal arrays of synthase terminal complexes
(TCs) whose secreted, chemically pure cellulose chains
bundle into so-called cellulose ribbons that lead to the
formation of thick biofilm mats with a high cellulose
crystallinity index [28,29]. Many enterobacteria, such as
*E. coli*, *S. enterica* serovar Typhimurium, and others, se-
crete a chemically modified cellulose where up to half of
the glycosyl moieties feature pEtN residues at the C⁶
position [30]. The pEtN-transferase function is carried
out in the periplasm by BcsG, an inner-membrane-em-
bedded, Zn-dependent enzyme from the alkaline
phosphatase superfamily that likely partakes in direct
but more dynamic interactions with the biosynthetic
BcsAB platform [7,30–33]. Indeed, bacterial BcsG was
found to interact with an *E. coli*-specific N-terminal
domain (NTD) of the BcsA synthase [17] and a separate
cryo-EM study of the Bcs macrocomplex attributed
tubular densities within the detergent micelle to two
copies of BcsGNTD, whose poorer resolution and lack of
corresponding resolved densities for the C-terminal
catalytic domain indicate highly heterogeneous occu-
pancy [33]. Interestingly, whereas in some species and
strains BcsG is dispensable for secretion of non-modified
cellulose in vivo (e.g. in AR3110, UTI89, or the naturally
BcsG-deficient rUT12 [30,34]), in others, it appears re-
quired for cellulose biogenesis. For example, in *E. coli*
1094, a nonpolar *bcsG* deletion effectively abolishes cel-
lulose secretion [17]; similarly, in *S. enterica* serovar Ty-
phimurium not only does the transmembrane NTD of
BcsG appear to influence the integrity of the cellulose
synthase, but also BcsG point mutants carrying a cata-
lytically inactive BcsG C-terminal domain do not secrete
cellulose even in the presence of wild-type BcsA levels,
thus suggesting direct requirement for the pEtN mod-
ification itself [32]. The pEtN-derivatized cellulose has
been further shown to determine biofilm architecture
and tensile strength by directly interacting with and af-
fecting the polymerization dynamics of the other major
enterobacterial matrix component, amyloid curli [35,36].
Finally, some species such as *Pseudomonas fluorescens*
SBW25 and *Orella dioscoreae* feature wss gene clusters,
whose products are proposed to introduce a different
chemical modification — cellulose acetylation — that
can affect the biofilm strength, architecture, and/or the
overall ecological success of the species [26,37,38]. The
predicted Wss subunits share structural and functional
homology with the AlgIFJX components from the algi-
nate acetylation complex and include a membrane-
bound O-acyltransferase (MBOATs WssH and AlgI, respectively) proposed to flip acetyl moieties from as-yet uncharacterized cytosolic donors through the IM, one or more immunoglobulin-like adaptor proteins (WssG/AlgF), and SGNH hydrolase-like O-acyltransferases proposed to act on the nascent polymers in the periplasm (WssI/AlgJ and WssF/AlgX) [7,9,11,26,39,40]. Indeed, recent studies on purified WssI homologs from *P. fluorescens* and the multi-organ pathogen *Achromobacter in-suavis* have demonstrated in vitro acetylesterase and acetyltransferase activities using a variety of rationally designed acetyl donors and cellooligosaccharidic substrates [39]. Nevertheless, the exact structures, stoichiometry, and functional roles of the putative cellulose acetylation complexes, as well as the exact composition of the modified extracellular polymers, remain to be further experimentally examined.

## Periplasmic and outer membrane export

Once exported in the periplasm, the secreted EPS need to cross the peptidoglycan mesh and outer membrane, which is generally achieved through a tandem of a TPR-rich periplasmic scaffold and an outer membrane β-barrel porin [7,11]. The two modules can be part of the same (e.g. BcsC in cellulose, PgaA in PNAG, and PelB in Pel secretion) or different proteins (e.g. AlgK–AlgE in alginate secretion), and the number of TPR motifs and porin β-strands can vary across systems [7,11]. In some cases, export can be further facilitated by additional components such as the PelC lipoprotein, which is proposed to form an OM-proximal dodecameric ring whose negatively charged lumen would funnel the cationic Pel EPS through to the PelB porin domain [41].

The TPR-rich periplasmic scaffolds likely adopt flexible solenoid folds that extend across the periplasmic space [7,11,42] and can recruit additional functional partners. Whereas functional data on BcsC's NTD are currently limited, studies on its functional homolog PgaA from the PNAG secretion system have shown that the protein binds both PNAG and the periplasmic enzyme PgaB to stimulate the latter's deacetylase and glycoside hydrolase activities [43]. Similarly, AlgK recruits the periplasmic O-acetyltransferase AlgX in a stable, alginate-binding complex that is key to periplasmic polymer modification and downstream biofilm attachment [44]. In *E. coli*, BcsC features 19 TPR repeats in its N-terminal periplasmic region and a 16-stranded β-barrel porin domain with a large, electronegative lumen constricted near the extracellular surface and lined with conserved polar and aromatic residues. An ~15-residue-long proline-rich C-terminal extension folds into the channel to position the last aromatic (W¹¹⁵⁷) residue midway across the lumen [45]. These features, conserved across pEtN-cellulose-secreting bacteria, likely prevent permeability for solutes in the resting state while facilitating the take-

up and outward gliding of hydrated, zwitterionic pEtN-cellulose [7,45].

Interestingly, many *bcs* clusters do not feature a *bcsC* gene but instead encode BcsK, which has multiple TPR motifs but no obvious porin domain, so how the polymer crosses the outer membrane has remained enigmatic [7,10] (Figure 1). Finally, some cyanobacterial systems likely feature a very different, Type-I protein secretion system-like Bcs architecture where the BcsB copolymerase and BcsC are substituted by proteins similar to the membrane fusion protein HlyD and the outer membrane efflux protein TolC, respectively [46].

## Intracellular cytoskeletal scaffolds

Crystalline cellulose secretion by bacteria from the *Gluconacetobacter* lineage has long attracted interest as a source of chemically pure polymer for a variety of biotechnological applications [47]. Almost half a century ago, freeze-fracture and negative-stain electron microscopy experiments visualized the longitudinal linear assembly of synthase TCs, their colocalization with the exit points of secreted cellulose microfibrils, and the latter's extracellular bundling into a single crystalline cellulose ribbon per cell [28]. Two different Bcs proteins were subsequently identified as cellulose crystallinity factors: BcsD (CesD) and BcsH (CcpAx). BcsD is a small, ~17-kDa protein, which in *G. hansenii* assembles into donut-shaped D4-symmetric octamers featuring 4 independent luminal passages capable to bind cellulosic oligosaccharides [48,49]. This peculiar architecture had led to a functional model in which BcsD serves as a periplasmic guide for individual cellulose chains exiting the BcsA translocation pores and fluorescence-based imaging studies showed that indeed BcsD localizes in a longitudinal line along the cell body, similarly to the synthase TCs [48,50]. Importantly, this linear BcsD localization is required for crystalline cellulose biogenesis and depends on direct interactions with the second crystallinity factor BcsH, initially proposed to be a short, ~8-kDa periplasmic peptide [50].

Recently, an in situ cryoelectron tomography study of *Gluconacetobacter* cells and biofilms revealed a cytoskeletal structure, dubbed the ‘cortical belt’ [29] (Figure 3). The cytosolic assembly, tens of nanometers wide and hundreds of nanometers long, was shown to run in one to several stacked sheets parallel to the extracellular cellulose ribbon and at a fixed distance (~24 nm) from the IM [29]. In addition, cryo-EM and functional studies on the BcsHD tandem showed that BcsH is in fact an ~37-kDa protein that assembles the BcsD octamers into ‘beads-on-a-string’ filaments via its short C-terminal domain (Figure 3), whereas the N-terminal proline-rich region is capable of self-oligomerization, likely driving the BcsD filaments into three-dimensional sheets and
bound O-acyltransferase (MBOATs WssH and AlgI, respectively) proposed to flip acetyl moieties from as-yet uncharacterized cytosolic donors through the IM, one or more immunoglobulin-like adaptor proteins (WssG/AlgF), and SGNH hydrolase-like O-acyltransferases proposed to act on the nascent polymers in the periplasm (WssI/AlgJ and WssF/AlgX) [7,9,11,26,39,40]. Indeed, recent studies on purified WssI homologs from *P. fluorescens* and the multi-organ pathogen *Achromobacter in-suavis* have demonstrated in vitro acetylesterase and acetyltransferase activities using a variety of rationally designed acetyl donors and cellooligosaccharidic substrates [39]. Nevertheless, the exact structures, stoichiometry, and functional roles of the putative cellulose acetylation complexes, as well as the exact composition of the modified extracellular polymers, remain to be further experimentally examined.

## Periplasmic and outer membrane export

Once exported in the periplasm, the secreted EPS need to cross the peptidoglycan mesh and outer membrane, which is generally achieved through a tandem of a TPR-rich periplasmic scaffold and an outer membrane β-barrel porin [7,11]. The two modules can be part of the same (e.g. BcsC in cellulose, PgaA in PNAG, and PelB in Pel secretion) or different proteins (e.g. AlgK–AlgE in alginate secretion), and the number of TPR motifs and porin β-strands can vary across systems [7,11]. In some cases, export can be further facilitated by additional components such as the PelC lipoprotein, which is proposed to form an OM-proximal dodecameric ring whose negatively charged lumen would funnel the cationic Pel EPS through to the PelB porin domain [41].

The TPR-rich periplasmic scaffolds likely adopt flexible solenoid folds that extend across the periplasmic space [7,11,42] and can recruit additional functional partners. Whereas functional data on BcsC's NTD are currently limited, studies on its functional homolog PgaA from the PNAG secretion system have shown that the protein binds both PNAG and the periplasmic enzyme PgaB to stimulate the latter's deacetylase and glycoside hydrolase activities [43]. Similarly, AlgK recruits the periplasmic O-acetyltransferase AlgX in a stable, alginate-binding complex that is key to periplasmic polymer modification and downstream biofilm attachment [44]. In *E. coli*, BcsC features 19 TPR repeats in its N-terminal periplasmic region and a 16-stranded β-barrel porin domain with a large, electronegative lumen constricted near the extracellular surface and lined with conserved polar and aromatic residues. An ~15-residue-long proline-rich C-terminal extension folds into the channel to position the last aromatic (W¹¹⁵⁷) residue midway across the lumen [45]. These features, conserved across pEtN-cellulose-secreting bacteria, likely prevent permeability for solutes in the resting state while facilitating the take-

up and outward gliding of hydrated, zwitterionic pEtN-cellulose [7,45].

Interestingly, many *bcs* clusters do not feature a *bcsC* gene but instead encode BcsK, which has multiple TPR motifs but no obvious porin domain, so how the polymer crosses the outer membrane has remained enigmatic [7,10] (Figure 1). Finally, some cyanobacterial systems likely feature a very different, Type-I protein secretion system-like Bcs architecture where the BcsB copolymerase and BcsC are substituted by proteins similar to the membrane fusion protein HlyD and the outer membrane efflux protein TolC, respectively [46].

## Intracellular cytoskeletal scaffolds

Crystalline cellulose secretion by bacteria from the *Gluconacetobacter* lineage has long attracted interest as a source of chemically pure polymer for a variety of biotechnological applications [47]. Almost half a century ago, freeze-fracture and negative-stain electron microscopy experiments visualized the longitudinal linear assembly of synthase TCs, their colocalization with the exit points of secreted cellulose microfibrils, and the latter's extracellular bundling into a single crystalline cellulose ribbon per cell [28]. Two different Bcs proteins were subsequently identified as cellulose crystallinity factors: BcsD (CesD) and BcsH (CcpAx). BcsD is a small, ~17-kDa protein, which in *G. hansenii* assembles into donut-shaped D4-symmetric octamers featuring 4 independent luminal passages capable to bind cellulosic oligosaccharides [48,49]. This peculiar architecture had led to a functional model in which BcsD serves as a periplasmic guide for individual cellulose chains exiting the BcsA translocation pores and fluorescence-based imaging studies showed that indeed BcsD localizes in a longitudinal line along the cell body, similarly to the synthase TCs [48,50]. Importantly, this linear BcsD localization is required for crystalline cellulose biogenesis and depends on direct interactions with the second crystallinity factor BcsH, initially proposed to be a short, ~8-kDa periplasmic peptide [50].

Recently, an in situ cryoelectron tomography study of *Gluconacetobacter* cells and biofilms revealed a cytoskeletal structure, dubbed the ‘cortical belt’ [29] (Figure 3). The cytosolic assembly, tens of nanometers wide and hundreds of nanometers long, was shown to run in one to several stacked sheets parallel to the extracellular cellulose ribbon and at a fixed distance (~24 nm) from the IM [29]. In addition, cryo-EM and functional studies on the BcsHD tandem showed that BcsH is in fact an ~37-kDa protein that assembles the BcsD octamers into ‘beads-on-a-string’ filaments via its short C-terminal domain (Figure 3), whereas the N-terminal proline-rich region is capable of self-oligomerization, likely driving the BcsD filaments into three-dimensional sheets and
<figure><img src="image_7.png" /><figcaption>Figure 3</figcaption></figure>

Cytoskeletal scaffolds for cellulose secretion. Left, cortical microtubules and plant cellulose synthase complexes (CSCs or rosettes); CSI, cellulose synthase-interacting protein 1; KOR, KORRIGAN β-1,4-endoglucanases; COB, COBRA cellulose-binding proteins in the apoplast, membrane-anchored; CesA, a eukaryotic cellulose synthase.

(adapted from reference [52]). Right, bacterial cytoskeletal scaffolds of octameric or tetrameric BcsD and proline-rich partners. Structural data from references [29,37,48,49,51].

stacks [49]. Importantly, bacterial two-hybrid (BACTH) functional complementation assays revealed interactions of the crystallinity factors with the cytosolic BcsA<sup>PilZ</sup> domain, whereas in situ cryo-EM visualization at higher resolution revealed striking similarity between the cortical belt profile and the BcsHD ‘beads-on-a-string’ filaments [49]. These findings, together with the lack of secretion signals on either BcsD or BcsH, support an updated model of inside-out regulation, where the two proteins assemble into the intracellular cortical belt to drive longitudinal TC array formation and crystalline cellulose biogenesis [49] (Figure 3).

Whether BcsD and BcsH use an as-yet uncharacterized export process to provide additional roles as polysaccharide conduits in the periplasm remains to be further investigated. The inside-out hypothesis was recently further enforced by structure-function studies on BcsD homologs from diverse bacteria, including species expected to secrete acetylated (*O. dioscoreae*) or pEtN-modified (*Enterobacter* sp. 638) cellulose [37]. Cryo-EM structures of BcsD from several β- and γ-Proteobacteria revealed that through a conserved additional N-terminal helix, BcsD can switch from D4-symmetric octamers to D2-symmetric tetramers [37] (Figure 3). The latter in turn can interact with previously uncharacterized proline-rich partners (BcsP in β- and BcsO

in γ-Proteobacteria), as well as with the synthase-binding ATPase BcsQ, to form intracellular supramolecular scaffolds that determine the efficiency of cellulose secretion and the overall biofilm strength and architecture [37]. Unlike the BcsHD filaments that drive the assembly of longitudinal TC arrays and a crystalline cellulose ribbon in *Gluconacetobacter*, the BcsD-BcsP complexes from *O. dioscoreae* were shown to array into triangular tiling modules likely determined by the tetrahedron-like architecture of the individual BcsD tetramers [37] (Figure 3).

How in each case these atypical cytoskeletal scaffolds assemble and whether they serve to recruit additional regulatory components remains to be further examined. Nevertheless, enlisting cytoskeletal elements into the regulation and spatial organization of cellulose synthase activity appears to have evolved multiple times in evolution. Indeed, in plants, the mature cellulose synthase complexes (CSCs) are delivered from the *trans*-Golgi network to the cell membrane, where they are linked to cortical microtubules via CSI1 [51,52], among others, and activated by phosphorylation (Figure 3). Whereas in plants the ensuing synthesis of crystalline cellulose is proposed to generate forces propelling the CSC rosettes within the membrane and along the cortical cytoskeleton in a process required for cell wall biogenesis [52], in
<figure><img src="image_7.png" /><figcaption>Figure 3</figcaption></figure>

Cytoskeletal scaffolds for cellulose secretion. Left, cortical microtubules and plant cellulose synthase complexes (CSCs or rosettes); CSI, cellulose synthase-interacting protein 1; KOR, KORRIGAN β-1,4-endoglucanases; COB, COBRA cellulose-binding proteins in the apoplast, membrane-anchored; CesA, a eukaryotic cellulose synthase. (adapted from reference [52]). Right, bacterial cytoskeletal scaffolds of octameric or tetrameric BcsD and proline-rich partners. Structural data from references [29,37,48,49,51].

stacks [49]. Importantly, bacterial two-hybrid (BACTH) functional complementation assays revealed interactions of the crystallinity factors with the cytosolic BcsA<sup>PilZ</sup> domain, whereas in situ cryo-EM visualization at higher resolution revealed striking similarity between the cortical belt profile and the BcsHD ‘beads-on-a-string’ filaments [49]. These findings, together with the lack of secretion signals on either BcsD or BcsH, support an updated model of inside-out regulation, where the two proteins assemble into the intracellular cortical belt to drive longitudinal TC array formation and crystalline cellulose biogenesis [49] (Figure 3).

Whether BcsD and BcsH use an as-yet uncharacterized export process to provide additional roles as polysaccharide conduits in the periplasm remains to be further investigated. The inside-out hypothesis was recently further enforced by structure-function studies on BcsD homologs from diverse bacteria, including species expected to secrete acetylated (*O. dioscoreae*) or pEtN-modified (*Enterobacter* sp. 638) cellulose [37]. Cryo-EM structures of BcsD from several β- and γ-Proteobacteria revealed that through a conserved additional N-terminal helix, BcsD can switch from D4-symmetric octamers to D2-symmetric tetramers [37] (Figure 3). The latter in turn can interact with previously uncharacterized proline-rich partners (BcsP in β- and BcsO

in γ-Proteobacteria), as well as with the synthase-binding ATPase BcsQ, to form intracellular supramolecular scaffolds that determine the efficiency of cellulose secretion and the overall biofilm strength and architecture [37]. Unlike the BcsHD filaments that drive the assembly of longitudinal TC arrays and a crystalline cellulose ribbon in *Gluconacetobacter*, the BcsD-BcsP complexes from *O. dioscoreae* were shown to array into triangular tiling modules likely determined by the tetrahedron-like architecture of the individual BcsD tetramers [37] (Figure 3).

How in each case these atypical cytoskeletal scaffolds assemble and whether they serve to recruit additional regulatory components remains to be further examined. Nevertheless, enlisting cytoskeletal elements into the regulation and spatial organization of cellulose synthase activity appears to have evolved multiple times in evolution. Indeed, in plants, the mature cellulose synthase complexes (CSCs) are delivered from the *trans*-Golgi network to the cell membrane, where they are linked to cortical microtubules via CSI1 [51,52], among others, and activated by phosphorylation (Figure 3). Whereas in plants the ensuing synthesis of crystalline cellulose is proposed to generate forces propelling the CSC rosettes within the membrane and along the cortical cytoskeleton in a process required for cell wall biogenesis [52], in
*Gluconacetobacter* similar forces and cortical belt-anchored, static TCs are believed to convert the secreted cellulose ribbon in a *bona fide* motility organelle during substrate colonization [28,29].

## Conclusions and outlook

Secreted cellulosic polymers represent key architectural and functional constituents of the three-dimensional biofilms of many free-living and host-associated bacteria and are produced in both temporally and spatially regulated manner by dedicated multicomponent Bcs secretion systems [7]. Although these demonstrate functional similarities with other synthase-dependent EPS-secretory assemblies and even evolutionary distant eukaryotic systems for cellulose biogenesis, they also present important mechanistic idiosyncrasies that have only recently begun to emerge. Although bacterial cellulose biogenesis represents the longest-studied process for synthase- and c-di-GMP-dependent EPS secretion [53], it remains to be fully understood how subsets of biofilm cells switch to an energetically expensive, sugar-consuming anabolic process in typically nutrient-limited conditions to provide the scaffold and ramparts of the mature macrocolony; how in each system activating c-di-GMP is relayed to and recycled by the cellulose synthase; how the latter interacts with polymer-modifying enzymes that heterogenize the nascent chains for novel functionalities, and/or with cytoskeletal elements to form higher-order biosynthetic arrays for increased polymer crystallinity; how these flexible polysaccharides are guided through the periplasmic peptidoglycan mesh and to the cell surface; or how they interact with additional extracellular matrix components from the parent bacteria, synergistic or competing community microbes, or colonized eukaryotic hosts. Advancing our current understanding of cellulose biogenesis and modifications could allow us not only to design strategies for biofilm prevention and dispersal, but also to engineer cellulose superproducers and/or chemi-enzymatic systems with enhanced probiotic, biocontrol, or industrially relevant properties.

## CRediT authorship contribution statement

Petya V. Krasteva: Conceptualization, Data curation, Visualization, Funding acquisition, Writing – original draft, Writing – review & editing.

## Data Availability

No data were used for the research described in the article.

## Declaration of Competing Interest

The author declares that she has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

The author would like to acknowledge all past and present members of the "Structural Biology of Biofilms team" for their important contributions and stimulating discussions. She apologizes to colleagues whose works are not covered in this short review or are cited indirectly. Structure-function research on EPS secretion in the Krasteva lab is supported by the ERC Executive Agency under grant agreement 757507-BioMatrix-ERC-2017-StG (to P.V.K.) and the *Agence Nationale de la Recherche* (ANR, France) under grant agreements CelluSec (to P.V.K.).

## Data availability

No new or unpublished data were reported in this work.

## References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

* of special interest

* of outstanding interest

1. Jo J, Price-Whelan A, Dietrich LEP: **Gradients and consequences of heterogeneity in biofilms.** *Nat Rev Microbiol* 2022, 20:593-607, https://doi.org/10.1038/s41579-022-00692-2

2. Serra DO, Hengge R: **Bacterial multicellularity: the biology of**
*Escherichia coli* building large-scale biofilm communities. *Annu
Rev Microbiol* 2021, 75:269-290, https://doi.org/10.1146/annurev-
micro-031921-055801.

An in-depth review on metabolic, signaling and 3D-differentiation events in *E. coli* multicellular biofilms.

3. Flemming H-C, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S: **Biofilms: an emergent form of bacterial life.** *Nat Rev Microbiol* 2016, 14:563-575, https://doi.org/10.1038/nrmicro.2016.94

4. Whitfield C, Wear SS, Sande C: **Assembly of bacterial capsular polysaccharides and exopolysaccharides.** *Annu Rev Microbiol* 2020, 74:521-543, https://doi.org/10.1146/annurev-micro-011420-075607

5. Caffalette CA, Kuklewicz J, Spellmon N, Zimmer J: **Biosynthesis and export of bacterial glycolipids.** *Annu Rev Biochem* 2020, 89:741-768, https://doi.org/10.1146/annurev-biochem-011520-104707

6. Moradali MF, Rehm BHA: **Bacterial biopolymers: from pathogenesis to advanced materials.** *Nat Rev Microbiol* 2020, 18:195-210, https://doi.org/10.1038/s41579-019-0313-3

7. Abidi W, Torres-Sánchez L, Siroy A, Krasteva PV: **Weaving of**

*bacterial cellulose by the Bcs secretion systems.* FEMS Microbiol Rev 2022, 46:fuab051, https://doi.org/10.1093/femsre/fuab051.

An in-depth review on Bcs systems preceding, however, the identification of the cortical belt components.

8. Low KE, Howell PL: **Gram-negative synthase-dependent exopolysaccharide biosynthetic machines.** *Curr Opin Struct Biol* 2018, 53:32-44, https://doi.org/10.1016/j.sbi.2018.05.001

9. Bundalovic-Torma C, Whitfield GB, Marmont LS, Howell PL, Parkinson J: **A systematic pipeline for classifying bacterial operons reveals the evolutionary landscape of biofilm machineries.** PLoS Comput Biol 2020, 16:e1007721, https://doi.org/10.1371/journal.pcbi.1007721

10. Römling U, Galperin MY: **Bacterial cellulose biosynthesis:**

*diversity of operons, subunits, products, and functions.* *Trends Microbiol* 2015, 23:545-557, https://doi.org/10.1016/j.tim.2015.05.005.

An in-depth review on the diversity and distribution of bcs operons.

11. Gheorghita AA, Wozniak DJ, Parsek MR, Howell PL:

*Pseudomonas aeruginosa* biofilm exopolysaccharides: assembly, function, and degradation. *FEMS Microbiol Rev* 2023, 47:fuad060, https://doi.org/10.1093/femsre/fuad060.
*Gluconacetobacter* similar forces and cortical belt-anchored, static TCs are believed to convert the secreted cellulose ribbon in a *bona fide* motility organelle during substrate colonization [28,29].

## Conclusions and outlook

Secreted cellulosic polymers represent key architectural and functional constituents of the three-dimensional biofilms of many free-living and host-associated bacteria and are produced in both temporally and spatially regulated manner by dedicated multicomponent Bcs secretion systems [7]. Although these demonstrate functional similarities with other synthase-dependent EPS-secretory assemblies and even evolutionary distant eukaryotic systems for cellulose biogenesis, they also present important mechanistic idiosyncrasies that have only recently begun to emerge. Although bacterial cellulose biogenesis represents the longest-studied process for synthase- and c-di-GMP-dependent EPS secretion [53], it remains to be fully understood how subsets of biofilm cells switch to an energetically expensive, sugar-consuming anabolic process in typically nutrient-limited conditions to provide the scaffold and ramparts of the mature macrocolony; how in each system activating c-di-GMP is relayed to and recycled by the cellulose synthase; how the latter interacts with polymer-modifying enzymes that heterogenize the nascent chains for novel functionalities, and/or with cytoskeletal elements to form higher-order biosynthetic arrays for increased polymer crystallinity; how these flexible polysaccharides are guided through the periplasmic peptidoglycan mesh and to the cell surface; or how they interact with additional extracellular matrix components from the parent bacteria, synergistic or competing community microbes, or colonized eukaryotic hosts. Advancing our current understanding of cellulose biogenesis and modifications could allow us not only to design strategies for biofilm prevention and dispersal, but also to engineer cellulose superproducers and/or chemi-enzymatic systems with enhanced probiotic, biocontrol, or industrially relevant properties.

## CRediT authorship contribution statement

Petya V. Krasteva: Conceptualization, Data curation, Visualization, Funding acquisition, Writing – original draft, Writing – review & editing.

## Data Availability

No data were used for the research described in the article.

## Declaration of Competing Interest

The author declares that she has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

The author would like to acknowledge all past and present members of the "Structural Biology of Biofilms team" for their important contributions and stimulating discussions. She apologizes to colleagues whose works are not covered in this short review or are cited indirectly. Structure-function research on EPS secretion in the Krasteva lab is supported by the ERC Executive Agency under grant agreement 757507-BioMatrix-ERC-2017-StG (to P.V.K.) and the *Agence Nationale de la Recherche* (ANR, France) under grant agreements CelluSec (to P.V.K.).

## Data availability

No new or unpublished data were reported in this work.

## References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

* of special interest

* of outstanding interest

1. Jo J, Price-Whelan A, Dietrich LEP: *Gradients and consequences of heterogeneity in biofilms*. *Nat Rev Microbiol* 2022, 20:593-607, https://doi.org/10.1038/s41579-022-00692-2

2. Serra DO, Hengge R: *Bacterial multicellularity: the biology of Escherichia coli building large-scale biofilm communities*. *Annu Rev Microbiol* 2021, 75:269-290, https://doi.org/10.1146/annurev-micro-031921-055801.

An in-depth review on metabolic, signaling and 3D-differentiation events in *E. coli* multicellular biofilms.

3. Flemming H-C, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S: *Biofilms: an emergent form of bacterial life*. *Nat Rev Microbiol* 2016, 14:563-575, https://doi.org/10.1038/nrmicro.2016.94

4. Whitfield C, Wear SS, Sande C: *Assembly of bacterial capsular polysaccharides and exopolysaccharides*. *Annu Rev Microbiol* 2020, 74:521-543, https://doi.org/10.1146/annurev-micro-011420-075607

5. Caffalette CA, Kuklewicz J, Spellmon N, Zimmer J: *Biosynthesis and export of bacterial glycolipids*. *Annu Rev Biochem* 2020, 89:741-768, https://doi.org/10.1146/annurev-biochem-011520-104707

6. Moradali MF, Rehm BHA: *Bacterial biopolymers: from pathogenesis to advanced materials*. *Nat Rev Microbiol* 2020, 18:195-210, https://doi.org/10.1038/s41579-019-0313-3

7. Abidi W, Torres-Sánchez L, Siroy A, Krasteva PV: *Weaving of bacterial cellulose by the Bcs secretion systems*. *FEMS Microbiol Rev* 2022, 46:fuab051, https://doi.org/10.1093/femsre/fuab051.

An in-depth review on Bcs systems preceding, however, the identification of the cortical belt components.

8. Low KE, Howell PL: *Gram-negative synthase-dependent exopolysaccharide biosynthetic machines*. *Curr Opin Struct Biol* 2018, 53:32-44, https://doi.org/10.1016/j.sbi.2018.05.001

9. Bundalovic-Torma C, Whitfield GB, Marmont LS, Howell PL, Parkinson J: *A systematic pipeline for classifying bacterial operons reveals the evolutionary landscape of biofilm machineries*. *PLoS Comput Biol* 2020, 16:e1007721, https://doi.org/10.1371/journal.pcbi.1007721

10. Römling U, Galperin MY: *Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions*. *Trends Microbiol* 2015, 23:545-557, https://doi.org/10.1016/j.tim.2015.05.005.

An in-depth review on the diversity and distribution of bcs operons.

11. Gheorghita AA, Wozniak DJ, Parsek MR, Howell PL:

*Pseudomonas aeruginosa biofilm exopolysaccharides: assembly, function, and degradation*. *FEMS Microbiol Rev* 2023, 47:fuad060, https://doi.org/10.1093/femsre/fuad060.
An in-depth review on the structure and function of *P. aeruginosa* EPS secretion systems.

12. Morgan JLW, Strumillo J, Zimmer J: *Crystallographic snapshot of cellulose synthesis and membrane translocation*. *Nature* 2013, **493**:181-186, https://doi.org/10.1038/nature11744.

The first structure by X-ray crystallography of a bacterial cellulose synthase BcsAB tandem from *Rhodobacter sphaeroides*.

13. Morgan JLW, McNamara JT, Fischer M, Rich J, Chen H-M, Withers
* SG, Zimmer J: Observing cellulose biosynthesis and membrane
translocation in crystallo. *Nature* 2016, **531**:329-334, https://doi.
org/10.1038/nature16966.

A study exploring conformational changes along the *R. sphaeroides*
BcsAB catalytic cycle captured in saturating c-di-GMP conditions in
crystallo.

14. Morgan JLW, McNamara JT, Zimmer J: *Mechanism of activation of bacterial cellulose synthase by cyclic di-GMP*. *Nat Struct Mol Biol* 2014, **21**:489-496, https://doi.org/10.1038/nsmb.2803

15. Richter AM, Possling A, Malysheva N, Yousef KP, Herbst S, von

* Kleist M, Hengge R: *Local c-di-GMP signaling in the control of synthesis of the E. coli biofilm exopolysaccharide pEtN-cellulose*. *J Mol Biol* 2020, **432**:4576-4595, https://doi.org/10.1016/j.jmb.2020.06.006.

A study reporting local control of synthase-activating c-di-GMP con-
centrations via direct interactions between a dedicated diguanylate
cyclase/phosphodiesterase tandem and the cellulose secretion ma-
chinery.

16. Da Re S, Ghigo J-M: *A CsgD-independent pathway for cellulose production and biofilm formation in Escherichia coli*. *J Bacteriol* 2006, **188**:3073-3087, https://doi.org/10.1128/JB.188.8.3073-3087.2006

17. Krasteva PV, Bernal-Bayard J, Travier L, Martin FA, Kaminski P-A,

* Karimova G, Fronzes R, Ghigo PV: *Insights into the structure and assembly of a bacterial cellulose secretion system*. *Nat Commun* 2017, **8**:2065, https://doi.org/10.1038/s41467-017-01523-2.

The first structure of a multicomponent Bcs macrocomplex from *E. coli* in negative-stain electron microscopy, revealing essential for secretion roles for multiple accessory subunits.

18. Abidi W, Zouhir S, Caleechurn M, Roche S, Krasteva PV:

* Architecture and regulation of an enterobacterial cellulose secretion system. *Sci Adv* 2021, 7:eabd8049, https://doi.org/10.1126/sciadv.abd8049.

An in-depth structure-function study of the *E. coli* Bcs secretion mac-
rocomplex including cryo-EM structures of the multicomponent as-
sembly, revealing non-canonical BcsAB stoichiometry and superhelical
crown polymerization, and multiple crystallographic snapshots of reg-
ulatory subcomplexes, revealing synthase-proximal c-di-GMP enrich-
ment and ATP-dependent regulation.

19. Fang X, Ahmad I, Blanka A, Schottkowski M, Cimdins A, Galperin MY, Römling U, Gomelsky M: *GIL*, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria: *GIL*, a new c-di-GMP-binding domain. *Mol Microbiol* 2014, **93**:439-452, https://doi.org/10.1111/mmi.12672

20. Zouhir S, Abidi W, Caleechurn M, Krasteva PV: *Structure and*

* multitasking of the c-di-GMP-sensing cellulose secretion regulator BcsE. *mBio* 2020, 11:e01303-20, https://doi.org/10.1128/mBio.01303-20.

A structure-function study on the structure, c-di-GMP-binding and
functional multitasking of the cellulose secretion regulator BcsE.

21. Omadjela O, Narahari A, Strumillo J, Mélida H, Mazur O, Bulone V, Zimmer J: *BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis*. *Proc Natl Acad Sci USA* 2013, **110**:17856-17861, https://doi.org/10.1073/pnas.1314063110

22. Du J, Vepachedu V, Cho SH, Kumar M, Nixon BT: *Structure of the cellulose synthase complex of Gluconacetobacter hansenii at 23.4 Å resolution*. *PLoS One* 2016, **11**:e0155886, https://doi.org/10.1371/journal.pone.0155886

23. Le Quéré B, Ghigo J-M: *BcsQ is an essential component of the Escherichia coli cellulose biosynthesis apparatus that localizes at the bacterial cell pole*. *Mol Microbiol* 2009, **72**:724-740, https://doi.org/10.1111/j.1365-2958.2009.06678.x

24. Serra DO, Richter AM, Hengge R: *Cellulose as an architectural*

* element in spatially structured Escherichia coli biofilms. *J Bacteriol* 2013, **195**:5540-5554, https://doi.org/10.1128/JB.00946-13.

Original research article on the spatial differentiation of *E. coli* biofilms.

25. Pérez-Mendoza D, Rodríguez-Carvajal MÁ, Romero-Jiménez L, Farias G de A, Lloret J, Gallegos MT, Sanjuán J: *Novel mixed-linkage β-glucan activated by c-di-GMP in Sinorhizobium meliloti*. *Proc Natl Acad Sci USA* 2015, **112**:E757-E765, https://doi.org/10.1073/pnas.1421748112

26. Spiers AJ, Bohannon J, Gehrig SM, Rainey PB: Biofilm formation

* at the air-liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose: Biofilm formation by P. fluorescens SBW25. *Mol Microbiol* 2003, **50**:15-27, https://doi.org/10.1046/j.1365-2958.2003.03670.x.

To my knowledge the first report of covalently modified bacterial cel-
lulose.

27. Jennings LK, Storek KM, Ledvina HE, Coulon C, Marmont LS,

* Sadovskaya I, Secor PR, Tseng BS, Scian M, Filloux A, et al.: *Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix*. *Proc Natl Acad Sci USA* 2015, **112**:11353-11358, https://doi.org/10.1073/pnas.1503058112.

Characterization of the Pel EPS as a cationic, eDNA-crosslinking ma-
terial in the biofilms of *P. aeruginosa*.

28. Brown RM, Willson JH, Richardson CL: *Cellulose biosynthesis in*

* Acetobacter xylinum: visualization of the site of synthesis and direct measurement of the in vivo process*. *Proc Natl Acad Sci USA* 1976, **73**:4565-4569, https://doi.org/10.1073/pnas.73.12.4565.

An almost half-century-old report on the linear synthase arrangement and synthesis of crystalline cellulose ribbon by Gluconacetobacter.

29. Nicolas WJ, Ghosal D, Tocheva EI, Meyerowitz EM, Jensen GJ:

* Structure of the bacterial cellulose ribbon and its assembly-guiding cytoskeleton by electron cryotomography. *J Bacteriol* 2021, **203**:e00371-20, https://doi.org/10.1128/JB.00371-20.

A cryoelectron tomography study of the Gluconacetobacter crystalline
ribbon secretion with the discovery of the cortical belt cytoskeleton.

30. Thongsomboon W, Serra DO, Possling A, Hadjineophytou C,

Hengge R, Cegelski L: *Phosphoethanolamine cellulose: a naturally produced chemically modified cellulose*. *Science* 2018, **359**:334-338, https://doi.org/10.1126/science.aao4096

31. Anderson AC, Burnett AJN, Hiscock L, Maly KE, Weadge JT: *The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase*. *J Biol Chem* 2020, **295**:6225-6235, https://doi.org/10.1074/jbc.RA119.011668

32. Sun L, Vella P, Schnell R, Polyakova A, Bourenkov G, Li F, Cimdins A, Schneider TR, Lindqvist Y, Galperin MY, et al.: *Structural and functional characterization of the BcsG subunit of the cellulose synthase in Salmonella typhimurium*. *J Mol Biol* 2018, **430**:3170-3189, https://doi.org/10.1016/j.jmb.2018.07.008

33. Acheson JF, Ho R, Goularte NF, Cegelski L, Zimmer J: *Molecular organization of the E. coli cellulose synthase macrocomplex*. *Nat Struct Mol Biol* 2021, **28**:310-318, https://doi.org/10.1038/s41594-021-00569-7

34. Thongsomboon W, Werby SH, Cegelski L: *Evaluation of*

* phosphoethanolamine cellulose production among bacterial communities using congo red fluorescence. *J Bacteriol* 2020, **202**:e00030-20, https://doi.org/10.1128/JB.00030-20.

The first report of secretion of pEtN-modified bacterial cellulose and identification of BcsG as the pEtN- transferase.

35. Hollenbeck EC, Antonoplis A, Chai C, Thongsomboon W, Fuller

* GG, Cegelski L: Phosphoethanolamine cellulose enhances curli-mediated adhesion of uropathogenic *Escherichia coli* to bladder epithelial cells. *Proc Natl Acad Sci USA* 2018, **115**:10106-10111, https://doi.org/10.1073/pnas.1801564115.

An initial report on the pEtN modification as promoting curli-mediated
adhesion.

36. Tyrikos-Ergas T, Gim S, Huang J-Y, Pinzón Martín S, Varón Silva D,

* Seeberger PH, Delbianco M: *Synthetic phosphoethanolamine-modified oligosaccharides reveal the importance of glycan*
An in-depth review on the structure and function of *P. aeruginosa* EPS secretion systems.

12. Morgan JLW, Strumillo J, Zimmer J: *Crystallographic snapshot of cellulose synthesis and membrane translocation*. *Nature* 2013, **493**:181-186, https://doi.org/10.1038/nature11744.

The first structure by X-ray crystallography of a bacterial cellulose synthase BcsAB tandem from *Rhodobacter sphaeroides*.

13. Morgan JLW, McNamara JT, Fischer M, Rich J, Chen H-M, Withers
* SG, Zimmer J: Observing cellulose biosynthesis and membrane
translocation in crystallo. *Nature* 2016, **531**:329-334, https://doi.
org/10.1038/nature16966.

A study exploring conformational changes along the *R. sphaeroides*
BcsAB catalytic cycle captured in saturating c-di-GMP conditions in
crystallo.

14. Morgan JLW, McNamara JT, Zimmer J: *Mechanism of activation of bacterial cellulose synthase by cyclic di-GMP*. *Nat Struct Mol Biol* 2014, **21**:489-496, https://doi.org/10.1038/nsmb.2803

15. Richter AM, Possling A, Malysheva N, Yousef KP, Herbst S, von
Kleist M, Hengge R: *Local c-di-GMP signaling in the control of
synthesis of the E. coli biofilm exopolysaccharide pEtN-
cellulose*. *J Mol Biol* 2020, **432**:4576-4595, https://doi.org/10.
1016/j.jmb.2020.06.006.

A study reporting local control of synthase-activating c-di-GMP con-
centrations via direct interactions between a dedicated diguanylate
cyclase/phosphodiesterase tandem and the cellulose secretion ma-
chinery.

16. Da Re S, Ghigo J-M: *A CsgD-independent pathway for cellulose production and biofilm formation in Escherichia coli*. *J Bacteriol* 2006, **188**:3073-3087, https://doi.org/10.1128/JB.188.8.3073-3087.2006

17. Krasteva PV, Bernal-Bayard J, Travier L, Martin FA, Kaminski P-A,
* Karimova G, Fronzes R, Ghigo PV: *Insights into the structure and
assembly of a bacterial cellulose secretion system*. *Nat Commun* 2017, **8**:2065, https://doi.org/10.1038/s41467-017-01523-2.

The first structure of a multicomponent Bcs macrocomplex from *E. coli* in negative-stain electron microscopy, revealing essential for secretion roles for multiple accessory subunits.

18. Abidi W, Zouhir S, Caleechurn M, Roche S, Krasteva PV:
* Architecture and regulation of an enterobacterial cellulose
secretion system. *Sci Adv* 2021, 7:eabd8049, https://doi.org/10.
1126/sciadv.abd8049.

An in-depth structure-function study of the *E. coli* Bcs secretion mac-
rocomplex including cryo-EM structures of the multicomponent as-
sembly, revealing non-canonical BcsAB stoichiometry and superhelical
crown polymerization, and multiple crystallographic snapshots of reg-
ulatory subcomplexes, revealing synthase-proximal c-di-GMP enrich-
ment and ATP-dependent regulation.

19. Fang X, Ahmad I, Blanka A, Schottkowski M, Cimdins A, Galperin MY, Römling U, Gomelsky M: **GIL**, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria: **GIL**, a new c-di-GMP-binding domain. *Mol Microbiol* 2014, **93**:439-452, https://doi.org/10.1111/mmi.12672

20. Zouhir S, Abidi W, Caleechurn M, Krasteva PV: *Structure and
* multitasking of the c-di-GMP-sensing cellulose secretion
regulator BcsE. mBio* 2020, 11:e01303-20, https://doi.org/10.
1128/mBio.01303-20.

A structure-function study on the structure, c-di-GMP-binding and
functional multitasking of the cellulose secretion regulator BcsE.

21. Omadjela O, Narahari A, Strumillo J, Mélida H, Mazur O, Bulone V, Zimmer J: *BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis*. *Proc Natl Acad Sci USA* 2013, **110**:17856-17861, https://doi.org/10.1073/pnas.1314063110

22. Du J, Vepachedu V, Cho SH, Kumar M, Nixon BT: *Structure of the cellulose synthase complex of Gluconacetobacter hansenii at 23.4 Å resolution*. *PLoS One* 2016, **11**:e0155886, https://doi.org/10.1371/journal.pone.0155886

23. Le Quéré B, Ghigo J-M: *BcsQ is an essential component of the Escherichia coli cellulose biosynthesis apparatus that localizes at the bacterial cell pole*. *Mol Microbiol* 2009, **72**:724-740, https://doi.org/10.1111/j.1365-2958.2009.06678.x

24. Serra DO, Richter AM, Hengge R: *Cellulose as an architectural element in spatially structured Escherichia coli biofilms*. *J Bacteriol* 2013, **195**:5540-5554, https://doi.org/10.1128/JB.00946-13.

Original research article on the spatial differentiation of *E. coli* biofilms.

25. Pérez-Mendoza D, Rodríguez-Carvajal MÁ, Romero-Jiménez L, Farias G de A, Lloret J, Gallegos MT, Sanjuán J: *Novel mixed-linkage β-glucan activated by c-di-GMP in Sinorhizobium meliloti*. *Proc Natl Acad Sci USA* 2015, **112**:E757-E765, https://doi.org/10.1073/pnas.1421748112

26. Spiers AJ, Bohannon J, Gehrig SM, Rainey PB: *Biofilm formation at the air-liquid interface by the Pseudomonas fluorescens SBW25* wrinkly spreader requires an acetylated form of cellulose: *Biofilm formation by P. fluorescens SBW25*. *Mol Microbiol* 2003, **50**:15-27, https://doi.org/10.1046/j.1365-2958.2003.03670.x.

To my knowledge the first report of covalently modified bacterial cel-
lulose.

27. Jennings LK, Storek KM, Ledvina HE, Coulon C, Marmont LS,
* Sadovskaya I, Secor PR, Tseng BS, Scian M, Filloux A, et al.: Pel is
a cationic exopolysaccharide that cross-links extracellular
DNA in the Pseudomonas aeruginosa biofilm matrix. *Proc Natl
Acad Sci USA* 2015, **112**:11353-11358, https://doi.org/10.1073/
pnas.1503058112.

Characterization of the Pel EPS as a cationic, eDNA-crosslinking ma-
terial in the biofilms of *P. aeruginosa*.

28. Brown RM, Willson JH, Richardson CL: *Cellulose biosynthesis in Acetobacter xylinum*: visualization of the site of synthesis and direct measurement of the *in vivo* process. *Proc Natl Acad Sci USA* 1976, **73**:4565-4569, https://doi.org/10.1073/pnas.73.12.4565.

An almost half-century-old report on the linear synthase arrangement and synthesis of crystalline cellulose ribbon by Gluconacetobacter.

29. Nicolas WJ, Ghosal D, Tocheva EI, Meyerowitz EM, Jensen GJ:
* Structure of the bacterial cellulose ribbon and its assembly-
guiding cytoskeleton by electron cryotomography. *J Bacteriol* 2021, **203**:e00371-20, https://doi.org/10.1128/JB.00371-20.

A cryoelectron tomography study of the Gluconacetobacter crystalline
ribbon secretion with the discovery of the cortical belt cytoskeleton.

30. Thongsomboon W, Serra DO, Possling A, Hadjineophytou C, Hengge R, Cegelski L: *Phosphoethanolamine cellulose: a naturally produced chemically modified cellulose*. *Science* 2018, **359**:334-338, https://doi.org/10.1126/science.aao4096

31. Anderson AC, Burnett AJN, Hiscock L, Maly KE, Weadge JT: *The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase*. *J Biol Chem* 2020, **295**:6225-6235, https://doi.org/10.1074/jbc.RA119.011668

32. Sun L, Vella P, Schnell R, Polyakova A, Bourenkov G, Li F, Cimdins A, Schneider TR, Lindqvist Y, Galperin MY, et al.: *Structural and functional characterization of the BcsG subunit of the cellulose synthase in Salmonella typhimurium*. *J Mol Biol* 2018, **430**:3170-3189, https://doi.org/10.1016/j.jmb.2018.07.008

33. Acheson JF, Ho R, Goularte NF, Cegelski L, Zimmer J: *Molecular organization of the E. coli cellulose synthase macrocomplex*. *Nat Struct Mol Biol* 2021, **28**:310-318, https://doi.org/10.1038/s41594-021-00569-7

34. Thongsomboon W, Werby SH, Cegelski L: *Evaluation of
* phosphoethanolamine cellulose production among bacterial
communities using congo red fluorescence*. *J Bacteriol* 2020,
**202**:e00030-20, https://doi.org/10.1128/JB.00030-20.

The first report of secretion of pEtN-modified bacterial cellulose and identification of BcsG as the pEtN- transferase.

35. Hollenbeck EC, Antonoplis A, Chai C, Thongsomboon W, Fuller
* GG, Cegelski L: Phosphoethanolamine cellulose enhances
curli-mediated adhesion of uropathogenic Escherichia coli to
bladder epithelial cells. *Proc Natl Acad Sci USA* 2018,
**115**:10106-10111, https://doi.org/10.1073/pnas.1801564115.

An initial report on the pEtN modification as promoting curli-mediated
adhesion.

36. Tyrikos-Ergas T, Gim S, Huang J-Y, Pinzón Martín S, Varón Silva D,
* Seeberger PH, Delbianco M: Synthetic phosphoethanolamine-
modified oligosaccharides reveal the importance of glycan
length and substitution in biofilm-inspired assemblies. *Nat Commun* 2022, **13**:3954, https://doi.org/10.1038/s41467-022-31633-5

A more mechanistic article examining the influence of the pEtN mod-
ification on curli amyloid fiber polymerization using a synthetic model
system of pEtN-modified oligosaccharides and the amyogenic R5
peptide.

37. Sana TG, Notopoulou A, Puygrenier L, Decossas M, Moreau S, Carlier
* A, Krasteva PV: *Structures and roles of BcsD and partner scaffold
proteins in proteobacterial cellulose secretion*. *Curr Biol* 2024,
**34**:106-116.e6, https://doi.org/10.1016/j.cub.2023.11.057.

The first report of cellulose secretion-promoting BcsD-based cytoske-
letal elements in a variety of Bcs systems for non-crystalline cellulose
secretion and identification of BcsD oligomerization-driving structural
motifs leading to different supramolecular assemblies.

38. Gal M, Preston GM, Massey RC, Spiers AJ, Rainey PB: *Genes encoding a cellulosic polymer contribute toward the ecological success of Pseudomonas fluorescens SBW25 on plant surfaces*. *Mol Ecol* 2003, **12**:3109-3121, https://doi.org/10.1046/j.1365-294X.2003.01953.x

39. Burnett AJN, Rodriguez E, Constable S, Lowrance B, Fish M,
* Weadge JT: *Wssl from the gram-negative bacterial cellulose
synthase is an O-acetyltransferase that acts on cello-
oligomers with several acetyl donor substrates*. *J Biol Chem*
2023, **299**:104849, https://doi.org/10.1016/j.jbc.2023.104849.

In vitro enzymatic study on Wssl homologs providing validation of the
previously proposed cellulose acetylation activity of Wss components.

40. Varadi M, Anyango S, Deshpande M, Nair S, Natassia C, Yordanova G, Yuan D, Stroe O, Wood G, Laydon A, et al.: *AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models*. *Nucleic Acids Res* 2022, **50**:D439-D444, https://doi.org/10.1093/nar/gkab1061

41. Marmont LS, Rich JD, Whitney JC, Whitfield GB, Almblad H,
* Robinson H, Parsek MR, Harrison JJ, Howell PL: *Oligomeric
lipoprotein PelC guides Pel polysaccharide export across the
outer membrane of Pseudomonas aeruginosa*. *Proc Natl Acad
Sci USA* 2017, **114**:2892-2897, https://doi.org/10.1073/pnas.
1613606114.

Structure-function study of the PelC subunit acting as an electrostatic funnel for Pel guidance towards the outer membrane porin.

42. Nojima S, Fujishima A, Kato K, Ohuchi K, Shimizu N, Yonezawa K,
* Tajima K, Yao M: *Crystal structure of the flexible tandem repeat
domain of bacterial cellulose synthesis subunit C*. *Sci Rep* 2017,
**7**:13018, https://doi.org/10.1038/s41598-017-12530-0.

This study combines X-ray crystallography and small-angle X-ray
scattering (SAXS) to visualize the flexibility and likely peptidoglycan-
spanning roles of the TPR-rich N-terminal region of BcsC.

43. Pfoh R, Subramanian AS, Huang J, Little DJ, Forman A, DiFrancesco BR, Balouchestani-Asli N, Kitova EN, Klassen JS, Pomès R, et al.: *The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing*. *PLoS Pathog* 2022, **18**:e1010750, https://doi.org/10.1371/journal.ppat.1010750

44. Gheorghita AA, Li YE, Kitova EN, Bui DT, Pfoh R, Low KE, Whitfield GB, Walvoort MTC, Zhang Q, Codée JDC, et al.: *Structure of the AlgKX modification and secretion complex required for alginate production and biofilm attachment in Pseudomonas aeruginosa*. *Nat Commun* 2022, **13**:7631, https://doi.org/10.1038/s41467-022-35131-6

45. Acheson JF, Derewenda ZS, Zimmer J: Architecture of the
cellulose synthase outer membrane channel and its

association with the periplasmic TPR domain. *Structure* 2019,
*27*:1855-1861.e3, https://doi.org/10.1016/j.str.2019.09.008.

Crystal structure of the outer membrane porin of BcsC revealing pos-
sible role of C-terminal in modified cellulose secretion as reviewed in
reference 7.

46. Maeda K, Tamura J, Okuda Y, Narikawa R, Midorikawa T, Ikeuchi
* M: *Genetic identification of factors for extracellular cellulose
accumulation in the thermophilic cyanobacterium
*Thermosynechococcus vulcanus*: proposal of a novel tripartite
secretion system: *Cyanobacterial genes for cellulose
accumulation*. *Mol Microbiol* 2018, **109**:121-134, https://doi.org/
**10.1111/mmi.13977**.

Report of an atypical tripartite system for cellulose secretion relying on a TolC-like protein for outer membrane export

47. Li T, Chen C, Brozena AH, Zhu JY, Xu L, Driemeier C, Dai J, Rojas OJ, Isogai A, Wågberg L, et al.: *Developing fibrillated cellulose as a sustainable technological material*. *Nature* 2021, **590**:47-56, https://doi.org/10.1038/s41586-020-03167-7

48. Hu S-Q, Gao Y-G, Tajima K, Sunagawa N, Zhou Y, Kawano S,
* Fujiwara T, Yoda T, Shimura D, Satoh Y, et al.: *Structure of
bacterial cellulose synthase subunit D octamer with four inner
passageways*. *Proc Natl Acad Sci USA* 2010, **107**:17957-17961,
https://doi.org/10.1073/pnas.1000601107.

This study reports crystal structures of the Gluconacetobacter BcsD subunit and its characteristic donut-like architecture with four independent passageways, leading to proposed roles in periplasmic cellulose extrusion. While recent studies have placed BcsD in the cytosol as a major component of the cortical belt, they do not exclude that the protein is also secreted to prevent cellulose strand aggregation in the periplasm.

49. Abidi W, Decossas M, Torres-Sánchez L, Puygrenier L, Létoffé S,
* Ghigo J-M, Krasteva PV: *Bacterial crystalline cellulose secretion
via a supramolecular BcsHD scaffold*. *Sci Adv* 2022,
**8**:eadd1170, https://doi.org/10.1126/sciadv.add1170.

Cryo-EM structure of the BcsH-driven BcsD polymerization in 'beads-
on-a-string' filaments and identification of the two proteins as the likely
constituents of the intracellular 'cortical belt' cytoskeleton for inside-out
control of cellulose secretion.

50. Sunagawa N, Fujiwara T, Yoda T, Kawano S, Satoh Y, Yao M,
* Tajima K, Dairi T: *Cellulose complementing factor (Ccp) is a new
member of the cellulose synthase complex (terminal complex)
in Acetobacter xylinum*. *J Biosci Bioeng* 2013, **115**:607-612,
https://doi.org/10.1016/j.jbiosc.2012.12.021.

A report on the BcsH-dependent BcsD linear array formation. The study suggests that BcsH is a short peptide, however recent work has shown that BcsH is a proline-rich self-oligomerizing protein driving arrangement of BcsD in the cortical belt cytoskeleton

51. Purushotham P, Ho R, Zimmer J: Architecture of a catalytically
**active homotrimeric plant cellulose synthase complex**. *Science*
2020, **369**:1089-1094, https://doi.org/10.1126/science.abb2978.

The first cryo-EM structure of a eukaryotic cellulose synthase, CesA8,
from Populus tremula x tremuloides.

52. Lampugnani ER, Flores-Sandoval E, Tan QW, Mutwil M, Bowman JL, Persson S: *Cellulose synthesis – central components and their evolutionary relationships*. *Trends Plant Sci* 2019, **24**:402-412, https://doi.org/10.1016/j.tplants.2019.02.011

53. Ross P, Weinhouse H, Aloni Y, Michaeli D, Weinberger-Ohana P, Mayer R, Braun S, de Vroom E, van der Marel GA, van Boom JH, et al.: *Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid*. *Nature* 1987, **325**:279-281, https://doi.org/10.1038/325279a0
length and substitution in biofilm-inspired assemblies. *Nat Commun* 2022, **13**:3954, https://doi.org/10.1038/s41467-022-31633-5

A more mechanistic article examining the influence of the pEtN mod-
ification on curli amyloid fiber polymerization using a synthetic model
system of pEtN-modified oligosaccharides and the amyogenic R5
peptide.

37. Sana TG, Notopoulou A, Puygrenier L, Decossas M, Moreau S, Carlier
* A, Krasteva PV: *Structures and roles of BcsD and partner scaffold
proteins in proteobacterial cellulose secretion*. *Curr Biol* 2024,
**34**:106-116.e6, https://doi.org/10.1016/j.cub.2023.11.057.

The first report of cellulose secretion-promoting BcsD-based cytoske-
letal elements in a variety of Bcs systems for non-crystalline cellulose
secretion and identification of BcsD oligomerization-driving structural
motifs leading to different supramolecular assemblies.

38. Gal M, Preston GM, Massey RC, Spiers AJ, Rainey PB: *Genes encoding a cellulosic polymer contribute toward the ecological success of Pseudomonas fluorescens SBW25 on plant surfaces*. *Mol Ecol* 2003, **12**:3109-3121, https://doi.org/10.1046/j.1365-294X.2003.01953.x

39. Burnett AJN, Rodriguez E, Constable S, Lowrance B, Fish M,
* Weadge JT: *Wssl from the gram-negative bacterial cellulose
synthase is an O-acetyltransferase that acts on cello-
oligomers with several acetyl donor substrates*. *J Biol Chem*
2023, **299**:104849, https://doi.org/10.1016/j.jbc.2023.104849.

In vitro enzymatic study on Wssl homologs providing validation of the
previously proposed cellulose acetylation activity of Wss components.

40. Varadi M, Anyango S, Deshpande M, Nair S, Natassia C, Yordanova G, Yuan D, Stroe O, Wood G, Laydon A, et al.: *AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models*. *Nucleic Acids Res* 2022, **50**:D439-D444, https://doi.org/10.1093/nar/gkab1061

41. Marmont LS, Rich JD, Whitney JC, Whitfield GB, Almblad H,
* Robinson H, Parsek MR, Harrison JJ, Howell PL: *Oligomeric
lipoprotein PelC guides Pel polysaccharide export across the
outer membrane of Pseudomonas aeruginosa*. *Proc Natl Acad
Sci USA* 2017, **114**:2892-2897, https://doi.org/10.1073/pnas.
1613606114.

Structure-function study of the PelC subunit acting as an electrostatic funnel for Pel guidance towards the outer membrane porin.

42. Nojima S, Fujishima A, Kato K, Ohuchi K, Shimizu N, Yonezawa K,
* Tajima K, Yao M: *Crystal structure of the flexible tandem repeat
domain of bacterial cellulose synthesis subunit C*. *Sci Rep* 2017,
**7**:13018, https://doi.org/10.1038/s41598-017-12530-0.

This study combines X-ray crystallography and small-angle X-ray
scattering (SAXS) to visualize the flexibility and likely peptidoglycan-
spanning roles of the TPR-rich N-terminal region of BcsC.

43. Pfoh R, Subramanian AS, Huang J, Little DJ, Forman A, DiFrancesco BR, Balouchestani-Asli N, Kitova EN, Klassen JS, Pomès R, et al.: *The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing*. *PLoS Pathog* 2022, **18**:e1010750, https://doi.org/10.1371/journal.ppat.1010750

44. Gheorghita AA, Li YE, Kitova EN, Bui DT, Pfoh R, Low KE, Whitfield GB, Walvoort MTC, Zhang Q, Codée JDC, et al.: *Structure of the AlgKX modification and secretion complex required for alginate production and biofilm attachment in Pseudomonas aeruginosa*. *Nat Commun* 2022, **13**:7631, https://doi.org/10.1038/s41467-022-35131-6

45. Acheson JF, Derewenda ZS, Zimmer J: Architecture of the
cellulose synthase outer membrane channel and its

association with the periplasmic TPR domain. *Structure* 2019,
**27**:1855-1861.e3, https://doi.org/10.1016/j.str.2019.09.008.

Crystal structure of the outer membrane porin of BcsC revealing pos-
sible role of C-terminal in modified cellulose secretion as reviewed in
reference 7.

46. Maeda K, Tamura J, Okuda Y, Narikawa R, Midorikawa T, Ikeuchi
* M: *Genetic identification of factors for extracellular cellulose
accumulation in the thermophilic cyanobacterium
*Thermosynechococcus vulcanus*: proposal of a novel tripartite
secretion system: *Cyanobacterial genes for cellulose
accumulation*. *Mol Microbiol* 2018, **109**:121-134, https://doi.org/
**10.1111/mmi.13977**.

Report of an atypical tripartite system for cellulose secretion relying on a TolC-like protein for outer membrane export

47. Li T, Chen C, Brozena AH, Zhu JY, Xu L, Driemeier C, Dai J, Rojas OJ, Isogai A, Wågberg L, et al.: *Developing fibrillated cellulose as a sustainable technological material*. *Nature* 2021, **590**:47-56, https://doi.org/10.1038/s41586-020-03167-7

48. Hu S-Q, Gao Y-G, Tajima K, Sunagawa N, Zhou Y, Kawano S,
* Fujiwara T, Yoda T, Shimura D, Satoh Y, et al.: *Structure of
bacterial cellulose synthase subunit D octamer with four inner
passageways*. *Proc Natl Acad Sci USA* 2010, **107**:17957-17961,
https://doi.org/10.1073/pnas.1000601107.

This study reports crystal structures of the Gluconacetobacter BcsD subunit and its characteristic donut-like architecture with four independent passageways, leading to proposed roles in periplasmic cellulose extrusion. While recent studies have placed BcsD in the cytosol as a major component of the cortical belt, they do not exclude that the protein is also secreted to prevent cellulose strand aggregation in the periplasm.

49. Abidi W, Decossas M, Torres-Sánchez L, Puygrenier L, Léstoffé S,
* Ghigo J-M, Krasteva PV: *Bacterial crystalline cellulose secretion
via a supramolecular BcsHD scaffold*. *Sci Adv* 2022,
**8**:eadd1170, https://doi.org/10.1126/sciadv.add1170.

Cryo-EM structure of the BcsH-driven BcsD polymerization in 'beads-
on-a-string' filaments and identification of the two proteins as the likely
constituents of the intracellular 'cortical belt' cytoskeleton for inside-out
control of cellulose secretion.

50. Sunagawa N, Fujiwara T, Yoda T, Kawano S, Satoh Y, Yao M,
* Tajima K, Dairi T: *Cellulose complementing factor (Ccp) is a new
member of the cellulose synthase complex (terminal complex)
in Acetobacter xylinum*. *J Biosci Bioeng* 2013, **115**:607-612,
https://doi.org/10.1016/j.jbiosc.2012.12.021.

A report on the BcsH-dependent BcsD linear array formation. The study suggests that BcsH is a short peptide, however recent work has shown that BcsH is a proline-rich self-oligomerizing protein driving arrangement of BcsD in the cortical belt cytoskeleton

51. Purushotham P, Ho R, Zimmer J: Architecture of a catalytically
**active homotrimeric plant cellulose synthase complex**. *Science*
2020, **369**:1089-1094, https://doi.org/10.1126/science.abb2978.

The first cryo-EM structure of a eukaryotic cellulose synthase, CesA8,
from Populus tremula x tremuloides.

52. Lampugnani ER, Flores-Sandoval E, Tan QW, Mutwil M, Bowman JL, Persson S: *Cellulose synthesis – central components and their evolutionary relationships*. *Trends Plant Sci* 2019, **24**:402-412, https://doi.org/10.1016/j.tplants.2019.02.011

53. Ross P, Weinhouse H, Aloni Y, Michaeli D, Weinberger-Ohana P, Mayer R, Braun S, de Vroom E, van der Marel GA, van Boom JH, et al.: *Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid*. *Nature* 1987, **325**:279-281, https://doi.org/10.1038/325279a0