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org/biochemistry Article

A Mechanistic Basis for Phosphoethanolamine Modification of the

Cellulose Biofilm Matrix in Escherichia coli
Alexander C. Anderson, Alysha J. N. Burnett, Shirley Constable, Lana Hiscock, Kenneth E. Maly,
and Joel T. Weadge*
Cite This: Biochemistry 2021, 60, 3659−3669 Read Online

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ABSTRACT: Biofilms are communities of self-enmeshed bacteria

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in a matrix of exopolysaccharides. The widely distributed human

pathogen and commensal Escherichia coli produces a biofilm matrix
composed of phosphoethanolamine (pEtN)-modified cellulose and
amyloid protein fibers, termed curli. The addition of pEtN to the
cellulose exopolysaccharide is accomplished by the action of the
pEtN transferase, BcsG, and is essential for the overall integrity of
the biofilm. Here, using the synthetic co-substrates p-nitrophenyl
phosphoethanolamine and β-D-cellopentaose, we demonstrate
using an in vitro pEtN transferase assay that full activity of the
pEtN transferase domain of BcsG from E. coli (EcBcsGΔN) requires
Zn2+ binding, a catalytic nucleophile/acid-base arrangement
(Ser278/Cys243/His396), disulfide bond formation, and other
newly uncovered essential residues. We further confirm that EcBcsGΔN catalysis proceeds by a ping-pong bisubstrate−biproduct
reaction mechanism and displays inefficient kinetic behavior (kcat/KM = 1.81 × 10−4 ± 2.81 × 10−5 M−1 s−1), which is typical of
exopolysaccharide-modifying enzymes in bacteria. Thus, the results presented, especially with respect to donor binding (as reflected
by KM), have importantly broadened our understanding of the substrate profile and catalytic mechanism of this class of enzymes,
which may aid in the development of inhibitors targeting BcsG or other characterized members of the pEtN transferase family,
including the intrinsic and mobile colistin resistance factors.

iofilms are dynamic communities of microorganisms fixed

B in a synthesized matrix of secreted polysaccharide
materials.1 This biofilm matrix serves as the interface between
its constituents and their outside environment, but also plays
many other roles to the benefit of the enclosed microorganisms
that may justify the large resource cost of producing copious
quantities of this extracellular matrix material.2 For example,
biofilms have been observed to serve in resource capture,3
accelerate cell growth,4 and improve tolerance to stressors or
disinfectants, such as shearing forces,2 desiccation,2 antimicro-
bial compounds,2,5 extreme temperature,2,5,6 or sanitizing
agents2,3,5 for the enclosed microorganisms. The persistent
colonization of surfaces by the widely distributed human and
animal pathogens Escherichia coli and Salmonella spp. has been
linked to the production of biofilms composed of the
polysaccharide cellulose and amyloid protein fibers termed
curli.7 In addition to these species, biofilms containing
cellulose have been reported in numerous other species,
including other pathogens of plants and humans, like
Agrobacterium tumefaciens, Cronobacter sakazakii, Acetobacter
xylinum, Clostridium (previously Sarcina) spp., Rhizobium spp.,
and some Pseudomonads.8−12 Although the biofilms of these
bacteria were long thought to contain a similar linear β-(1−4)-
glucan, the diversity of these biofilm polysaccharides is
© 2021 The Authors. Published by
American Chemical Society
3659
beginning to be understood.13 For example, the biofilm of
Pseudomonas f luorescens SBW25 was observed to contain an
acetylated form of cellulose,8 and more recently, the discovery
of phosphoethanolamine (pEtN) cellulose was reported in E.
coli and Salmonella enterica (Figure 1).14 Cellulose has also
been recently proposed in the Gram-positive human pathogen
Clostridioides dif f icile,12 and an operon for the production of
Pel exopolysaccharide was reported in Bacillus cereus,15 thereby
expanding the repertoire of bacteria known to produce these
exopolysaccharides beyond Gram-negative organisms.
At present, all Gram-negative bacteria known to produce
cellulose as a component of their biofilm possess an operon
encoding the essential and conserved protein complex
responsible for cellulose biosynthesis and export.16 Structural
and functional studies, informed by research on other
polysaccharide secretion systems, have provided evidence for
Received: September 10, 2021
Revised: October 22, 2021
Published: November 11, 2021
https://doi.org/10.1021/acs.biochem.1c00605
Biochemistry 2021, 60, 3659−3669
Biochemistry pubs.acs.org/biochemistry
Figure 1. Schematic of the reaction catalyzed by BcsG.
Phosphoethanolamine is added to approximately 50% of glucose
units of bacterial cellulose, exclusively at the C6 position with an
unknown pattern across the polymer.14,25
the roles of each gene product in this operon, often annotated
bcsABCZ.17 Biosynthesis of cellulose occurs by the action of
the processive glycosyltransferase BcsA at the cytoplasmic face
of the inner membrane, using UDP-glucose as a substrate.18
Regulation of cellulose biosynthesis occurs through the binding
of the second messenger molecule cyclic dimeric guanosine
monophosphate (c-di-GMP) to the PilZ domain of BcsA,
allosterically regulating the activity of the glycosyltransferase
domain.19 Translocation of the growing chain from the
cytoplasm to the periplasm occurs co-synthetically, through a
pore in BcsA, and is aided by a carbohydrate-binding domain
found in the periplasmic protein BcsB.18,20 Similar to structural
and functional studies of other polysaccharide systems,
cellulose export across the outer membrane was recently
shown to require an outer membrane β-barrel and periplasmic
tetratricopeptide repeat (TPR)-containing protein, encoded by
BcsC.21−24 Finally, a periplasmic polysaccharide lyase or
glycoside hydrolase is found in many known polysaccharide
biosynthesis operons.22 With respect to microbial cellulose,
structural and functional studies of BcsZ from E. coli and CcsZ
from C. dif f icile demonstrated that these terminal enzymes
were from glycoside hydrolase families 8 and 5, respectively,
and both possess endo-β-(1,4)-glucanase activity for mod-
ification of the polymer.12,26
Previously, the role of accessory operons or genes within
these operons remained unknown until the discovery of
modified polysaccharide materials within biofilms.17 For
example, in cellulose-producing organisms, the role of a type
II operon, containing bcsEFG, was unknown, and its gene
products showed little similarity to other characterized proteins
based on sequence.17 These genes were annotated as necessary
for maximal cellulose production,17 but more recently,
structural and functional characterization has shed light on
their true roles in processing of the cellulose polysacchar-
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ide.14,25,27−29 The BcsE protein was shown to comprise both a
degenerate receiver domain and a GGDEF domain responsible
for sensing c-di-GMP.28 BcsE has also recently been proposed
to be responsible for the recruitment of BcsQ and BcsR to the
membrane during early Bcs macrocomplex assembly.29 Other
recent efforts with cryo-electron microscopy have resolved the
native Bcs macrocomplex in several states and demonstrate its
piecewise assembly.30,31 In light of the discovery of pEtN
cellulose, bcsG was also shown to be necessary for the
architecture and assembly of the biofilm matrix.14 BcsG was
further proposed to be the pEtN transferase responsible for the
postsynthetic modification of cellulose in the periplasm of
organisms producing a pEtN cellulose polysaccharide.14,27 We
previously reported the structure and activity of BcsG from E.
coli (EcBcsG), demonstrating that it acts as a pEtN transferase
with substrate specificity for cellulose.25 Additionally, the
structure and function of BcsG from Salmonella typhimurium
(StBcsG) was independently reported, and the results support
the conclusion that EcBcsG and StBcsG are functionally
equivalent pEtN transferases acting on cellulose polysacchar-
ides.27
Both EcBcsG and StBcsG studies reported the functional
complementation of bcsG using a library of BcsG amino acid
variants.25,27 These combined results demonstrated the
essential catalytic residues of BcsG, which are partially
conserved among other characterized pEtN transferases,
including the intrinsic and mobile colistin resistance factors.
Disruption of these essential residues in EcBcsG resulted in a
fragile pellicle biofilm phenotype in E. coli AR3110 that was
indistinguishable from a strain bearing a chromosomal deletion
of bcsG.25 However, the enzymatic consequences of amino acid
substitutions that resulted in these phenotypes remain
unexplored. Using an in vitro pEtN transferase assay, we
show here that Zn2+ binding, disulfide bond formation, and the
catalytic nucleophile/acid-base arrangement (Ser278/Cys243/
His396) are essential for full transferase activity of the
recombinant catalytic domain of EcBcsG (herein EcBcsGΔN).
Furthermore, we expand the donor substrate profile to confirm
EcBcsGΔN is specific for pEtN transfer and report the
Michaelis−Menten kinetics of EcBcsG using the co-substrate
analogues p-nitrophenyl phosphoethanolamine (p-NPPE) and
cello-oligosaccharides. We also demonstrate that the kinetic
behavior of EcBcsG further supports a ping-pong bisubstrate−
biproduct (bibi) catalytic mechanism and expand the active
site to include new residues proposed by examination of the
structural model to be involved in catalysis.
■ EXPERIMENTAL PROCEDURES
Materials. Culture media, isopropyl β-D-thiogalactopyrano-
side (IPTG), and kanamycin sulfate were purchased from
BioBasic (Markham, ON). Cello-oligomers were purchased
from Megazyme (Dublin, Ireland). Nickel-nitrilotriacetic acid
(Ni-NTA) agarose was purchased from Qiagen (Valencia,
CA). Unless otherwise specified, all other materials were
purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON).
Cloning, Expression, and Purification of EcBcsGΔN.
The catalytic domain of the bcsG gene was cloned into the
pET28a expression vector, as described previously.25 Briefly,
bcsGΔN was amplified from E. coli K12 chromosomal DNA and
cloned into the pET28a expression vector using NdeI and XhoI
restriction sites. The polymerase chain reaction (PCR)
products were digested by the appropriate enzymes for 1 h,
followed by purification using a GeneJET PCR purification kit
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Biochemistry pubs.acs.org/biochemistry
(Thermo Fisher Scientific). The purified PCR product was
ligated into an NdeI/XhoI doubly digested vector using T4
DNA ligase (Thermo Fisher Scientific) at 22 °C for 4 h. The
ligation mixture was used to transform E. coli TOP10 (Thermo
Fisher Scientific), and pET28a-bcsGΔN was isolated from 16−
18 h grown cultures using an EZ-10 plasmid prep kit (Bio
Basic). The resulting recombinant BcsG C-terminal catalytic
domain, EcBcsGΔN, was comprised of residues Ala164−Gln559
from UniProt entry P37659 and included the addition of an N-
terminal hexahistidine tag conferred by the pET28a expression
plasmid.
Site-Directed Mutagenesis. Site-directed mutagenesis
was performed using the PCR primer method with pET28a-
bcsGΔN as a template. Amino acid variants of Arg458 and Ser244
were generated from site-directed mutagenesis of the wild-type
pET28a-bcsGΔN plasmid as a service provided by BioBasic. The
primers used to generate the alanine EcBcsGΔN amino acid
variants of Cys243, Tyr277, Ser278, His396, Glu442, and His443 are
available in Table S1. Following amplification of amino acid
variant plasmids, DpnI (1 unit) was added to reaction mixtures
to digest methylated template DNA. The reaction mixture was
transformed into E. coli TOP10, and the resulting clones were
sequenced to confirm the presence of the desired mutations
(The Centre for Applied Genomics).
Expression and Purification of EcBcsGΔN and Deriv-
atives. pET28a-bcsGΔN and derivative plasmids thereof were
transformed into E. coli BL21 (DE3), and cells were grown in 1
L volumes of rich medium (32 g of tryptone, 20 g of yeast
extract, and 10 g of NaCl) containing kanamycin (50 μg
mL−1). Cultures were incubated at 37 °C while being shaken,
until they reached an optical density at 600 nm of 0.6−0.8.
Expression of EcBcsGΔN and amino acid variants thereof was
induced by the addition of IPTG (1 mM), and growth was
allowed to continue for 16−20 h at 23 °C. The cells were
collected by centrifugation (4300 × g for 15 min at 4 °C) and
stored at −20 °C until use. Cell pellets were resuspended in
lysis buffer [50 mM Tris (pH 8.0) and 300 mM NaCl] and
supplemented with 0.5 mg of RNase A (BioBasic), 0.25 mg of
DNase I (BioBasic), and for some variants one EDTA-free
mini protease inhibitor tablet (Roche). The resulting cell
suspensions were lysed using a cell disruptor (Constant
Systems) operating at 17000 psi (117211 kPa) and 4 °C. The
lysate was separated by centrifugation (28000 × g for 45 min at
4 °C), and hexahistidine-tagged EcBcsGΔN (and amino acid
variants thereof) were purified from the soluble fraction using
nickel affinity chromatography. Ni-NTA resin (Thermo Fisher
Scientific) pre-equilibrated in lysis buffer (2 mL) was
suspended in the cell lysate and incubated for 1 h at 4 °C to
facilitate binding. This solution was filtered by being passed
over a chromatography column and washing of the resin in at
least three 10 mL volumes of lysis buffer, followed by three 10
mL volumes of lysis buffer with the addition of 25 mM
imidazole. Elution was achieved by addition of 10 mL of
elution buffer (lysis buffer with the addition of 250 mM
imidazole) to the chromatography resin. Buffer exchange was
performed by a two-step dialysis against 50 mM Tris (pH 8.0)
and 150 mM NaCl using two 2 L volumes for at least 1 h per
volume at 4 °C. Secondary purification was performed by
anion exchange chromatography when deemed necessary as
assessed by sodium dodecyl sulfate−polyacrylamide gel
electrophoresis. For secondary purification, proteins were
buffer exchanged by dialysis against 50 mM Tris (pH 8.0)
using two 2 L volumes for at least 1 h per volume at 4 °C. The
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resulting solutions were loaded onto an Ä KTA Start fast-
protein liquid chromatography instrument (Cytiva) equipped
with a HiTrap Q FF cartridge (1 mL, Cytiva). The solvents
were (A) 50 mM Tris (pH 8.0) and (B) 50 mM Tris and (pH
8.0) and 1 M NaCl. The protein was bound by passing sample
five times over the column at a flow rate of 1 mL/min in 100%
A. The column was washed for 20 min with 100% A, followed
by elution in a linear gradient from 0% to 100% B over 30 min.
Peaks of >50 mAU at 280 nm were collected using a Frac30
fraction collector (Cytiva). All purification buffers were
supplemented with 5 mM MgCl2, as we observed this to
improve the stability of recombinant EcBcsGΔN and deriva-
tives. Recombinant protein eluted from purification columns
was pooled and concentrated to between 30 and 45 mg mL−1
in a 30 kDa molecular weight cutoff centrifugal filter unit
(Cytiva) and stored at 4 or −20 °C for short- or long-term
storage, respectively.
Measurement of the Enzymatic Rate. The enzymatic
rate of EcBcsGΔN and derivatives was measured with the
chromogenic assay using the mock co-substrates p-nitrophenyl
phosphoethanolamine (p-NPPE), p-nitrophenyl phosphopro-
panolamine (p-NPPP), p-nitrophenyl phosphate (p-NPP)
(Sigma), and cellooligosaccharides, as described previously.25
The synthesis of p-NPPE was performed as described
previously.25 The detailed synthesis of p-NPPP is described
in the Supporting Information. The esterase activity of
EcBcsGΔN and amino acid variants thereof was measured
using 7 mM p-NPPE and 50 μM enzyme. The substrates p-
NPPP and p-NPP were tested at 2 mM with and without 3
mM cellopentaose. Transferase activity was measured under
the same conditions, but only in the presence of
cellooligosaccharides, as described previously.25 All reactions
were carried out in 50 mM HEPES buffer (pH 7.5) containing
50 mM NaCl at 37 °C using 100 μL volumes. Concentrations
of 1 mM ethylenediamine tetraacetic acid (EDTA) or
dithiothreitol (DTT) were exposed to EcBcsGΔN by buffer
exchange for no less than 24 h prior to the assay. Reactions
were monitored in a Cytation5 imaging plate reader (Biotek)
at 37 °C for 30 min at 414 nm. The specific activity and
velocity of the reactions were calculated from absorbance data
using a standard curve prepared with p-nitrophenol analytical
reference material (Sigma, catalog no. 241326). All rates are
reported as the mean ± the standard deviation (SD) of at least
three replicates. Figure preparation and statistical analysis were
performed using GraphPad Prism version 9.0.1.
Steady-State Kinetics. Kinetic assays were performed
using the same assay design described above but with p-NPPE
concentrations as specified that ranged from 0 to 14.5 mM.
Kinetics with the cellooligosaccharide acceptor were also
conducted with concentrations ranging from 0 to 6 mM with a
fixed p-NPPE concentration of 7 mM. Michaelis−Menten
parameters (kcat and KM) were determined by nonlinear
regression analysis of plots of initial velocity (nanomoles per
second) as a function of p-NPPE concentration. All model
fitting, analysis, and figure preparation were performed using
GraphPad Prism version 9.0.1.
Mass Spectrometry. Liquid chromatography−mass spec-
trometry analyses were performed at the Mass Spectrometry
Facility of the Advanced Analysis Centre (University of
Guelph, Guelph, ON) on an Agilent 1200 HPLC liquid
chromatograph interfaced with an Agilent UHD 6530 Q-TOF
mass spectrometer. A C18 column (Agilent Extend-C18, 50
mm × 2.1 mm, 1.8 μm) was used for chromatographic
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Figure 2. Amino acid replacements of essential catalytic residues abrogate EcBcsGΔN activity. (A) The structure of the EcBcsGΔN active site (PDB
entry 6PD0) is depicted here and was previously mapped by functional complementation in vivo.25 (B) The measured rates of esterase (i.e.,
carbohydrate acceptor-free conditions) and transferase (i.e., in the presence of a cellopentaose acceptor) activities of EcBcsGΔN amino acid variants
within the catalytic site demonstrate these residues are essential in vitro with the exception of Y277. Asterisks denote statistical significance [Tukey’s
multiple comparisons, q(33) > 8.40 and p < 0.0001 for all comparisons].

separation. The following solvents were used for separation:
water with 0.1% (v/v) formic acid (A) and acetonitrile with
0.1% (v/v) formic acid (B). The initial mobile phase
conditions were 10% B, hold for 1 min, and then increase to
100% B over 29 min.
in this study (C243A, S244A, Y277F, S278A, H396A, E442A,
H443A, R458A, and R458H) were generated using site-
directed mutagenesis of pET28a-bcsGΔN. Each of these
recombinant proteins were expressed in E. coli BL21
transformants with observed yields similar to that of wild-

■ RESULTS AND DISCUSSION

Amino Acid Replacements. To assess the catalytic
consequences of amino acid substitutions within the EcBcsG
active site, we constructed a library of amino acid EcBcsGΔN
variants with alanine substitutions of active site residues
observed to be essential for catalytic activity in vivo (Cys243,
Tyr277, Ser278, His396, Glu442, and His443). These residues
represent the structural equivalent to the active site of known
pEtN transferases. Interestingly, only His443 and His396 are
strictly conserved, although our previous work demonstrated
that the remainder are catalytically essential in vivo and
probably represent the functional equivalents to the conserved
residues of other pEtN transferases.25 However, the con-
sequences of these amino acid replacements remained untested
on an enzymatic level, so herein we explored these residues,
along with two new residues we suspected may be catalytically
important (Ser244 and Arg458) based on inspection of the BcsG
structure and that of its homologues. All of the constructs used
3662
type EcBcsGΔN. These variant proteins were purified to
apparent homogeneity using the protocol described previ-
ously,25 with additional washing steps as deemed necessary for
purification to homogeneity. Care was taken to use new
chromatography media for the purification of each respective
enzyme to prevent enzyme carryover and cross-contamination
of assay data with multiple enzyme forms.
As expected, replacement of the nucleophilic Ser278 (S278A)
resulted in a loss of any detectable activity. The observed rates
of both esterase (i.e., bulk solvent serves as the pEtN acceptor)
and transferase activity (i.e., in the presence of a defined
carbohydrate acceptor) of the S278A replacement were not
different from an enzyme-free control (Figure 2; t tests, t(4) =
0.3586 and p = 0.7830 for esterase; t(4) = 0.4935 and p =
0.6475 for transferase). This lack of activity supports the role
of this nucleophilic serine in the catalytic mechanism. His396 is
highly conserved and has also been shown to be catalytically
important in other pEtN transferases.32 Replacement of this
histidine in BcsG (H396A) also led to reductions in activity
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with a residual esterase activity of 16.6% and a residual
transferase activity of 7.0% (Figure 2), which is consistent with
results previously noted for this mutation in the in vivo biofilm
assays, where alanine substitution of this residue resulted in the
fragile pellicle phenotype.25
Substantial losses of activity were also observed with alanine
replacement of the metal-binding triad residues of the enzyme
(Figure 2). The Glu442 replacement (E442A) resulted in an
esterase activity indistinguishable from an enzyme-free control
(e.g., 1.96 ± 0.07 nmol min−1 mg−1), and only 2.3% residual
transferase activity was detected in the presence of β-D-
cellopentaose. Replacement of Cys243 with alanine (C243A)
also resulted in low residual specific activities of 4.7%
(esterase) and 2.4% (transferase). The His443 substitution
(H443A) also demonstrated reduced specific activities,
although to a lesser degree, representing 38.5% residual
esterase and 10.4% residual transferase activity. The retention
of some activity by His443 may be explained by the observation
of Ser278 also serving as a ligand for Zn2+ binding in some of
the structures when His443 is not. Completion of the Zn2+
coordination sphere in at least one of the structural models
shows the Zn2+ also bound to a water molecule, which is
consistent with the Zn2+ playing a catalytic and not a structural
role.33 Combined, these observations and the low residual
specific activities are consistent with the essential Zn2+ binding
roles of these residues that had previously been noted in the
structural model and assayed in vivo.25 These findings further
support the observation that Zn2+ binding is indispensable for
the catalytic activity of BcsG in vivo, suggesting the residues we
replaced here are truly equivalent to the catalytic residues of
other polymyxin resistance factors.25
Previous mass spectrometry and structural data we collected
suggested that an additional residue may contribute to the
catalytic fold and belonged to the loop that presents Cys243 to
the active site.25 Although we did not test a full-length BcsG
amino acid replacement in vivo, we propose that the identity of
this residue is Ser244 (Figure 2). The side chain hydroxyl of
Ser244 is oriented toward the Zn2+ at an atomic distance of 4.5
Å, and this residue is the only one that could plausibly serve an
essential function given both our structural and mass
spectrometry data sets. In support of our hypothesis, we
found that replacement of Ser244 with alanine (S244A) resulted
in 7.2% residual esterase and 5.8% residual transferase activity,
demonstrating that Ser244 is indeed part of the active site and is
required for full activity.
To our surprise, replacement of Tyr277 with Phe (Y277F)
produced an enzyme variant with greater esterase activity and
84% residual transferase activity compared to wild-type
EcBcsGΔN (Figure 2). This observation did not support our
earlier proposal that Tyr277 may function in cellulose
accommodation in the proximity of the Zn2+ ion at the active
center.25 An increase in solvent accessibility to the active site
may account for the improved rate of esterase activity and an
increased level of competition for transfer to oligosaccharides
that is reflected by the lower transferase activity. This
hypothesis is further supported by the fact that an equivalent
tyrosine residue is not observed in available structures of other
pEtN transferases,32,34,35 nor was a tyrosine residue observed
to participate in contact with the lipid-binding site in the
structure of the full-length pEtN transferase EptA from
Neisseria meningitidis [NmEptA, Protein Data Bank (PDB)
entry 5FGN].36 Instead, the residue equivalent to Tyr277 in
NmEptA, Ser279, appears to shape the lipid-binding pocket at
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the interface of the catalytic and membrane domains.36
Consequently, we rationalized that this disparity in our data
may indicate that the role of the Tyr277 hydroxyl may also
include the stability of the local folding and interaction of the
EcBcsG C-terminal catalytic domain relative to the N-terminal
transmembrane region and, thus, allow EcBcsG to sample
productive conformations during the catalytic cycle. This
hypothesis would account for the importance of Tyr277 in
EcBcsG activity observed in vivo, but was not needed for
EcBcsGΔN in vitro activity.25
The MCR-1 pEtN transferase has been resolved by X-ray
crystallography with both one Zn2+ ion and two Zn2+ ions at
the active site32 (PDB entries 5LRN and 5LRM, respectively).
Although the Zn2+ ion resolved in EcBcsGΔN is found at the
conserved location found in all pEtN transferases, it remains
unclear if the second Zn2+ site reported for MCR-1 is
catalytically important and whether this site is found in other
pEtN transferases. Two His residues in MCR-1 coordinate this
second Zn2+ ion and are equivalent to His396 and Arg458 in
EcBcsG. To assess if Arg458 plays a role in secondary Zn2+
binding or catalysis, we measured the enzymatic rates of
alanine (R458A) and histidine (R458H) replacements (Figure
2). We found that the R458A variant displayed 25.6% esterase
and no detectable transferase activity, while the R458H variant
displayed 11.4% esterase and also did not display detectable
transferase activity compared to that of the wild type. As the
histidine imidazole group would be expected to functionally
complement the arginine guanidino group in metal binding,
these results suggest that Arg458 does not participate in
secondary metal binding. Instead, because Arg458 contributes a
guanidino group at a similar distance and across the face of the
active site from His396 (i.e., 5 Å above the face of the Zn2+ ion),
this configuration is consistent with the possibility that Arg458
stabilizes the charged pEtN−enzyme intermediate formed
during the first mechanistic step that results in reduced esterase
activity in both mutants. Because replacement of Arg458 with
either Ala or His also abrogated transferase activity, this
residue likely also plays an essential role in accommodation of
the acceptor co-substrate during the second mechanistic step.
An in situ assay for biofilm phenotype in S. typhimurium using
BcsG variants suggested that both His and Ala replacements of
the equivalent to Arg458 resulted in reduced cellulose
production by S. typhimurium,27 corroborating the role of
this conserved Arg residue in substrate binding and catalysis
rather than Zn2+ binding.
Molecular Determinants. To assess if the losses of
activity we observed for substitutions of the Zn2+-binding
residues were in fact due to the loss of Zn2+ in the EcBcsGΔN
active site or instead due to structural changes in the enzyme,
we supplied the metal chelating agent ethylenediaminetetra-
acetic acid (EDTA) to EcBcsGΔN prior to assaying. We
observed that treatment with 1 mM EDTA significantly
reduced the specific esterase activity of EcBcsGΔN to 10.6% of
the untreated activity [Figure 3; Tukey’s test, q(14) = 12.12,
and p < 0.0001]. A matched enzyme-free control containing 1
mM EDTA displayed no difference in the rate of p-NPPE
turnover, suggesting the effect seen was due to loss of Zn2+ in
the active site. This significant loss of esterase activity agreed
with the large reduction in esterase activity observed for the
alanine replacements of Cys243, Glu442, and His443. To our
surprise, however, the loss of transferase activity observed for
EDTA treatment was markedly smaller than that observed for
single-amino acid substitutions of the Zn2+-binding residues
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Figure 3. Zn2+ binding and disulfide bond formation are essential
catalytic features of EcBcsGΔN. Treatment of EcBcsGΔN with the
metal chelating agent EDTA or the reducing agent DTT results in
impaired enzymatic activities, consistent with impaired biofilm-
forming phenotypes observed in vivo. Asterisks denote significant
differences from respective controls [Tukey’s multiple comparisons,
q(14) > 6.5 and p < 0.004 for all comparisons].
(i.e., 43% residual activity of the wild type for the EDTA-
treated form, compared to 2.4%, 2.3%, and 10.4% residual
transferase activity for alanine replacements of Cys243, Glu442,
and His443, respectively). Our data suggest either that BcsG has
an exceptional affinity for Zn2+, given that 1 mM EDTA is a
standard concentration used to assess the metal dependency of
enzymes, or that in addition to Zn2+ binding, Cys243, Glu442,
and His443 are also catalytically important residues, particularly
during the latter step of catalysis when pEtN is transferred to
cellulose. In support of this theory, any general mechanism
involving Ser278 as the catalytic nucleophile would also require
a general acid−base residue to abstract the serine hydroxyl
proton during the formation of the covalent enzyme
intermediate and then subsequently replace it following
transfer of the pEtN group to its ultimate acceptor. Our
results also mirror the findings that treatment of MCR-1-
expressing cells with 1 mM EDTA reduces but does not
abolish the colistin MIC to MCR-1 negative levels. Similarly,
replacement of the residue equivalent to Cys243 in MCR-1
(Glu246) reduces the colistin MIC to the same extent as the
vector control, which is beyond what is observed for EDTA
treatment. In the literature, there are examples of cysteine
residues acting as a catalytic base when proximal to a
histidine,37 but this would be unprecedented among pEtN
family members. Indeed, cysteine is an objectively poorer
acid−base catalyst, and the Glu442 residue seems intuitively
better suited for this function. However, given that alanine
replacements of both Glu442 and Cys243 are significantly less
active than either a single replacement of His443 or EDTA-
treated enzyme, these data suggest that these two amino acids
and/or His396 (as noted below) are the leading candidates to
participate as acid−base catalytic residues.
A disulfide bond that fixes the helical element presenting the
catalytic nucleophile is a conserved and essential feature of
pEtN transferases.25,38 We previously showed that BcsG
contains a disulfide bond between Cys290 and Cys306, and
3664
Article
that replacement of the Cys pair with Ala results in a loss of
catalytic activity in vivo.25 Surprisingly, this disulfide bond in
BcsG is not at the conserved location but serves to fix the same
helical element presenting the nucleophilic Ser278. We were
unable to isolate a C290A/C306A variant of EcBcsGΔN, further
suggesting that the disulfide bond is a critical structural feature
of BcsG. To assess the importance of disulfide bond formation
to enzymatic activity in vitro, we introduced the reducing agent
dithiothreitol (DTT) to EcBcsGΔN prior to measurement of
the enzymatic rate. An observed decrease in enzymatic activity,
representing 31.9% of residual esterase activity, was observed
(Figure 3). Surprisingly, however, the measured residual
transferase activity was 72.8% of that of wild-type EcBcsGΔN,
while a matched enzyme-free control showed no differences in
the rate of p-NPPE hydrolysis. These findings are in apparent
disagreement with the loss of function of the disulfide-deficient
EcBcsG variant we reported in vivo. These data may in part be
rationalized by the specific requirements of exopolysaccharide
modification for biofilm formation. For example, the biofilm
polysaccharide poly-β-(1,6)-N-acetyl-D-glucosamine (PNAG)
requires only partial deacetylation by the enzyme PgaB or its
orthologues for successful biofilm formation in various
bacteria.39−41 PNAG found in biofilms has been observed to
have approximately 15−20% of the N-acetyl-D-glucosamine
saccharide units deacetylated, with either more or less
extensive deacetylation causing disruption in biofilm for-
mation, probably due to the loss of a distributed cationic
charge on the polymer.40,42,43 Similarly, a loss of EcBcsGΔN
activity of approximately 30% measured in the presence of
DTT might be considered trivial for the biological function of
some enzymes; however, this loss may not be enough to
maintain the 50% level of pEtN modification of D-glucose
saccharide units that has been observed for pEtN cellulose
biofilms isolated from E. coli or S. enterica.14 Thus, even subtle
reductions in the enzymatic rate, such as those observed here,
could still plausibly alter the degree of pEtN substitution so
that it is not matched properly to the rate of cellulose
synthesis, thereby disrupting the otherwise uniform charge and
chemistry required for proper biofilm architecture and
assembly in vivo.
Donor Co-substrate Preference. Although it has been
established that the natural co-substrates are phosphatidyle-
thanolamine14,27 and bacterial cellulose,14 we demonstrated
that BcsG was not capable of transfer to equivalent linear β-
1,4-aminosugar polymers (i.e., chitin).25 However, the
sufficiency of BcsG to use alternative phosphatidylethanol-
amine mimetics as phospho-donors remains unexplored, which
may be of interest for materials science and development. To
investigate the phospho-donor preference of EcBcsG, we
assayed the enzyme in vitro using the commercially available
substrate analogue p-nitrophenyl phosphate (p-NPP). Addi-
tionally, using a synthetic approach similar to that of p-
NPPE,25 the related compound p-nitrophenyl phosphopropa-
nolamine (p-NPPP) was synthesized in two steps (see the
Supporting Information). BcsG demonstrated detectable
esterase activity on both the minimal substrate p-NPP and
the extended substrate p-NPPP, although to a lesser extent
than the preferred p-NPPE (Figure S1). We repeated these
experiments in the presence of cellopentaose as an acceptor
substrate and observed no significant increase in the rate of
turnover of p-NPP or p-NPPP that would suggest catalytic
transfer of the ester-linked phosphate or phosphopropanol-
amine. Analysis of the enzymatic products by LC-MS
https://doi.org/10.1021/acs.biochem.1c00605
Biochemistry 2021, 60, 3659−3669
Biochemistry
corroborated these results, as no evidence of phospho- or
phosphopropanolamine cellulose was detected, thereby in-
dicating that BcsG is exclusively capable of transferring the
pEtN functional group under these conditions but also further
confirming it as the true biological substrate.
Steady-State Esterase Kinetics. To investigate the
kinetic parameters of EcBcsGΔN, we measured the steady-
state esterase rate and calculated the specific activity at varied
concentrations of p-NPPE (0−14.5 mM) without the addition
of an acceptor co-substrate (Figure 4A). The resulting data
Figure 4. Steady-state kinetics of EcBcsGΔN. (A) Steady-state esterase
(WT) and transferase (mutant) kinetics using p-NPPE as the
phosphoethanolamine donor and 3 mM cellopentaose as the acceptor
co-substrate. (B) Steady-state transferase kinetics using 7 mM p-
NPPE as the donor and cellooligosaccharides with DP values of 4−6
as the acceptor co-substrates.
were fit in agreement with the Michaelis−Menten model, with
an R2 value of 0.9624 (Table 1). We calculated from our data
an apparent KM of 2.40 ± 0.28 mM, a maximal rate (Vmax) of
2.20 × 10−2 nmol s−1, and a kcat of 4.34 × 10−7 ± 1.30 × 10−8
s−1. The derived catalytic efficiency kcat/KM was 1.81 × 10−4 ±
2.81 × 10−5 M−1 s−1.
Ser244 and His396 Participate in Both Mechanistic
Steps but Not in Zn2+ Binding. While the roles of the other
Table 1. Measured Michaelis−Menten Parameters for EcBcsG and Its Variants
parameter WT
kcat (s−1) 4.34 × 10−7 ± 1.30 × 10−8
KM (mM) 2.40 ± 0.28
Vmax (nmol s−1) 0.022
kcat/KM (M−1 s−1) 1.81 × 10−4 ± 2.81 × 10−5
a
With 3 mM cellopenatose co-substrate.
pubs.acs.org/biochemistry Article
active site residues can be implied from the crystal structure
and from trapping of the covalent enzyme intermediate, the
catalytic importance of Ser244 and His396 was not obviated from
prior data, notably for His396 that may plausibly serve as the
complementary base for the reaction that has not otherwise
been identified.25,27 Although our specific activity data (noted
above) suggest that these variants of EcBcsGΔN display
impaired ability to transfer pEtN to cellulosic substrates, it
remained unclear if these residues are exclusively involved in
the latter half of the mechanism whereby pEtN is transferred to
cellulose, or if their roles might extend to cleavage of the pEtN
substrate. Therefore, we assessed the steady-state kinetics of
the S244A and H396A EcBcsGΔN variants in the presence of 3
mM cellopentaose.
The alanine replacement of Ser244 demonstrated a decrease
in turnover number (kcat) and a greatly reduced affinity for p-
NPPE (KM increases >3-fold) in the presence of acceptor,
suggesting this residue plays a role in both steps of the
proposed mechanism (Figure 4A and Table 1). While other
biochemically characterized pEtN transferases possess a
conserved Thr residue that is equivalent to Ser244 in BcsG,
none of the previous work has kinetically explored the role of
this residue in catalysis. Thus, the results presented herein,
especially with respect to donor binding (as reflected by KM),
have importantly broadened our understanding of the catalytic
mechanism of this class of enzymes, which may aid in the
development of inhibitors. Other biochemically characterized
pEtN transferases, including MCR-1, NmEptA, and CjEptC,
each possess a conserved histidine equivalent to the BcsG
His396 residue.32,34,35 Although this His residue has been
shown to be essential for at least MCR-1,32 it has also been
proposed to coordinate a second Zn2+ ion at the active site,
which is apparently required for catalysis (Figure 5C).32,44 In
addition to our specific activity results (Figure 2), our kinetic
analysis of His396 in EcBcsGΔN confirmed the importance of
this residue in catalysis and expanded our knowledge of its role
in the reaction mechanism. Specifically, H396A demonstrated
a lower turnover number (kcat decreases 2.6-fold) and a modest
reduction in affinity for p-NPPE in the presence of
cellopentaose, thereby indicating that this residue is important
in both steps of the proposed mechanism (Figure 4A and
Table 1). However, our group and others27 were not able to
support the existence of a second Zn2+ ion in the active site of
BcsG, suggesting the role of His396 is truly in catalysis rather
than secondary metal binding among the pEtN transferases. In
the structural models of BcsG, His396 is positioned above the
zinc ion (approximately 5 Å) and in some models is within
hydrogen bonding distance of the main chain carbonyl of
Asp397, as well as a water molecule when present (Figure 5A).
These results are consistent with the distances noted for the
equivalents to His396 and Asp397 in ICRMc (His429 and Gly430,
respectively). However, in ICRMc, the water molecule is instead
occupied by the phosphoryl group of the bound phosphoe-
S244Aa H396Aa
2.98 × 10−7 ± 2.45 × 10−8 1.64 × 10−7 ± 9.58 × 10−9
7.85 ± 1.30 3.20 ± 0.56
0.015 0.008
3.80 × 10−5 ± 9.42 × 10−6 5.13 × 10−5 ± 8.98 × 10−6
3665 https://doi.org/10.1021/acs.biochem.1c00605
Biochemistry 2021, 60, 3659−3669
Biochemistry pubs.acs.org/biochemistry Article

Figure 5. Conserved His396 functions in catalysis and not secondary metal binding. The active sites of (A) BcsG (PDB entry 6PD0), (B) IcrMc
(PDB entry 6BND), and (C) MCR-1 (PDB entry 5LRM) display a conserved His residue in the proximity of the active site. Although involved in
binding a second Zn2+ ion in some structures of MCR-1, this His residue is within H-bonding distance of the adjacent main chain carbonyl in each
of the three structures and likely functions in proton relay, supported by kinetic studies of the BcsG H396A variant.

Table 2. Measured Michaelis−Menten Parameters for EcBcsG Acceptor Co-substrates

parameter cellotetraose cellopentaose cellohexaose
kcat (s−1) 2.00 × 10−6 ± 1.42 × 10−7 3.80 × 10−6 ± 2.99 × 10−7 3.27 × 10−6 ± 1.97 × 10−7
KM (mM) 3.20 ± 0.47 3.08 ± 0.53 1.76 ± 0.21
Vmax (nmol s−1) 0.10 0.19 0.16
kcat/KM (M−1 s−1) (6.24 ± 1.41) × 10−4 1.23 × 10−3 ± 2.96 × 10−4 1.86 × 10−3 ± 3.18 × 10−4

thanolamine (PDB entry 6BND), which is further coordinated
from the opposite side by the zinc ion (Figure 5B). These
findings suggest that His396 is also positioned to be part of a
similar proton relay that is important for catalysis in BcsG.
While further work to explore this role is warranted, the
essential nature of His396 in the previously proposed ping-pong
bibi mechanism25 has been further evidenced by our kinetic
and structural results.
Steady-State Transferase Kinetics. We previously
reported that introducing high relative concentrations of
cellooligosaccharides with degrees of polymerization (DP)
from 4 to 6 significantly increased the specific activity of
EcBcsGΔN in a length-dependent manner.25 Under these
conditions, pEtN-modified cellooligosaccharides accumulated
over time, demonstrating EcBcsGΔN is capable of transferase
activity in vitro. However, the substrate preference of
EcBcsGΔN using these cellooligosaccharides remains to be
seen. To that end, we performed steady-state kinetics using
fixed and saturating concentrations of p-NPPE and varied the
concentration of cellotetraose, cellopentaose, and cellohexaose
to understand EcBcsGΔN acceptor preference (Table 2).
As our previous results suggested, EcBcsGΔN displays an
increasing affinity for cellooligosaccharides with an increasing
DP, although due to limited solubility, cellohexaose represents
the highest DP that can be assayed using our experimental
design (Figure 4B). We measured apparent KM values of 3.20
± 0.47, 3.08 ± 0.53, and 1.76 ± 0.21 mM for
celloligosaccharides with DP values of 4, 5, and 6, respectively
(Figure 4B and Table 2). A similar trend in calculated kcat
values was observed with values of 2.00 × 10−6 ± 1.41 × 10−7,
3.80 × 10−6 ± 3.00 × 10−7, and 3.27 × 10−6 ± 1.97 × 10−7 s−1,
respectively, and the resulting catalytic efficiencies (kcat/KM)
were then calculated to be (6.24 ± 1.41) × 10−4, 1.23 × 10−3
± 2.96 × 10−4, and 1.86 × 10−3 ± 3.18 × 10−4 M−1 s−1 for DP
values of 4, 5, and 6, respectively (Table 2). In the case of
3666
cellohexaose, we measured an order of magnitude increase in
catalytic efficiency in the presence of the acceptor (i.e., 1.86 ×
10−3 ± 3.18 × 10−4 compared to 1.81 × 10−4 ± 2.81 × 10−5
M−1 s−1 without an acceptor), suggesting that the enzyme is far
more active as a transferase than as an esterase, at least with the
artificial substrate p-NPPE.
Measurement of the kinetic parameters of other pEtN
transferases has not been reported at present, likely due to the
absence of commercially available substrate analogues with
which to do so. Accordingly, a comparison between the kinetic
parameters of EcBcsGΔN measured here and those of other
pEtN transferases is not possible. However, the measured kcat
and KM values we report are poor in comparison to those of
other biochemically characterized enzymes of virtually any
identity; a majority of enzymes have reported kcat (s−1) values
of >1 and catalytic efficiencies of at least 1.0 × 103 M−1 s−1.45
However, when examined against characterized enzymes that
modify other bacterial exopolysaccharides, the BcsG values are
more typical. For example, the PNAG de-N-acetylases PgaB
from E. coli and IcaB from Staphylococcus epidermidis have low
reported kcat (PgaB, 0.0013 s−1; IcaB, 0.0007 s−1) and kcat/KM
values (PgaB, 0.26 M−1 s−1; IcaB, 0.03 M−1 s−1), using a similar
assay design with a synthetic fluorogenic substrate 4-
methylumbelliferyl acetate.43,46 In addition to PNAG, the
alginate epimerase AlgG from Pseudomonas aeruginosa (kcat/KM
∼ 0.02 M−1 s−1) is also consistent with EcBcsGΔN.47 It is worth
noting that the analyses mentioned above were all performed
with artificial co-substrates that are not present in a cellular
context, which may also explain in part the catalytic
inefficiencies of these enzymes to act upon them. Regardless,
the generally low turnover numbers found in exopolysacchar-
ide-modifying enzymes have been rationalized previously by
the partial degree of modification achieved in the mature
polymer,39,43 which we mentioned above. Accordingly, only
50% of the saccharide units are C6-pEtN substituted in the
https://doi.org/10.1021/acs.biochem.1c00605
Biochemistry 2021, 60, 3659−3669
Biochemistry pubs.acs.org/biochemistry Article

Figure 6. Proposed mechanism of BcsG as a pEtN transferase. In the first mechanistic step, the hydroxyl proton is abstracted from Ser278 by His443
and Glu442 (2). Protonation and collapse of the resulting oxyanion intermediate are plausibly achieved by His396 and Cys243, resulting in the
covalent enzyme intermediate and release of the diacylglycerol co-product (3). In the second mechanistic step, the C6 hydroxyl of cellulose is
deprotonated, possibly by Cys243, and attacks the phosphoryl-Ser278 intermediate. The resulting Ser278 oxyanion is protonated, likely by the adjacent
His443, followed by release of the pEtN cellulose co-product (4). Our enzymatic data also suggest that Ser244 and Arg458 serve as important polar
contacts during both mechanistic steps, especially Arg458 in the second mechanistic step.

mature pEtN cellulose polymer,14 which is in better agreement
with the low EcBcsGΔN turnover number and catalytic
efficiency.
The rate of cellulose synthesis by BcsAB has been measured,
and surprisingly, Omadjela and co-workers reported a rate for
BcsAB that is orders of magnitude greater than that of BcsG.18
However, they similarly acknowledge for their in vitro assay, as
we did above for BcsG, that these assays can fail to capture
nuances of the biological complexity of the Bcs macrocomplex
and that the rate of cellulose synthesis that they report for
BcsAB is probably much lower in vivo. Direct rate comparisons
between these disparately designed assays should also be
interpreted with caution. However, it should be noted that the
native E. coli Bcs macrocomplex has been resolved with two
copies of BcsG associated with a single BcsA catalytic
subunit.23 Taken together, these observations may partly
reconcile the differences in rates between BcsG and BcsA in
such a way as to generate the level of pEtN modification
required for biofilm formation in vivo.
our data suggest a shared mono-Zn2+ mechanism for the pEtN
transferase family and that BcsG possesses an active site similar
to, but distinct from, that of the colistin resistance enzymes,
thereby pointing to new roles for the family.
On the basis of our current data, we propose a catalytic
mechanism for transfer of phosphoethanolamine to cellulose
by BcsG that involves activation of Ser278 by abstraction of the
hydroxyl proton by His443 and Glu442 (Figure 6). Following a
nucleophilic attack of the phosphate, the loss of the
diacylglycerol product is probably achieved by protonation of
the oxyanion intermediate, stabilized by the Zn2+ ion, possibly
through Cys243 and His396. Although a catalytic Cys base is
unprecedented in the pEtN family, the Glu442/His443 acid−
base pair would be too distant to serve this role, separated by
at least 5 Å. Our data presented herein support a catalytic role
for Cys243, and its presence in place of the typical glutamic acid
may be rationalized by the lower overall catalytic efficiency

■
required of BcsG in general. The essential substrate
CONCLUDING REMARKS recognition and catalytic features observed thus provide a
We have demonstrated that full EcBcsGΔN activity in vitro is molecular level understanding of BcsG and will facilitate the
dependent upon Zn 2+ binding, the putative catalytic design of biofilm inhibitors targeting this enzyme.

■
nucleophile and acid−base arrangement (Ser278/Cys243/
His396), the previously unidentified active site residues Arg458 ASSOCIATED CONTENT
and Ser244, and proper formation of a disulfide bond between
Cys290 and Cys306. A kinetic analysis of EcBcsGΔN demon- *
sı Supporting Information

strated that it is specific for the transfer of phosphoethanol-
amine over other donor substrates tested. Our data also
showed that EcBcsG shares equivalent catalytic residues with
the characterized colistin resistance enzymes, although our
model does not support a two-Zn2+ model for catalysis that
was suggested for one other group member, MCR-1. Instead,
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biochem.1c00605.
A complete list of plasmids, primers, and strains used in
this study, the synthesis of p-NPPP, and representative
1
H, 13C, and 31P NMR shifts for p-NPPP (PDF)

3667 https://doi.org/10.1021/acs.biochem.1c00605
Biochemistry 2021, 60, 3659−3669
Biochemistry
■
pubs.acs.org/biochemistry
AUTHOR INFORMATION
Corresponding Author
Joel T. Weadge − Department of Biology, Wilfrid Laurier
University, Waterloo, ON N2L3C5, Canada; orcid.org/
0000-0002-8020-0015; Phone: 519-884-0710, ext. 2161;
Email: jweadge@wlu.ca
Authors
Alexander C. Anderson − Department of Biology, Wilfrid
Laurier University, Waterloo, ON N2L3C5, Canada;
Present Address: A.C.A.: Department of Molecular and
Cellular Biology, University of Guelph, Guelph, ON
N1G2W1, Canada; orcid.org/0000-0002-1870-2903
Alysha J. N. Burnett − Department of Biology, Wilfrid Laurier
University, Waterloo, ON N2L3C5, Canada
Shirley Constable − Department of Biology, Wilfrid Laurier
University, Waterloo, ON N2L3C5, Canada
Lana Hiscock − Department of Biology and Department of
Chemistry & Biochemistry, Wilfrid Laurier University,
Waterloo, ON N2L3C5, Canada
Kenneth E. Maly − Department of Chemistry & Biochemistry,
Wilfrid Laurier University, Waterloo, ON N2L3C5,
Canada; orcid.org/0000-0002-3695-4995
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biochem.1c00605
Author Contributions
J.T.W. and A.C.A. conceived the research and acquired
funding. A.C.A. and A.J.N.B. designed and carried out the
enzymology experiments and prepared the manuscript. L.H.
and K.E.M. carried out the synthesis and validation of p-NPPE
and p-NPPP. S.C. carried out some of the mutant-based
enzymology. J.T.W., A.C.A., A.J.N.B., S.C., L.H., and K.E.M.
were involved in manuscript review and editing. All authors
have given approval to the final version of the manuscript.
Funding
The authors acknowledge the support of the Natural Sciences
and Engineering Research Council of Canada (NSERC) in the
form of a grant to J.T.W. (229971) and a graduate scholarship
(PGS-D) to A.C.A. A.C.A. and A.J.N.B. were also supported by
Ontario Graduate Scholarships via Wilfrid Laurier University.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors thank D. Brewer and A. Charchoglyan at the
University of Guelph for expert technical assistance with mass
spectrometry experiments.
■
ABBREVIATIONS
pEtN, phosphoethanolamine; c-di-GMP, cyclic dimeric
guanosine monophosphate; TPR, tetratricopeptide repeat; p-
NPPE, p-nitrophenyl phosphoethanolamine; p-NPPP, p-nitro-
phenyl phosphopropanolamine; p-NPP, p-nitrophenyl phos-
phate; Ni-NTA, nickel-nitrilotriacetic acid; IPTG, isopropyl β-
D-thiogalactopyranoside; EDTA, ethylenediaminetetraacetic
acid; DTT, dithiothreitol; DP, degree of polymerization.
■
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3669 https://doi.org/10.1021/acs.biochem.1c00605
Biochemistry 2021, 60, 3659−3669