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A Mechanistic Basis for Phosphoethanolamine Modification of the Cellulose Biofilm Matrix in Escherichia coli

Cite this: Biochemistry 2021, 60, 47, 3659–3669
Publication Date (Web):November 11, 2021
https://doi.org/10.1021/acs.biochem.1c00605

Copyright © 2021 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Biofilms are communities of self-enmeshed bacteria 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.

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Biofilms are dynamic communities of microorganisms fixed 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) antimicrobial compounds, (2,5) extreme temperature, (2,5,6) or sanitizing agents (2,3,5) 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 beginning to be understood. (13) For example, the biofilm of Pseudomonas fluorescens 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 difficile, (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.

Figure 1

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)

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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 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. difficile demonstrated that these terminal enzymes were from glycoside hydrolase families 8 and 5, respectively, and both possess endo-β-(1,4)-glucanase activity for modification 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 polysaccharide. (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 polysaccharides. (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

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Materials

Culture media, isopropyl β-d-thiogalactopyranoside (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 (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 Derivatives

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 resulting solutions were loaded onto an ÄKTA 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 derivatives. 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 phosphopropanolamine (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 spectrometry 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 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.

Results and Discussion

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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 consequences 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 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-type EcBcsGΔN. These variant proteins were purified to apparent homogeneity using the protocol described previously, (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 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)

Figure 2

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].

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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 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 EcBcsGΔNin 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 site (32) (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 ethylenediaminetetraacetic 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 (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.

Figure 3

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].

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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 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 formation, 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 phosphatidylethanolamine (14,27) 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 phosphatidylethanolamine 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). Additionally, using a synthetic approach similar to that of p-NPPE, (25) the related compound p-nitrophenyl phosphopropanolamine (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 phosphopropanolamine. Analysis of the enzymatic products by LC-MS corroborated these results, as no evidence of phospho- or phosphopropanolamine cellulose was detected, thereby indicating 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 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.

Figure 4

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.

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Ser244 and His396 Participate in Both Mechanistic Steps but Not in Zn2+ Binding

While the roles of the other 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 others (27) 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 phosphoethanolamine (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 mechanism (25) has been further evidenced by our kinetic and structural results.
Table 1. Measured Michaelis–Menten Parameters for EcBcsG and Its Variants
parameter WT S244Aa H396Aa
kcat (s–1) 4.34 × 10–7 ± 1.30 × 10–8 2.98 × 10–7 ± 2.45 × 10–8 1.64 × 10–7 ± 9.58 × 10–9
KM (mM) 2.40 ± 0.28 7.85 ± 1.30 3.20 ± 0.56
Vmax (nmol s–1) 0.022 0.015 0.008
kcat/KM (M–1 s–1) 1.81 × 10–4 ± 2.81 × 10–5 3.80 × 10–5 ± 9.42 × 10–6 5.13 × 10–5 ± 8.98 × 10–6
a

With 3 mM cellopenatose co-substrate.

Figure 5

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.

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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).
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
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 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 exopolysaccharide-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 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.

Concluding Remarks

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We have demonstrated that full EcBcsGΔN activity in vitro is dependent upon Zn2+ binding, the putative catalytic nucleophile and acid–base arrangement (Ser278/Cys243/His396), the previously unidentified active site residues Arg458 and Ser244, and proper formation of a disulfide bond between Cys290 and Cys306. A kinetic analysis of EcBcsGΔN demonstrated that it is specific for the transfer of phosphoethanolamine 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, 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 recognition and catalytic features observed thus provide a molecular level understanding of BcsG and will facilitate the design of biofilm inhibitors targeting this enzyme.

Figure 6

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.

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Supporting Information

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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 1H, 13C, and 31P NMR shifts for p-NPPP (PDF)

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Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
  • Authors
    • Alexander C. Anderson - Department of Biology and , 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;  Orcidhttps://orcid.org/0000-0002-1870-2903
    • Alysha J. N. Burnett - Department of Biology and , Wilfrid Laurier University, Waterloo, ON N2L3C5, Canada
    • Shirley Constable - Department of Biology and , Wilfrid Laurier University, Waterloo, ON N2L3C5, Canada
    • Lana Hiscock - Department of Biology  and  Department of Chemistry & Biochemistry and , Wilfrid Laurier University, Waterloo, ON N2L3C5, Canada
    • Kenneth E. Maly - Department of Chemistry & Biochemistry and , Wilfrid Laurier University, Waterloo, ON N2L3C5, Canada;  Orcidhttps://orcid.org/0000-0002-3695-4995
  • 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

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The authors thank D. Brewer and A. Charchoglyan at the University of Guelph for expert technical assistance with mass spectrometry experiments.

Abbreviations

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pEtN

phosphoethanolamine
c-di-GMP

cyclic dimeric guanosine monophosphate
TPR

tetratricopeptide repeat
p-NPPE

p-nitrophenyl phosphoethanolamine
p-NPPP

p-nitrophenyl phosphopropanolamine
p-NPP

p-nitrophenyl phosphate
Ni-NTA

nickel-nitrilotriacetic acid
IPTG

isopropyl β-d-thiogalactopyranoside
EDTA

ethylenediaminetetraacetic acid
DTT

dithiothreitol
DP

degree of polymerization.

References

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This article references 47 other publications.

  1. 1
    Mah, T.-F. C.; O’Toole, G. A. Mechanisms of Biofilm Resistance to Antimicrobial Agents. Trends Microbiol. 2001, 9 (1), 34– 39,  DOI: 10.1016/S0966-842X(00)01913-2
    Google Scholar
    1
    Mechanisms of biofilm resistance to antimicrobial agents
    Mah, Thien-Fah C.; O'Toole, George A.
    Trends in Microbiology (2001), 9 (1), 34-39CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Science Ltd.)
    A review with 46 refs. Biofilms are communities of microorganisms attached to a surface. It has become clear that biofilm-grown cells express properties distinct from planktonic cells, one of which is an increased resistance to antimicrobial agents. Recent work has indicated that slow growth and/or induction of an rpoS-mediated stress response could contribute to biocide resistance. The phys. and/or chem. structure of exopolysaccharides or other aspects of biofilm architecture could also confer resistance by exclusion of biocides from the bacterial community. Finally, biofilm-grown bacteria might develop a biofilm-specific biocide-resistant phenotype. Owing to the heterogeneous nature of the biofilm, it is likely that there are multiple resistance mechanisms at work within a single community. Recent research has begun to shed light on how and why surface-attached microbial communities develop resistance to antimicrobial agents.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXlvFChsr0%253D&md5=5c8ebbac46c7e8d1c5efa409d19e2093
  2. 2
    Flemming, H.-C.; Wingender, J.; Szewzyk, U.; Steinberg, P.; Rice, S. A.; Kjelleberg, S. Biofilms: An Emergent Form of Bacterial Life. Nat. Rev. Microbiol. 2016, 14 (9), 563– 575,  DOI: 10.1038/nrmicro.2016.94
    Google Scholar
    2
    Biofilms: an emergent form of bacterial life
    Flemming, Hans-Curt; Wingender, Jost; Szewzyk, Ulrich; Steinberg, Peter; Rice, Scott A.; Kjelleberg, Staffan
    Nature Reviews Microbiology (2016), 14 (9), 563-575CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
    A review. Bacterial biofilms are formed by communities that are embedded in a self-produced matrix of extracellular polymeric substances (EPS). Importantly, bacteria in biofilms exhibit a set of 'emergent properties' that differ substantially from free-living bacterial cells. In this Review, we consider the fundamental role of the biofilm matrix in establishing the emergent properties of biofilms, describing how the characteristic features of biofilms - such as social cooperation, resource capture and enhanced survival of exposure to antimicrobials - all rely on the structural and functional properties of the matrix. Finally, we highlight the value of an ecol. perspective in the study of the emergent properties of biofilms, which enables an appreciation of the ecol. success of biofilms as habitat formers and, more generally, as a bacterial lifestyle.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhtlejur%252FO&md5=66cc69a8cc3bebd666245b5a7ced5132
  3. 3
    Marsh, P. Dental Plaque as a Microbial Biofilm Dental Plaque – Existing Perspective. Caries Res. 2004, 38, 204– 211,  DOI: 10.1159/000077756
    Google Scholar
    3
    Dental plaque as a microbial biofilm
    Marsh, P. D.
    Caries Research (2004), 38 (3), 204-211CODEN: CAREBK; ISSN:0008-6568. (S. Karger AG)
    A review. New technologies have provided novel insights into how dental plaque functions as a biofilm. Confocal microscopy has confirmed that plaque has an open architecture similar to other biofilms, with channels and voids. Gradients develop in areas of dense biomass over short distances in key parameters that influence microbial growth and distribution. Bacteria exhibit an altered pattern of gene expression either as a direct result of being on a surface or indirectly as a response to the local environmental heterogeneity within the biofilm. Bacteria communicate via small diffusible signalling mols. (e.g. competence-stimulating peptide, CSP; autoinducer 2); CSP induces both genetic competence and acid tolerance in recipient sessile cells. Thus, rates of gene transfer increase in biofilm communities, and this is one of several mechanisms (others include: diffusion-reaction, neutralization/inactivation, slow growth rates, novel phenotype) that contribute to the increased antimicrobial resistance exhibited by bacteria in biofilms. Oral bacteria in plaque do not exist as independent entities but function as a coordinated, spatially organized and fully metabolically integrated microbial community, the properties of which are greater than the sum of the component species. A greater understanding of the significance of dental plaque as a mixed culture biofilm will lead to novel control strategies.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXktFKmurs%253D&md5=7bd5386addb414d09060b753df03d095
  4. 4
    Flickinger, S. T.; Copeland, M. F.; Downes, E. M.; Braasch, A. T.; Tuson, H. H.; Eun, Y.-J.; Weibel, D. B. Quorum Sensing between Pseudomonas Aeruginosa Biofilms Accelerates Cell Growth. J. Am. Chem. Soc. 2011, 133 (15), 5966– 5975,  DOI: 10.1021/ja111131f
    Google Scholar
    4
    Quorum sensing between Pseudomonas aeruginosa biofilms accelerates cell growth
    Flickinger, Shane T.; Copeland, Matthew F.; Downes, Eric M.; Braasch, Andrew T.; Tuson, Hannah H.; Eun, Ye-Jin; Weibel, Douglas B.
    Journal of the American Chemical Society (2011), 133 (15), 5966-5975CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    This paper describes the fabrication of arrays of spatially confined chambers embossed in a layer of poly(ethylene glycol) diacrylate (PEGDA) and their application to studying quorum sensing between communities of Pseudomonas aeruginosa. The authors hypothesized that biofilms may produce stable chem. signaling gradients in close proximity to surfaces, which influence the growth and development of nearby microcolonies into biofilms. To test this hypothesis, they embossed a layer of PEGDA with 1.5-mm wide chambers in which P. aeruginosa biofilms grew, secreted homoserine lactones (HSLs, small mol. regulators of quorum sensing), and formed spatial and temporal gradients of these compds. In static growth conditions (i.e., no flow), nascent biofilms secreted N-(3-oxododecanoyl) HSL that formed a gradient in the hydrogel and was detected by P. aeruginosa cells that were ≤8 mm away. Diffusing HSLs increased the growth rate of cells in communities that were < 3 mm away from the biofilm, where the concn. of HSL was > 1 μM, and had little effect on communities farther away. The HSL gradient had no observable influence on biofilm structure. Surprisingly, 0.1-10 μM of N-(3-oxododecanoyl) HSL had no effect on cell growth in liq. culture. The results suggest that the secretion of HSLs from a biofilm enhances the growth of neighboring cells in contact with surfaces into communities and may influence their compn., organization, and diversity.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjslektb8%253D&md5=7adecd7bcc6612a13242208094991b98
  5. 5
    Fux, C. A.; Costerton, J. W.; Stewart, P. S.; Stoodley, P. Survival Strategies of Infectious Biofilms. Trends Microbiol. 2005, 13 (1), 34– 40,  DOI: 10.1016/j.tim.2004.11.010
    Google Scholar
    5
    Survival strategies of infectious biofilms
    Fux, C. A.; Costerton, J. W.; Stewart, P. S.; Stoodley, P.
    Trends in Microbiology (2005), 13 (1), 34-40CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)
    A review. Modern medicine is facing the spread of biofilm-related infections. Bacterial biofilms are difficult to detect in routine diagnostics and are inherently tolerant to host defenses and antibiotic therapies. In addn., biofilms facilitate the spread of antibiotic resistance by promoting horizontal gene transfer. The authors review current concepts of biofilm tolerance with special emphasis on the role of the biofilm matrix and the physiol. of biofilm-embedded cells. The heterogeneity in metabolic and reproductive activity within a biofilm correlates with a non-uniform susceptibility of enclosed bacteria. Recent studies have documented similar heterogeneity in planktonic cultures. Nutritional starvation and high cell d., 2 key characteristics of biofilm physiol., also mediate antimicrobial tolerance in stationary-phase planktonic cultures. Advances in characterizing the role of stress response genes, quorum sensing and phase variation in stationary-phase planktonic cultures have shed new light on tolerance mechanisms within biofilm communities.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhvF2ntA%253D%253D&md5=a8e5c4e4090484582faebdaecae5dc07
  6. 6
    Römling, U.; Bokranz, W.; Rabsch, W.; Zogaj, X.; Nimtz, M.; Tschäpe, H. Occurrence and Regulation of the Multicellular Morphotype in Salmonella Serovars Important in Human Disease. Int. J. Med. Microbiol. 2003, 293 (4), 273– 285,  DOI: 10.1078/1438-4221-00268
    Google Scholar
    6
    Occurrence and regulation of the multicellular morphotype in Salmonella serovars important in human disease
    Romling Ute; Bokranz Werner; Rabsch Wolfgang; Zogaj Xhavit; Nimtz Manfred; Tschape Helmut
    International journal of medical microbiology : IJMM (2003), 293 (4), 273-85 ISSN:1438-4221.
    Multicellular behavior in Salmonella Typhimurium ATCC14028 called the rdar morphotype is characterized by the expression of the extracellular matrix components cellulose and curli fimbriae. Over 90% of S. Typhimurium and S. Enteritidis strains from human disease, food and animals expressed the rdar morphotype at 28 degrees C. Regulation of the rdar morphotype occurred via the response regulator ompR, which activated transcription of csgD required for production of cellulose and curli fimbriae. Serovar-specific regulation of csgD required rpoS in S. Typhimurium, but was partially independent of rpoS in S. Enteritidis. Rarely, strain-specific temperature-deregulated expression of the rdar morphotype was observed. The host-restricted serovars S. Typhimurium var. Copenhagen phage type DT2 and DT99, Salmonella Typhi and Salmonella Choleraesuis did not express the rdar morphotype, while in Salmonella Gallinarum cellulose expression at 37 degrees C was seen in some strains. Therefore, the expression pattern of the rdar morphotype is serovar specific and correlates with a disease phenotype breaching the intestinal epithelial cell lining.
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  7. 7
    Saldaña, Z.; Xicohtencatl-Cortes, J.; Avelino, F.; Phillips, A. D.; Kaper, J. B.; Puente, J. L.; Girón, J. A. Synergistic Role of Curli and Cellulose in Cell Adherence and Biofilm Formation of Attaching and Effacing Escherichia Coli and Identification of Fis as a Negative Regulator of Curli. Environ. Microbiol. 2009, 11 (4), 992– 1006,  DOI: 10.1111/j.1462-2920.2008.01824.x
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    7
    Synergistic role of curli and cellulose in cell adherence and biofilm formation of attaching and effacing Escherichia coli and identification of Fis as a negative regulator of curli
    Saldana, Zeus; Xicohtencatl-Cortes, Juan; Avelino, Fabiola; Phillips, Alan D.; Kaper, James B.; Puente, Jose L.; Giron, Jorge A.
    Environmental Microbiology (2009), 11 (4), 992-1006CODEN: ENMIFM; ISSN:1462-2912. (Wiley-Blackwell)
    Curli are adhesive fimbriae of Escherichia coli and Salmonella enterica. Expression of curli (csgA) and cellulose (bcsA) is co-activated by the transcriptional activator CsgD. In this study, we investigated the contribution of curli and cellulose to the adhesive properties of enterohemorrhagic (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) O127:H6. While single mutations in csgA, csgD or bcsA in EPEC and EHEC had no dramatic effect on cell adherence, double csgAbcsA mutants were significantly less adherent than the single mutants or wild-type strains to human colonic HT-29 epithelial cells or to cow colon tissue in vitro. Overexpression of csgD (carried on plasmid pCP994) in a csgD mutant, but not in the single csgA or bscA mutants, led to significant increase in adherence and biofilm formation in EPEC and EHEC, suggesting that synchronized over-prodn. of curli and cellulose enhances bacterial adherence. In line with this finding, csgD transcription was activated significantly in the presence of cultured epithelial cells as compared with growth in tissue culture medium. Anal. of the influence of virulence and global regulators in the prodn. of curli in EPEC identified Fis (factor for inversion stimulation) as a, heretofore unrecognized, neg. transcriptional regulator of csgA expression. An EPEC E2348/69Δfis produced abundant amts. of curli whereas a double fis/csgD mutant yielded no detectable curli prodn. Our data suggest that curli and cellulose act in concert to favor host colonization, biofilm formation and survival in different environments.
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  8. 8
    Spiers, A. J.; Bohannon, J.; Gehrig, S. M.; Rainey, P. B. Biofilm Formation at the Air-Liquid Interface by the Pseudomonas Fluorescens SBW25 Wrinkly Spreader Requires an Acetylated Form of Cellulose. Mol. Microbiol. 2003, 50 (1), 15– 27,  DOI: 10.1046/j.1365-2958.2003.03670.x
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    8
    Biofilm formation at the air-liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose
    Spiers, Andrew J.; Bohannon, John; Gehrig, Stefanie M.; Rainey, Paul B.
    Molecular Microbiology (2003), 50 (1), 15-27CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)
    The wrinkly spreader (WS) genotype of Pseudomonas fluorescens SBW25 colonizes the air-liq. interface of spatially structured microcosms resulting in formation of a thick biofilm. Its ability to colonize this niche is largely due to overprodn. of a cellulosic polymer, the product of the wss operon. Chem. anal. of the biofilm matrix shows that the cellulosic polymer is partially acetylated cellulose, which is consistent with predictions of gene function based on in silico anal. of wss. Both polar and non-polar mutations in the sixth gene of the wss operon (wssF) or adjacent downstream genes (wssGHIJ) generated mutants that overproduce non-acetylated cellulose, thus implicating WssFGHIJ in acetylation of cellulose. WssGHI are homologs of AlgFIJ from P. aeruginosa, which together are necessary and sufficient to acetylate alginate polymer. WssF belongs to a newly established Pfam family and is predicted to provide acyl groups to WssGHI. The role of WssJ is unclear, but its similarity to MinD-like proteins suggests a role in polar localization of the acetylation complex. Fluorescent microscopy of Calcofluor-stained biofilms revealed a matrix structure composed of networks of cellulose fibers, sheets and clumped material. Quant. analyses of biofilm structure showed that acetylation of cellulose is important for effective colonization of the air-liq. interface: mutants identical to WS, but defective in enzymes required for acetylation produced biofilms with altered phys. properties. In addn., mutants producing non-acetylated cellulose were unable to spread rapidly across solid surfaces. Inclusion in these assays of a WS mutant with a defect in the GGDEF regulator (WspR) confirmed the requirement for this protein in expression of both acetylated cellulose polymer and bacterial attachment. These results suggest a model in which WspR regulation of cellulose expression and attachment plays a role in the co-ordination of surface colonization.
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  9. 9
    Hu, L.; Grim, C. J.; Franco, A. A.; Jarvis, K. G.; Sathyamoorthy, V.; Kothary, M. H.; McCardell, B. A.; Tall, B. D. Analysis of the Cellulose Synthase Operon Genes, BcsA, BcsB, and BcsC in Cronobacter Species: Prevalence among Species and Their Roles in Biofilm Formation and Cell-Cell Aggregation. Food Microbiol. 2015, 52, 97– 105,  DOI: 10.1016/j.fm.2015.07.007
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    9
    Analysis of the cellulose synthase operon genes, bcsA, bcsB, and bcsC in Cronobacter species: Prevalence among species and their roles in biofilm formation and cell-cell aggregation
    Hu, Lan; Grim, Christopher J.; Franco, Augusto A.; Jarvis, Karen G.; Sathyamoorthy, Vengopal; Kothary, Mahendra H.; McCardell, Barbara A.; Tall, Ben D.
    Food Microbiology (2015), 52 (), 97-105CODEN: FOMIE5; ISSN:0740-0020. (Elsevier Ltd.)
    Cronobacter species are emerging food-borne pathogens that cause severe sepsis, meningitis, and necrotizing entercolitis in neonates and infants. Bacterial pathogens such as Escherichia coli and Salmonella species produce extracellular cellulose which has been shown to be involved in rugosity, biofilm formation, and host colonization. In this study the distribution and prevalence of cellulose synthase operon genes (bcsABZC) were detd. by polymerase chain reaction (PCR) anal. in 231 Cronobacter strains isolated from clin., food, environmental, and unknown sources. Furthermore, bcsA and bcsB isogenic mutants were constructed in Cronobacter sakazakii BAA894 to det. their roles. In calcofluor binding assays bcsA and bcsB mutants did not produce cellulose, and their colonial morphotypes were different to that of the parent strain. Biofilm formation and bacterial cell-cell aggregation were significantly reduced in bcsA and bcsB mutants compared to the parental strain. bcsA or bcsAB PCR-neg. strains of C. sakazakii did not bind calcofluor, and produced less biofilm and cell-cell aggregation compared to strains possessing bcsAB genes. These data indicated that Cronobacter bcsABZC were present in all clin. isolates and most of food and environmental isolates. bcsA and bcsB genes of Cronobacter were necessary to produce cellulose, and were involved in biofilm formation and cell-cell aggregation.
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  10. 10
    Ross, P.; Mayer, R.; Benziman, M. Cellulose Biosynthesis and Function in Bacteria. Microbiol. Rev. 1991, 55 (1), 35– 58,  DOI: 10.1128/mr.55.1.35-58.1991
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    10
    Cellulose biosynthesis and function in bacteria
    Ross, Peter; Mayer, Raphael; Benziman, Moshe
    Microbiological Reviews (1991), 55 (1), 35-58CODEN: MBRED3; ISSN:0146-0749.
    A review with >150 refs., including regulation and comparative biochem. of cellulose formation.
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  11. 11
    Canale-Parola, E.; Borasky, R.; Wolfe, R. S. Studies on Sarcina Ventriculi III. Localization of Cellulose. J. Bacteriol. 1961, 81 (2), 311– 318,  DOI: 10.1128/jb.81.2.311-318.1961
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    11
    Studies on Sarcina ventriculi. III. Localization of cellulose
    CANALE-PAROLA E; BORASKY R; WOLFE R S
    Journal of bacteriology (1961), 81 (), 311-8 ISSN:0021-9193.
    There is no expanded citation for this reference.
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  12. 12
    Scott, W.; Lowrance, B.; Anderson, A. C.; Weadge, J. T. Identification of the Clostridial Cellulose Synthase and Characterization of the Cognate Glycosyl Hydrolase, CcsZ. PLoS One 2020, 15 (12), e0242686,  DOI: 10.1371/journal.pone.0242686
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    12
    Identification of the Clostridial cellulose synthase and characterization of the cognate glycosyl hydrolase, CcsZ
    Scott, William; Lowrance, Brian; Anderson, Alexander C.; Weadge, Joel T.
    PLoS One (2020), 15 (12), e0242686CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Biofilms are community structures of bacteria enmeshed in a self-produced matrix of exopolysaccharides. The biofilm matrix serves numerous roles, including resilience and persistence, making biofilms a subject of research interest among persistent clin. pathogens of global health importance. Our current understanding of the underlying biochem. pathways responsible for biosynthesis of these exopolysaccharides is largely limited to Gram-neg. bacteria. Clostridia are a class of Gram-pos., anaerobic and spore-forming bacteria and include the important human pathogens Clostridium perfringens, Clostridium botulinum and Clostridioides difficile, among numerous others. Several species of Clostridia have been reported to produce a biofilm matrix that contains an acetylated glucan linked to a series of hypothetical genes. Here, we propose a model for the function of these hypothetical genes, which, using homol. modeling, we show plausibly encode a synthase complex responsible for polymn., modification and export of an O-acetylated cellulose exopolysaccharide. Specifically, the cellulose synthase is homologous to that of the known exopolysaccharide synthases in Gram-neg. bacteria. The remaining proteins represent a mosaic of evolutionary lineages that differ from the described Gram-neg. cellulose exopolysaccharide synthases, but their predicted functions satisfy all criteria required for a functional cellulose synthase operon. Accordingly, we named these hypothetical genes ccsZABHI, for the Clostridial cellulose synthase (Ccs), in keeping with naming conventions for exopolysaccharide synthase subunits and to distinguish it from the Gram-neg. Bcs locus with which it shares only a single one-to-one ortholog. To test our model and assess the identity of the exopolysaccharide, we subcloned the putative glycoside hydrolase encoded by ccsZ and solved the X-ray crystal structure of both apo- and product-bound CcsZ, which belongs to glycoside hydrolase family 5 (GH-5). Although not homologous to the Gram-neg. cellulose synthase, which instead encodes the structurally distinct BcsZ belonging to GH-8, we show CcsZ displays specificity for cellulosic materials. This specificity of the synthase-assocd. glycosyl hydrolase validates our proposal that these hypothetical genes are responsible for biosynthesis of a cellulose exopolysaccharide. The data we present here allowed us to propose a model for Clostridial cellulose synthesis and serves as an entry point to an understanding of cellulose biofilm formation among class Clostridia.
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  13. 13
    Whitfield, G. B.; Marmont, L. S.; Howell, P. L. Enzymatic Modifications of Exopolysaccharides Enhance Bacterial Persistence. Front. Microbiol. 2015, 6, 471,  DOI: 10.3389/fmicb.2015.00471
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    13
    Enzymatic modifications of exopolysaccharides enhance bacterial persistence
    Whitfield Gregory B; Marmont Lindsey S; Howell P Lynne
    Frontiers in microbiology (2015), 6 (), 471 ISSN:1664-302X.
    Biofilms are surface-attached communities of bacterial cells embedded in a self-produced matrix that are found ubiquitously in nature. The biofilm matrix is composed of various extracellular polymeric substances, which confer advantages to the encapsulated bacteria by protecting them from eradication. The matrix composition varies between species and is dependent on the environmental niche that the bacteria inhabit. Exopolysaccharides (EPS) play a variety of important roles in biofilm formation in numerous bacterial species. The ability of bacteria to thrive in a broad range of environmental settings is reflected in part by the structural diversity of the EPS produced both within individual bacterial strains as well as by different species. This variability is achieved through polymerization of distinct sugar moieties into homo- or hetero-polymers, as well as post-polymerization modification of the polysaccharide. Specific enzymes that are unique to the production of each polymer can transfer or remove non-carbohydrate moieties, or in other cases, epimerize the sugar units. These modifications alter the physicochemical properties of the polymer, which in turn can affect bacterial pathogenicity, virulence, and environmental adaptability. Herein, we review the diversity of modifications that the EPS alginate, the Pel polysaccharide, Vibrio polysaccharide, cepacian, glycosaminoglycans, and poly-N-acetyl-glucosamine undergo during biosynthesis. These are EPS produced by human pathogenic bacteria for which studies have begun to unravel the effect modifications have on their physicochemical and biological properties. The biological advantages these polymer modifications confer to the bacteria that produce them will be discussed. The expanding list of identified modifications will allow future efforts to focus on linking these modifications to specific biosynthetic genes and biofilm phenotypes.
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  14. 14
    Thongsomboon, W.; Serra, D. O.; Possling, A.; Hadjineophytou, C.; Hengge, R.; Cegelski, L. Phosphoethanolamine Cellulose: A Naturally Produced Chemically Modified Cellulose. Science (Washington, DC, U. S.) 2018, 359 (6373), 334– 338,  DOI: 10.1126/science.aao4096
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    14
    Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose
    Thongsomboon, Wiriya; Serra, Diego O.; Possling, Alexandra; Hadjineophytou, Chris; Hengge, Regine; Cegelski, Lynette
    Science (Washington, DC, United States) (2018), 359 (6373), 334-338CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Cellulose is a major contributor to the chem. and mech. properties of plants and assumes structural roles in bacterial communities termed biofilms. We find that Escherichia coli produces chem. modified cellulose that is required for extracellular matrix assembly and biofilm architecture. Solid-state NMR spectroscopy of the intact and insol. material elucidates the zwitterionic phosphoethanolamine modification that had evaded detection by conventional methods. Installation of the phosphoethanolamine group requires BcsG, a proposed phosphoethanolamine transferase, with biofilm-promoting cyclic diguanylate monophosphate input through a BcsE-BcsF-BcsG transmembrane signaling pathway. The bcsEFG operon is present in many bacteria, including Salmonella species, that also produce the modified cellulose. The discovery of phosphoethanolamine cellulose and the genetic and mol. basis for its prodn. offers opportunities to modulate its prodn. in bacteria and inspires efforts to biosynthetically engineer alternatively modified cellulosic materials.
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  15. 15
    Whitfield, G. B.; Marmont, L. S.; Bundalovic-Torma, C.; Razvi, E.; Roach, E. J.; Khursigara, C. M.; Parkinson, J.; Howell, P. L. Discovery and Characterization of a Gram- Positive Pel Polysaccharide Biosynthetic Gene Cluster. PLoS Pathog. 2020, 16 (4), e1008281,  DOI: 10.1371/journal.ppat.1008281
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    15
    Discovery and characterization of a Gram positive Pel polysaccharide biosynthetic gene cluster
    Whitfield, Gregory B.; Marmont, Lindsey S.; Bundalovic-Torma, Cedoljub; Razvi, Erum; Roach, Elyse J.; Khursigara, Cezar M.; Parkinson, John; Howell, P. Lynne
    PLoS Pathogens (2020), 16 (4), e1008281CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)
    Our understanding of the biofilm matrix components utilized by Gram-pos.bacteria, and the signaling pathways that regulate their prodn.are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the anal. of synthase-dependent exopolysaccharide biosynthetic systems. Here, we explore the finding that many species of Grampos.bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B.cereus PelD ortholog binds cyclic-3',5-dimeric guanosine monophosphate (c-diGMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the prodn. of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signaling in Gram-pos.organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of bacilli, clostridia, streptococci, and actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-pos.bacteria.
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  16. 16
    Römling, U. Molecular Biology of Cellulose Production in Bacteria. Res. Microbiol. 2002, 153 (4), 205– 212,  DOI: 10.1016/S0923-2508(02)01316-5
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    16
    Molecular biology of cellulose production in bacteria
    Romling Ute
    Research in microbiology (2002), 153 (4), 205-12 ISSN:0923-2508.
    Cellulose biosynthesis has recently been established for a variety of bacteria of diverse origin at the phenotypic and genetic levels. Novel regulatory pathways, which involve the second messenger bis-(3',5') cyclic diguanylic acid and several proteins with the GGDEF domain, participate in the regulation of cellulose biosynthesis. The biological significance of cellulose production in environmental, commensal and pathogenic bacteria is only punctually resolved. This review summarizes current knowledge on cellulose biosynthesis, its regulation and biological function.
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  17. 17
    Römling, U.; Galperin, M. Y. Bacterial Cellulose Biosynthesis: Diversity of Operons, Subunits, Products, and Functions. Trends Microbiol. 2015, 23 (9), 545– 557,  DOI: 10.1016/j.tim.2015.05.005
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    17
    Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions
    Romling Ute; Galperin Michael Y
    Trends in microbiology (2015), 23 (9), 545-57 ISSN:.
    Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits - which differ among various taxa - affect the enzymatic activity and product yield in vivo by modulating (i) the expression of the biosynthesis apparatus, (ii) the export of the nascent β-D-glucan polymer to the cell surface, and (iii) the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of resulting biofilms, which is particularly important for the interactions of bacteria with higher organisms - leading to rhizosphere colonization and modulating the virulence of cellulose-producing bacterial pathogens inside and outside of host cells. We review the organization of four principal types of cellulose synthase operon found in various bacterial genomes, identify additional bcs genes that encode components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms and in the choice between acute infection and persistence in the host.
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  18. 18
    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. U. S. A. 2013, 110 (44), 17856– 17861,  DOI: 10.1073/pnas.1314063110
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    18
    BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis
    Omadjela, Okako; Narahari, Adishesh; Strumillo, Joanna; Melida, Hugo; Mazur, Olga; Bulone, Vincent; Zimmer, Jochen
    Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (44), 17856-17861,S17856/1-S17856/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromol. complexes contg. several different enzyme isoforms, prokaryotic synthases assoc. with addnl. subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-assocd. bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochem. studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a d.p. in the range 200-300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation anal. of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-assocd. domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP.
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  19. 19
    Morgan, J. L. W.; McNamara, J. T.; Zimmer, J. Mechanism of Activation of Bacterial Cellulose Synthase by Cyclic Di-GMP. Nat. Struct. Mol. Biol. 2014, 21 (5), 489– 496,  DOI: 10.1038/nsmb.2803
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    19
    Mechanism of activation of bacterial cellulose synthase by cyclic di-GMP
    Morgan, Jacob L. W.; McNamara, Joshua T.; Zimmer, Jochen
    Nature Structural & Molecular Biology (2014), 21 (5), 489-496CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
    The bacterial signaling mol. cyclic di-GMP (c-di-GMP) stimulates the synthesis of bacterial cellulose, which is frequently found in biofilms. Bacterial cellulose is synthesized and translocated across the inner membrane by a complex of cellulose synthase BcsA and BcsB subunits. Here we present crystal structures of the c-di-GMP-activated BcsA-BcsB complex. The structures reveal that c-di-GMP releases an autoinhibited state of the enzyme by breaking a salt bridge that otherwise tethers a conserved gating loop that controls access to and substrate coordination at the active site. Disrupting the salt bridge by mutagenesis generates a constitutively active cellulose synthase. Addnl., the c-di-GMP-activated BcsA-BcsB complex contains a nascent cellulose polymer whose terminal glucose unit rests at a new location above BcsA's active site and is positioned for catalysis. Our mechanistic insights indicate how c-di-GMP allosterically modulates enzymic functions.
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  20. 20
    Morgan, J. L. W.; Strumillo, J.; Zimmer, J. Crystallographic Snapshot of Cellulose Synthesis and Membrane Translocation. Nature 2013, 493 (7431), 181– 186,  DOI: 10.1038/nature11744
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    20
    Crystallographic snapshot of cellulose synthesis and membrane translocation
    Morgan, Jacob L. W.; Strumillo, Joanna; Zimmer, Jochen
    Nature (London, United Kingdom) (2013), 493 (7431), 181-186CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Cellulose, the most abundant biol. macromol., is an extracellular, linear polymer of glucose mols. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose prodn. frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB subunits of Rhodobacter sphaeroides cellulose synthase (CESA) contg. a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose mol. at a time.
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  21. 21
    Whitfield, C.; Mainprize, I. L. TPR Motifs: Hallmarks of a New Polysaccharide Export Scaffold. Structure 2010, 18 (2), 151– 153,  DOI: 10.1016/j.str.2010.01.006
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    21
    TPR Motifs: Hallmarks of a New Polysaccharide Export Scaffold
    Whitfield, Chris; Mainprize, Iain L.
    Structure (Cambridge, MA, United States) (2010), 18 (2), 151-153CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
    A review. Bacteria produce a remarkable range of surface and secreted polysaccharides. Two pathways have been defined for the biosynthesis and export of capsular polysaccharides and exopolysaccharides in Gram-neg. bacteria. A structure of AlgK described in this issue provides structural insight into a third previously unrecognized pathway assocd. with important biopolymers.
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  22. 22
    Low, K. E.; Howell, P. L. Gram-Negative Synthase-Dependent Exopolysaccharide Biosynthetic Machines. Curr. Opin. Struct. Biol. 2018, 53, 32– 44,  DOI: 10.1016/j.sbi.2018.05.001
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    22
    Gram-negative synthase-dependent exopolysaccharide biosynthetic machines
    Low, Kristin E.; Howell, P. Lynne
    Current Opinion in Structural Biology (2018), 53 (), 32-44CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)
    A review. Bacteria predominantly exist as matrix embedded communities of cells called biofilms. The biofilm matrix is made up of a variety of self-produced extracellular components including DNA, proteins, and exopolysaccharides. Bacterial exopolysaccharides have been implicated in surface adhesion, resistance to antibiotics, and protection from host immune systems. Herein review the structure and function of the proteins involved in the prodn. of the Gram-neg. synthase-dependent exopolysaccharides: alginate, poly-β(1,6)-N-acetyl-D-glucosamine (PNAG), cellulose, and the Pel polysaccharide. This study highlight the similarities and differences that exist at the mol. level in these synthase systems.
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  23. 23
    Acheson, J. F.; Derewenda, Z. S.; Zimmer, J. Architecture of the Cellulose Synthase Outer Membrane Channel and Its Association with the Periplasmic TPR Domain. Structure 2019, 27 (12), 1855– 1861.e3,  DOI: 10.1016/j.str.2019.09.008
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    23
    Architecture of the cellulose synthase outer membrane channel and its association with the periplasmic TPR domain
    Acheson, Justin F.; Derewenda, Zygmunt S.; Zimmer, Jochen
    Structure (Oxford, United Kingdom) (2019), 27 (12), 1855-1861.e3CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
    Extracellular bacterial cellulose contributes to biofilm stability and to the integrity of the bacterial cell envelope. In Gram-neg. bacteria, cellulose is synthesized and secreted by a multi-component cellulose synthase complex. The BcsA subunit synthesizes cellulose and also transports the polymer across the inner membrane. Translocation across the outer membrane occurs through the BcsC porin, which extends into the periplasm via 19 tetra-tricopeptide repeats (TPR). We present the crystal structure of a truncated BcsC, encompassing the last TPR repeat and the complete outer membrane channel domain, revealing a 16-stranded, β barrel pore architecture. The pore is blocked by an extracellular gating loop, while the extended C terminus inserts deeply into the channel and positions a conserved Trp residue near its extracellular exit. The channel is lined with hydrophilic and arom. residues suggesting a mechanism for facilitated cellulose diffusion based on arom. stacking and hydrogen bonding.
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  24. 24
    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 (1), 13018,  DOI: 10.1038/s41598-017-12530-0
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    24
    Crystal structure of the flexible tandem repeat domain of bacterial cellulose synthesis subunit C
    Nojima Shingo; Fujishima Ayumi; Kato Koji; Ohuchi Kayoko; Yao Min; Kato Koji; Yao Min; Shimizu Nobutaka; Yonezawa Kento; Tajima Kenji
    Scientific reports (2017), 7 (1), 13018 ISSN:.
    Bacterial cellulose (BC) is synthesized and exported through the cell membrane via a large protein complex (terminal complex) that consists of three or four subunits. BcsC is a little-studied subunit considered to export BC to the extracellular matrix. It is predicted to have two domains: a tetratrico peptide repeat (TPR) domain and a β-barrelled outer membrane domain. Here we report the crystal structure of the N-terminal part of BcsC-TPR domain (Asp24-Arg272) derived from Enterobacter CJF-002. Unlike most TPR-containing proteins which have continuous TPR motifs, this structure has an extra α-helix between two clusters of TPR motifs. Five independent molecules in the crystal had three different conformations that varied at the hinge of the inserted α-helix. Such structural feature indicates that the inserted α-helix confers flexibility to the chain and changes the direction of the TPR super-helix, which was also suggested by structural analysis of BcsC-TPR (Asp24-Leu664) in solution by size exclusion chromatography-small-angle X-ray scattering. The flexibility at the α-helical hinge may play important role for exporting glucan chains.
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  25. 25
    Anderson, A. C.; Burnett, A. J. N.; Hiscock, L.; Maly, K. E.; Weadge, J. T. The Escherichia Coli Cellulose Synthase Subunit G (BcsG) Is a Zn2+-Dependent Phosphoethanolamine Transferase. J. Biol. Chem. 2020, 295 (18), 6225– 6235,  DOI: 10.1074/jbc.RA119.011668
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    25
    The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase
    Anderson, Alexander C.; Burnett, Alysha J. N.; Hiscock, Lana; Maly, Kenneth E.; Weadge, Joel T.
    Journal of Biological Chemistry (2020), 295 (18), 6225-6235CODEN: JBCHA3; ISSN:1083-351X. (American Society for Biochemistry and Molecular Biology)
    A review. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrixes has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane-localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alk. phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-neg. bacteria. Informed by our structural studies, we present a functional complementation expt. in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes.
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  26. 26
    Mazur, O.; Zimmer, J. Apo- and Cellopentaose-Bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ. J. Biol. Chem. 2011, 286 (20), 17601– 17606,  DOI: 10.1074/jbc.M111.227660
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    26
    Apo- and Cellopentaose-bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ
    Mazur, Olga; Zimmer, Jochen
    Journal of Biological Chemistry (2011), 286 (20), 17601-17606CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Cellulose, a very abundant extracellular polysaccharide, is synthesized in a finely tuned process that involves the activity of glycosyl-transferases and hydrolases. The cellulose microfibril consists of bundles of linear β-1,4-glucan chains that are synthesized inside the cell; however, the mechanism by which these polymers traverse the cell membrane is currently unknown. In Gram-neg. bacteria, the cellulose synthase complex forms a trans-envelope complex consisting of at least four subunits. Although three of these subunits account for the synthesis and translocation of the polysaccharide, the fourth subunit, BcsZ, is a periplasmic protein with endo-β-1,4-glucanase activity. BcsZ belongs to family eight of glycosyl-hydrolases, and its activity is required for optimal synthesis and membrane translocation of cellulose. In this study we report two crystal structures of BcsZ from Escherichia coli. One structure shows the wild-type enzyme in its apo form, and the second structure is for a catalytically inactive mutant of BcsZ in complex with the substrate cellopentaose. The structures demonstrate that BcsZ adopts an (α/α)6-barrel fold and that it binds four glucan moieties of cellopentaose via highly conserved residues exclusively on the nonreducing side of its catalytic center. Thus, the BcsZ-cellopentaose structure most likely represents a posthydrolysis state in which the newly formed nonreducing end has already left the substrate binding pocket while the enzyme remains attached to the truncated polysaccharide chain. We further show that BcsZ efficiently degrades β-1,4-glucans in in vitro cellulase assays with carboxymethyl-cellulose as substrate.
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  27. 27
    Sun, L.; Vella, P.; Schnell, R.; Polyakova, A.; Bourenkov, G.; Li, F.; Cimdins, A.; Schneider, T. R.; Lindqvist, Y.; Galperin, M. Y.; Schneider, G.; Römling, U. Structural and Functional Characterization of the BcsG Subunit of the Cellulose Synthase in Salmonella Typhimurium. J. Mol. Biol. 2018, 430 (18), 3170– 3189,  DOI: 10.1016/j.jmb.2018.07.008
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    27
    Structural and functional characterization of the BcsG subunit of the cellulose synthase in Salmonella typhimurium
    Sun, Lei; Vella, Peter; Schnell, Robert; Polyakova, Anna; Bourenkov, Gleb; Li, Fengyang; Cimdins, Annika; Schneider, Thomas R.; Lindqvist, Ylva; Galperin, Michael Y.; Schneider, Gunter; Roemling, Ute
    Journal of Molecular Biology (2018), 430 (18_Part_B), 3170-3189CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Many bacteria secrete cellulose, which forms the structural basis for bacterial multicellular aggregates, termed biofilms. The cellulose synthase complex of S. typhimurium consists of catalytic subunits BcsA and BcsB and several auxiliary subunits that are encoded by 2 divergently transcribed operons, bcsRQABZC and bcsEFG. Expression of the bcsEFG operon is required for full-scale cellulose prodn., but the functions of its products are not fully understood. This work aimed to characterize the BcsG subunit of cellulose synthase, which consists of an N-terminal transmembrane fragment and a C-terminal domain in the periplasm. Deletion of the bcsG gene substantially decreased the total amt. of BcsA and cellulose prodn. BcsA levels were partially restored by the expression of the transmembrane segment, whereas restoration of cellulose prodn. required the presence of the C-terminal periplasmic domain and its characteristic metal-binding residues. The high-resoln. crystal structure of the periplasmic domain characterized BcsG as a member of the alk. phosphatase/sulfatase superfamily of metalloenzymes, contg. a conserved Zn2+-binding site. Sequence and structural comparisons showed that BcsG belongs to a specific family within alk. phosphatase-like enzymes, which includes bacterial Zn2+-dependent lipopolysaccharide phosphoethanolamine transferases such as MCR-1 (colistin resistance protein), EptA, and EptC and Mn2+-dependent lipoteichoic acid synthase (phosphoglycerol transferase) LtaS. These enzymes use the phospholipids, phosphatidylethanolamine and phosphatidylglycerol, resp., as substrates. These data were consistent with the recently discovered phosphoethanolamine modification of cellulose by BcsG and showed that its membrane-bound and periplasmic parts play distinct roles in the assembly of the functional cellulose synthase and cellulose prodn.
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  28. 28
    Fang, X.; Ahmad, I.; Blanka, A.; Schottkowski, M.; Cimdins, A.; Galperin, M. Y.; Römling, U.; Gomelsky, M. GIL, a New C-di-GMP-binding Protein Domain Involved in Regulation of Cellulose Synthesis in Enterobacteria. Mol. Microbiol. 2014, 93 (3), 439– 452,  DOI: 10.1111/mmi.12672
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    28
    GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria
    Fang, Xin; Ahmad, Irfan; Blanka, Andrea; Schottkowski, Marco; Cimdins, Annika; Galperin, Michael Y.; Roemling, Ute; Gomelsky, Mark
    Molecular Microbiology (2014), 93 (3), 439-452CODEN: MOMIEE; ISSN:0950-382X. (Wiley-Blackwell)
    In contrast to numerous enzymes involved in c-di-GMP synthesis and degrdn. in enterobacteria, only a handful of c-di-GMP receptors/effectors have been identified. In search of new c-di-GMP receptors, the authors screened the Escherichia coli ASKA overexpression gene library using the Differential Radial Capillary Action of Ligand Assay (DRaCALA) with fluorescently and radioisotope-labeled c-di-GMP. The authors uncovered three new candidate c-di-GMP receptors in E. coli and characterized one of them, BcsE. The bcsE gene is encoded in cellulose synthase operons in representatives of Gammaproteobacteria and Betaproteobacteria. The purified BcsE proteins from E. coli, Salmonella enterica and Klebsiella pneumoniae bind c-di-GMP via the domain of unknown function, DUF2819, which is hereby designated GIL, GGDEF I-site like domain. The RxGD motif of the GIL domain is required for c-di-GMP binding, similar to the c-di-GMP-binding I-site of the diguanylate cyclase GGDEF domain. Thus, GIL is the second protein domain, after PilZ, dedicated to c-di-GMP-binding. In S. enterica, BcsE is not essential for cellulose synthesis but is required for maximal cellulose prodn., and c-di-GMP binding is crit. for BcsE function. It appears that cellulose prodn. in enterobacteria is controlled by a two-tiered c-di-GMP-dependent system involving BcsE and the PilZ domain contg. glycosyltransferase BcsA.
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  29. 29
    Zouhir, S.; Abidi, W.; Caleechurn, M.; Krasteva, P. V. Structure and Multitasking of the C-Di-GMP-Sensing Cellulose Secretion Regulator BcsE. mBio 2020, 11, 4,  DOI: 10.1128/mBio.01303-20
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    There is no corresponding record for this reference.
  30. 30
    Acheson, J. F.; Ho, R.; Goularte, N. F.; Cegelski, L.; Zimmer, J. Molecular Organization of the E. Coli Cellulose Synthase Macrocomplex. Nat. Struct. Mol. Biol. 2021, 28, 310,  DOI: 10.1038/s41594-021-00569-7
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    30
    Molecular organization of the E. coli cellulose synthase macrocomplex
    Acheson, Justin F.; Ho, Ruoya; Goularte, Nicolette F.; Cegelski, Lynette; Zimmer, Jochen
    Nature Structural & Molecular Biology (2021), 28 (3), 310-318CODEN: NSMBCU; ISSN:1545-9993. (Nature Research)
    Abstr.: Cellulose is frequently found in communities of sessile bacteria called biofilms. Escherichia coli and other enterobacteriaceae modify cellulose with phosphoethanolamine (pEtN) to promote host tissue adhesion. The E. coli pEtN cellulose biosynthesis machinery contains the catalytic BcsA-B complex that synthesizes and secretes cellulose, in addn. to five other subunits. The membrane-anchored periplasmic BcsG subunit catalyzes pEtN modification. Here we present the structure of the roughly 1 MDa E. coli Bcs complex, consisting of one BcsA enzyme assocd. with six copies of BcsB, detd. by single-particle cryo-electron microscopy. BcsB homo-oligomerizes primarily through interactions between its carbohydrate-binding domains as well as intermol. beta-sheet formation. The BcsB hexamer creates a half spiral whose open side accommodates two BcsG subunits, directly adjacent to BcsA's periplasmic channel exit. The cytosolic BcsE and BcsQ subunits assoc. with BcsA's regulatory PilZ domain. The macrocomplex is a fascinating example of cellulose synthase specification.
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  31. 31
    Abidi, W.; Zouhir, S.; Caleechurn, M.; Roche, S.; Krasteva, P. V. Architecture and Regulation of an Enterobacterial Cellulose Secretion System. Sci. Adv. 2021, 7 (5), 1– 16,  DOI: 10.1126/sciadv.abd8049
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    There is no corresponding record for this reference.
  32. 32
    Hinchliffe, P.; Yang, Q. E.; Portal, E.; Young, T.; Li, H.; Tooke, C. L.; Carvalho, M. J.; Paterson, N. G.; Brem, J.; Niumsup, P. R.; Tansawai, U.; Lei, L.; Li, M.; Shen, Z.; Wang, Y.; Schofield, C. J.; Mulholland, A. J.; Shen, J.; Fey, N.; Walsh, T. R.; Spencer, J. Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1. Sci. Rep. 2017, 7, 39392,  DOI: 10.1038/srep39392
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    32
    Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1
    Hinchliffe, Philip; Yang, Qiu E.; Portal, Edward; Young, Tom; Li, Hui; Tooke, Catherine L.; Carvalho, Maria J.; Paterson, Neil G.; Brem, Jurgen; Niumsup, Pannika R.; Tansawai, Uttapoln; Lei, Lei; Li, Mei; Shen, Zhangqi; Wang, Yang; Schofield, Christopher J.; Mulholland, Adrian J.; Shen, Jianzhong; Fey, Natalie; Walsh, Timothy R.; Spencer, James
    Scientific Reports (2017), 7 (), 39392CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    The polymixin colistin is a "last line" antibiotic against extensively-resistant Gram-neg. bacteria. Recently, the mcr-1 gene was identified as a plasmid-mediated resistance mechanism in human and animal Enterobacteriaceae, with a wide geog. distribution and many producer strains resistant to multiple other antibiotics. Mcr-1 encodes a membrane-bound enzyme catalyzing phosphoethanolamine transfer onto bacterial lipid A. Here we present crystal structures revealing the MCR-1 periplasmic, catalytic domain to be a zinc metalloprotein with an alk. phosphatase/sulphatase fold contg. three disulfide bonds. One structure captures a phosphorylated form representing the first intermediate in the transfer reaction. Mutation of residues implicated in zinc or phosphoethanolamine binding, or catalytic activity, restores colistin susceptibility of recombinant E. coli. Zinc deprivation reduces colistin MICs in MCR-1-producing lab., environmental, animal and human E. coli. Conversely, over-expression of the disulfide isomerase DsbA increases the colistin MIC of lab. E. coli. Preliminary d. functional theory calcns. on cluster models suggest a single zinc ion may be sufficient to support phosphoethanolamine transfer. These data demonstrate the importance of zinc and disulfide bonds to MCR-1 activity, suggest that assays under zinc-limiting conditions represent a route to phenotypic identification of MCR-1 producing E. coli, and identify key features of the likely catalytic mechanism.
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  33. 33
    McCall, K. A.; Huang, C.; Fierke, C. A. Function and Mechanism of Zinc Metalloenzymes. J. Nutr. 2000, 130 (5), 1437S– 1446S,  DOI: 10.1093/jn/130.5.1437S
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    33
    Function and mechanism of zinc metalloenzymes
    McCall, Keith A.; Huang, Chih-Chinx; Fierke, Carol A.
    Journal of Nutrition (2000), 130 (5S), 1437S-1446SCODEN: JONUAI; ISSN:0022-3166. (American Society for Nutritional Sciences)
    A review with 115 refs. Zn is required for the activity of >300 enzymes, covering all 6 classes of enzymes. Zn-binding sites in proteins are often distorted tetrahedral or trigonal bipyramidal geometry, made up of the S atom of Cys, the N atom of His, or the O atom of Asp and Glu residues, or a combination. Zn in proteins can either participate directly in chem. catalysis or be important for maintaining protein structure and stability. In all catalytic sites, the Zn ion functions as a Lewis acid. Researchers in the authors' lab. are dissecting the determinants of mol. recognition and catalysis in the Zn-binding site of carbonic anhydrase. These studies demonstrate that the chem. nature of the direct ligands and the structure of the surrounding H-bond network are crucial for both the activity of carbonic anhydrase and the metal cation affinity of the Zn-binding site. An understanding of naturally occurring Zn-binding sites will aid in creating de novo Zn-binding proteins and in designing new metal sites in existing proteins for novel purposes such as to serve as metal ion biosensors.
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  34. 34
    Fage, C. D.; Brown, D. B.; Boll, J. M.; Keatinge-Clay, A. T.; Trent, M. S. Crystallographic Study of the Phosphoethanolamine Transferase EptC Required for Polymyxin Resistance and Motility in Campylobacter Jejuni. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2014, 70 (10), 2730– 2739,  DOI: 10.1107/S1399004714017623
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    34
    Crystallographic study of the phosphoethanolamine transferase EptC required for polymyxin resistance and motility in Campylobacter jejuni
    Fage, Christopher D.; Brown, Dusty B.; Boll, Joseph M.; Keatinge-Clay, Adrian T.; Trent, M. Stephen
    Acta Crystallographica, Section D: Biological Crystallography (2014), 70 (10), 2730-2739CODEN: ABCRE6; ISSN:1399-0047. (International Union of Crystallography)
    The food-borne enteric pathogen Campylobacter jejuni decorates a variety of its cell-surface structures with phosphoethanolamine (pEtN). Modifying lipid A with pEtN promotes cationic antimicrobial peptide resistance, whereas post-translationally modifying the flagellar rod protein FlgG with pEtN promotes flagellar assembly and motility, which are processes that are important for intestinal colonization. EptC, the pEtN transferase required for all known pEtN cell-surface modifications in C. jejuni, is a predicted inner-membrane metalloenzyme with a five-helix N-terminal transmembrane domain followed by a sol. sulfatase-like catalytic domain in the periplasm. The at. structure of the catalytic domain of EptC (cEptC) was crystd. and solved to a resoln. of 2.40 Å. cEptC adopts the α/β/α fold of the sulfatase protein family and harbors a zinc-binding site. A phosphorylated Thr-266 residue was obsd. that was hypothesized to mimic a covalent pEtN-enzyme intermediate. The requirement for Thr-266 as well as the nearby residues Asn-308, Ser-309, His-358 and His-440 was ascertained via in vivo activity assays on mutant strains. The results establish a basis for the design of pEtN transferase inhibitors.
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  35. 35
    Wanty, C.; Anandan, A.; Piek, S.; Walshe, J.; Ganguly, J.; Carlson, R. W.; Stubbs, K. A.; Kahler, C. M.; Vrielink, A. The Structure of the Neisserial Lipooligosaccharide Phosphoethanolamine Transferase A (LptA) Required for Resistance to Polymyxin. J. Mol. Biol. 2013, 425 (18), 3389– 3402,  DOI: 10.1016/j.jmb.2013.06.029
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    35
    The Structure of the Neisserial Lipooligosaccharide Phosphoethanolamine Transferase A (LptA) Required for Resistance to Polymyxin
    Wanty, Christopher; Anandan, Anandhi; Piek, Susannah; Walshe, James; Ganguly, Jhuma; Carlson, Russell W.; Stubbs, Keith A.; Kahler, Charlene M.; Vrielink, Alice
    Journal of Molecular Biology (2013), 425 (18), 3389-3402CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Gram-neg. bacteria possess an outer membrane envelope consisting of an outer leaflet of lipopolysaccharides, also called endotoxins, which protect the pathogen from antimicrobial peptides and have multifaceted roles in virulence. Lipopolysaccharide consists of a glycan moiety attached to lipid A, embedded in the outer membrane. Modification of the lipid A headgroups by phosphoethanolamine (PEA) or 4-amino-arabinose residues increases resistance to the cationic cyclic polypeptide antibiotic, polymyxin. Lipid A PEA transferases are members of the YhjW/YjdB/YijP superfamily and usually consist of a transmembrane domain anchoring the enzyme to the periplasmic face of the cytoplasmic membrane attached to a sol. catalytic domain. The crystal structure of the sol. domain of the protein of the lipid A PEA transferase from Neisseria meningitidis has been detd. crystallog. and refined to 1.4 Å resoln. The structure reveals a core hydrolase fold similar to that of alk. phosphatase. Loop regions in the structure differ, presumably to enable interaction with the membrane-localized substrates and to provide substrate specificity. A phosphorylated form of the putative nucleophile, Thr280, is obsd. Metal ions present in the active site are coordinated to Thr280 and to residues conserved among the family of transferases. The structure reveals the protein components needed for the transferase chem.; however, substrate-binding regions are not evident and are likely to reside in the transmembrane domain of the protein.
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  36. 36
    Anandan, A.; Evans, G. L.; Condic-Jurkic, K.; O’Mara, M. L.; John, C. M.; Phillips, N. J.; Jarvis, G. A.; Wills, S. S.; Stubbs, K. A.; Moraes, I.; Kahler, C. M.; Vrielink, A. Structure of a Lipid A Phosphoethanolamine Transferase Suggests How Conformational Changes Govern Substrate Binding. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (9), 2218– 2223,  DOI: 10.1073/pnas.1612927114
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    Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding
    Anandan, Anandhi; Evans, Genevieve L.; Condic-Jurkic, Karmen; O'Mara, Megan L.; John, Constance M.; Phillips, Nancy J.; Jarvis, Gary A.; Wills, Siobhan S.; Stubbs, Keith A.; Moraes, Isabel; Kahler, Charlene M.; Vrielink, Alice
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (9), 2218-2223CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Multidrug-resistant (MDR) gram-neg. bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin contg. lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addn. of phosphoethanolamine to the 1 and 4' headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from Neisseria meningitidis, detd. to 2.75-Å resoln. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing sol. domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helixes located in a periplasmic loop between two transmembrane helixes contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and mol. dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different-sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.
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  37. 37
    Moynihan, M. M.; Murkin, A. S. Cysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3-Nitropropionate. Biochemistry 2014, 53 (1), 178– 187,  DOI: 10.1021/bi401432t
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    37
    Cysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3-Nitropropionate
    Moynihan, Margaret M.; Murkin, Andrew S.
    Biochemistry (2014), 53 (1), 178-187CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Isocitrate lyase (ICL) catalyzes the reversible cleavage of isocitrate into succinate and glyoxylate. It is the first committed step in the glyoxylate cycle used by some organisms, including Mycobacterium tuberculosis, where it has been shown to be essential for cell survival during chronic infection. The pH-rate and pD-rate profiles measured in the direction of isocitrate synthesis revealed solvent kinetic isotope effects (KIEs) of 1.7 ± 0.4 for D2OV and 0.56 ± 0.07 for D2O(V/Ksuccinate). Whereas the D2OV is consistent with partially rate-limiting proton transfer during formation of the hydroxyl group of isocitrate, the large inverse D2O(V/Ksuccinate) indicates that substantially different kinetic parameters exist when the enzyme is satd. with succinate. Inhibition by 3-nitropropionate (3-NP), a succinate analog, was found to proceed through an unusual double slow-onset process featuring formation of a complex with a Ki of 3.3 ± 0.2 μM during the first minute, followed by formation of a final complex with a Ki* of 44 ± 10 nM over the course of several minutes to hours. Stopped-flow measurements during the first minute revealed an apparent solvent KIE of 0.40 ± 0.03 for assocn. and unity for dissocn. In contrast, itaconate, a succinate analog lacking an acidic α-proton, did not display slow-binding behavior and yielded a D2OKi of 1.0 ± 0.2. These results support a common mechanism for catalysis with succinate and inhibition by 3-NP featuring (1) an unfavorable prebinding isomerization of the active site Cys191-His193 pair to the thiolate-imidazolium form, a process that is favored in D2O, and (2) the transfer of a proton from succinate or 3-NP to Cys191. These findings also indicate that propionate-3-nitronate, which is the conjugate base of 3-NP and the "true inhibitor" of ICL, does not bind directly and must be generated enzymically.
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  38. 38
    Zhao, Y.; Meng, Q.; Lai, Y.; Wang, L.; Zhou, D.; Dou, C.; Gu, Y.; Nie, C.; Wei, Y.; Cheng, W. Structural and Mechanistic Insights into Polymyxin Resistance Mediated by EptC Originating from Escherichia Coli. FEBS J. 2019, 286 (4), 750– 764,  DOI: 10.1111/febs.14719
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    38
    Structural and mechanistic insights into polymyxin resistance mediated by EptC originating from Escherichia coli
    Zhao, Yanqun; Meng, Qiang; Lai, Yujie; Wang, Li; Zhou, Dan; Dou, Chao; Gu, Yijun; Nie, Chunlai; Wei, Yuquan; Cheng, Wei
    FEBS Journal (2019), 286 (4), 750-764CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
    Gram-neg. bacteria defend against the toxicity of polymyxins by modifying their outer membrane lipopolysaccharide (LPS). This modification mainly occurs through the addn. of cationic mols. such as phosphoethanolamine (PEA). EcEptC is a PEA transferase from Escherichia coli (E. coli). However, unlike its homologs CjEptC (Campylobacter jejuni) and MCR-1, EcEptC is unable to mediate polymyxin resistance when overexpressed in E. coli. Here, we report crystal structures of the C-terminal putative catalytic domain (EcEptCΔN, 205-577 aa) of EcEptC in apo and Zn2+-bound states at 2.10 and 2.60 Å, resp. EcEptCΔN is arranged into an α-β-α fold and equipped with the zinc ion in a conserved mode. Coupled with isothermal titrn. calorimetry (ITC) data, we provide insights into the mechanism by which EcEptC recognizes Zn2+. Furthermore, structure comparison anal. indicated that disulfide bonds, which play a key role in polymyxin resistance, were absent in EcEptCΔN. Supported by structural and biochem. evidence, we reveal mechanistic implications for disulfide bonds in PEA transferase-mediated polymyxin resistance. Significantly, because the structural effects exhibited by disulfide bonds are absent in EcEptC, it is impossible for this protein to participate in polymyxin resistance in E. coli.
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  39. 39
    Arciola, C. R.; Campoccia, D.; Ravaioli, S.; Montanaro, L. Polysaccharide Intercellular Adhesin in Biofilm: Structural and Regulatory Aspects. Front. Cell. Infect. Microbiol. 2015, 5, 7,  DOI: 10.3389/fcimb.2015.00007
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    39
    Polysaccharideintercellularadhesininbiofilm:structuralandregulatoryaspects
    Arciola, Carla Renata; Campoccia, Davide; Ravaioli, Stefano; Montanaro, Lucio
    Frontiers in Cellular and Infection Microbiology (2015), 5 (), 7/1-7/10CODEN: FCIMAB; ISSN:2235-2988. (Frontiers Media S.A.)
    Staphylococcus aureus and Staphylococcus epidermidis are the leading etiol. agents of implant-related infections. Biofilm formation is the main pathogenetic mechanism leading to the chronicity and irreducibility of infections. The extracellular polymeric substances of staphylococcal biofilms are the polysaccharide intercellular adhesin (PIA), extracellular-DNA, proteins, and amyloid fibrils. PIA is a poly-β(1-6)-N-acetylglucosamine (PNAG), partially deacetylated, pos. charged, whose synthesis is mediated by the icaADBC locus. DNA sequences homologous to icaADBC locus are present in many coagulase-neg. staphylococcal species, among which S. lugdunensis, however, produces a biofilm prevalently consisting of proteins. The product of icaA is an N-acetylglucosaminyltransferase that synthesizes PIA oligomers from UDP-N-acetylglucosamine. The product of icaD gives optimal efficiency to IcaA. The product of icaC is involved in the externalization of the nascent polysaccharide. The product of icaB is an N-deacetylase responsible for the partial deacetylation of PIA. The expression of ica locus is affected by environmental conditions. In S. aureus and S. epidermidis ica-independent alternative mechanisms of biofilm prodn. have been described. S.epidermidis and S. aureus undergo to a phase variation for the biofilm prodn. that has been ascribed, in turn, to the transposition of an insertion sequence in the icaC gene or to the expansion/contraction of a tandem repeat naturally harbored within icaC. A role is played by the quorum sensing system, which neg. regulates biofilm formation, favoring the dispersal phase that disseminates bacteria to new infection sites. Interfering with the QS system is a much debated strategy to combat biofilm-related infections. In the search of vaccines against staphylococcal infections deacetylated PNAG retained on the surface of S. aureus favors opsonophagocytosis and is a potential candidate for immune-protection.
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  40. 40
    Little, D. J.; Milek, S.; Bamford, N. C.; Ganguly, T.; Difrancesco, B. R.; Nitz, M.; Deora, R.; Howell, P. L. The Protein BpsB Is a Poly-β-1,6-N-Acetyl-D-Glucosamine Deacetylase Required for Biofilm Formation in Bordetella Bronchiseptica. J. Biol. Chem. 2015, 290 (37), 22827– 22840,  DOI: 10.1074/jbc.M115.672469
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    40
    The protein BpsB is a poly-β-1,6-N-acetyl-D-glucosamine deacetylase required for biofilm formation in Bordetella bronchiseptica
    Little, Dustin J.; Milek, Sonja; Bamford, Natalie C.; Ganguly, Tridib; Di Francesco, Benjamin R.; Nitz, Mark; Deora, Rajendar; Howell, P. Lynne
    Journal of Biological Chemistry (2015), 290 (37), 22827-22840CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Bordetella pertussis and Bordetella bronchiseptica are the causative agents of whooping cough in humans and a variety of respiratory diseases in animals, resp. Bordetella species produce an exopolysaccharide, known as the Bordetella polysaccharide (Bps), which is encoded by the bpsABCD operon. Bps is required for Bordetella biofilm formation, colonization of the respiratory tract, and confers protection from complement-mediated killing. Here, the authors investigated the role of BpsB in the biosynthesis of Bps and biofilm formation by B. bronchiseptica. BpsB is a two-domain protein that localizes to the periplasm and outer membrane. BpsB displays metal- and length-dependent deacetylation on poly-β-1,6-N-acetyl-D-glucosamine (PNAG) oligomers, supporting previous immunogenic data that suggests Bps is a PNAG polymer. BpsB can use a variety of divalent metal cations for deacetylase activity and showed highest activity in the presence of Ni2+ and Co2+. The structure of the BpsB deacetylase domain is similar to the PNAG deacetylases PgaB and IcaB and contains the same circularly permuted family four carbohydrate esterase motifs. Unlike PgaB from Escherichia coli, BpsB is not required for polymer export and has unique structural differences that allow the N-terminal deacetylase domain to be active when purified in isolation from the C-terminal domain. The enzymic characterizations here highlight the importance of conserved active site residues in PNAG deacetylation and demonstrate that the C-terminal domain is required for maximal deacetylation of longer PNAG oligomers. Furthermore, the authors show that BpsB is crit. for the formation and complex architecture of B. bronchiseptica biofilms.
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  41. 41
    Little, D. J.; Poloczek, J.; Whitney, J. C.; Robinson, H.; Nitz, M.; Howell, P. L. The Structure- and Metal-Dependent Activity of Escherichia Coli PgaB Provides Insight into the Partial de-N-Acetylation of Poly-β-1,6-N-Acetyl- D-Glucosamine. J. Biol. Chem. 2012, 287 (37), 31126– 31137,  DOI: 10.1074/jbc.M112.390005
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    41
    The structure- and metal-dependent activity of Escherichia coli PgaB provides insight into the partial de-N-acetylation of poly-β-1,6-N-acetyl-D-glucosamine
    Little, Dustin J.; Poloczek, Joanna; Whitney, John C.; Robinson, Howard; Nitz, Mark; Howell, P. Lynne
    Journal of Biological Chemistry (2012), 287 (37), 31126-31137CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In Escherichia coli, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-D-glucosamine (PNAG) by the periplasmic protein PgaB is required for polysaccharide intercellular adhesin-dependent biofilm formation. To understand the mol. basis for PNAG de-N-acetylation, the structure of PgaB in complex with Ni2+ and Fe3+ have been detd. to 1.9 and 2.1 Å resoln., resp., and its activity on β-1,6-GlcNAc oligomers has been characterized. The structure of PgaB reveals two (β/α)x barrel domains: a metal-binding de-N-acetylase that is a member of the family 4 carbohydrate esterases (CE4s) and a domain structurally similar to glycoside hydrolases. PgaB displays de-N-acetylase activity on β-1,6-GlcNAc oligomers but not on the β-1,4-(GlcNAc)4 oligomer chitotetraose and is the first CE4 member to exhibit this substrate specificity. De-N-acetylation occurs in a length-dependent manner, and specificity is obsd. for the position of de-N-acetylation. A key aspartic acid involved in de-N-acetylation, normally seen in other CE4s, is missing in PgaB, suggesting that the activity of PgaB is attenuated to maintain the low levels of de-N-acetylation of PNAG obsd. in vivo. The metal dependence of PgaB is different from most CE4s, because PgaB shows increased rates of de-N-acetylation with Co2+ and Ni2+ under aerobic conditions, and Co2+, Ni2+ and Fe2+ under anaerobic conditions, but decreased activity with Zn2+. The work presented herein will guide inhibitor design to combat biofilm formation by E. coli and potentially a wide range of medically relevant bacteria producing polysaccharide intercellular adhesin-dependent biofilms.
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  42. 42
    Little, D. J.; Li, G.; Ing, C.; DiFrancesco, B. R.; Bamford, N. C.; Robinson, H.; Nitz, M.; Pomes, R.; Howell, P. L. Modification and Periplasmic Translocation of the Biofilm Exopolysaccharide Poly- −1,6-N-Acetyl-D-Glucosamine. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (30), 11013– 11018,  DOI: 10.1073/pnas.1406388111
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    42
    Modification and periplasmic translocation of the biofilm exopolysaccharide poly-β-1,6-N-acetyl-D-glucosamine
    Little, Dustin J.; Li, Grace; Ing, Christopher; Di Francesco, Benjamin R.; Bamford, Natalie C.; Robinson, Howard; Nitz, Mark; Pomes, Regis; Howell, P. Lynne
    Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (30), 11013-11018CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Poly-β-1,6-N-acetyl-D-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of medically important bacteria. Polyglucosamine subunit B (PgaB) is responsible for the de-N-acetylation of PNAG, a process required for polymer export and biofilm formation. PgaB is located in the periplasm and likely bridges the inner membrane synthesis and outer membrane export machinery. Here, we present structural, functional, and mol. simulation data that suggest PgaB assocs. with PNAG continuously during periplasmic transport. We show that the assocn. of PgaB's N- and C-terminal domains forms a cleft required for the binding and de-N-acetylation of PNAG. Mol. dynamics (MD) simulations of PgaB show a binding preference for N-acetylglucosamine (GlcNAc) to the N-terminal domain and glucosammonium to the C-terminal domain. Continuous ligand binding d. is obsd. that extends around PgaB from the N-terminal domain active site to an electroneg. groove on the C-terminal domain that would allow for a processive mechanism. PgaB's C-terminal domain (PgaB310-672) directly binds PNAG oligomers with dissocn. consts. of ∼1-3 mM, and the structures of PgaB310-672 in complex with β-1,6-(GlcNAc)6, GlcNAc, and glucosamine reveal a unique binding mode suitable for interaction with de-N-acetylated PNAG (dPNAG). Furthermore, PgaB310-672 contains a β-hairpin loop (βHL) important for binding PNAG that was disordered in previous PgaB32-655 structures and is highly dynamic in the MD simulations. We propose that conformational changes in PgaB32-655 mediated by the βHL on binding of PNAG/dPNAG play an important role in the targeting of the polymer for export and its release.
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  43. 43
    Pokrovskaya, V.; Poloczek, J.; Little, D. J.; Griffiths, H.; Howell, P. L.; Nitz, M. Functional Characterization of Staphylococcus Epidermidis IcaB, a De-N-Acetylase Important for Biofilm Formation. Biochemistry 2013, 52 (32), 5463– 5471,  DOI: 10.1021/bi400836g
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    43
    Functional characterization of Staphylococcus epidermidis IcaB, a de-N-acetylase important for biofilm formation
    Pokrovskaya, Varvara; Poloczek, Joanna; Little, Dustin J.; Griffiths, Heather; Howell, P. Lynne; Nitz, Mark
    Biochemistry (2013), 52 (32), 5463-5471CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    A polymer of partially de-N-acetylated β-1,6-linked N-acetylglucosamine (dPNAG), also known as the polysaccharide intercellular adhesin (PIA), is an important component of many bacterial biofilm matrixes. In S. epidermidis, the poly-N-acetylglucosamine polymer is partially de-N-acetylated by the extracellular protein, polysaccharide deacetylase IcaB. To understand the mechanism of action of IcaB, the enzyme was overexpressed and purified. IcaB demonstrated metal-dependent de-N-acetylase activity on β-1,6-linked N-acetylglucosamine oligomers with a broad preference for divalent metals. Steady-state kinetic anal. revealed the low catalytic efficiency (pentasaccharide kcat/Km = 0.03 M-1 s-1) of the enzyme toward the oligomeric substrates. While IcaB displays similar rates of de-N-acetylation with tri- through hexasaccharide PNAG oligomers, position-specific de-N-acetylation was only obsd. with penta- and hexasaccharides. The enzyme preferentially de-N-acetylated the 2nd residue from the reducing terminus in the pentasaccharide and 2nd and 3rd residues from the reducing terminus in the hexasaccharide. The data described here represent an important step toward a detailed understanding of dPNAG biosynthesis in S. epidermidis, an important nosocomial pathogen, as well as in other Gram-pos. bacteria. The low catalytic activity of IcaB was consistent with reports of other enzymes which act on biofilm-related polysaccharides, and this emerging trend may indicate a common feature among this group of polysaccharide-processing enzymes.
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  44. 44
    Suardíaz, R.; Lythell, E.; Hinchliffe, P.; Van Der Kamp, M.; Spencer, J.; Fey, N.; Mulholland, A. J. Catalytic Mechanism of the Colistin Resistance Protein MCR-1. Org. Biomol. Chem. 2021, 19, 3813– 3819,  DOI: 10.1039/D0OB02566F
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    44
    Catalytic mechanism of the colistin resistance protein MCR-1
    Suardiaz, Reynier; Lythell, Emily; Hinchliffe, Philip; van der Kamp, Marc; Spencer, James; Fey, Natalie; Mulholland, Adrian J.
    Organic & Biomolecular Chemistry (2021), 19 (17), 3813-3819CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    The mcr-1 gene encodes a membrane-bound Zn2+-metalloenzyme, MCR-1, which catalyzes phosphoethanolamine transfer onto bacterial lipid A, making bacteria resistant to colistin, a last-resort antibiotic. Mechanistic understanding of this process remains incomplete. Here, we investigate possible catalytic pathways using DFT and ab initio calcns. on cluster models and identify a complete two-step reaction mechanism. The first step, formation of a covalent phosphointermediate via transfer of phosphoethanolamine from a membrane phospholipid donor to the acceptor Thr285, is rate-limiting and proceeds with a single Zn2+ ion. The second step, transfer of the phosphoethanolamine group to lipid A, requires an addnl. Zn2+. The calcns. suggest the involvement of the Zn2+ orbitals directly in the reaction is limited, with the second Zn2+ acting to bind incoming lipid A and direct phosphoethanolamine addn. The new level of mechanistic detail obtained here, which distinguishes these enzymes from other phosphotransferases, will aid in the development of inhibitors specific to MCR-1 and related bacterial phosphoethanolamine transferases.
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  45. 45
    Bar-Even, A.; Noor, E.; Savir, Y.; Liebermeister, W.; Davidi, D.; Tawfik, D. S.; Milo, R. The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters. Biochemistry 2011, 50 (21), 4402– 4410,  DOI: 10.1021/bi2002289
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    45
    The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters
    Bar-Even, Arren; Noor, Elad; Savir, Yonatan; Liebermeister, Wolfram; Davidi, Dan; Tawfik, Dan S.; Milo, Ron
    Biochemistry (2011), 50 (21), 4402-4410CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The kinetic parameters of enzymes are key to understanding the rate and specificity of most biol. processes. Although specific trends are frequently studied for individual enzymes, global trends are rarely addressed. We performed an anal. of kcat and KM values of several thousand enzymes collected from the literature. We found that the "av. enzyme" exhibits a kcat of ∼10 s-1 and a kcat/KM of ∼ 105 s-1 M-1, much below the diffusion limit and the characteristic textbook portrayal of kinetically superior enzymes. Why do most enzymes exhibit moderate catalytic efficiencies Maximal rates may not evolve in cases where weaker selection pressures are expected. We find, for example, that enzymes operating in secondary metab. are, on av., ∼ 30-fold slower than those of central metab. We also find indications that the physicochem. properties of substrates affect the kinetic parameters. Specifically, low mol. mass and hydrophobicity appear to limit KM optimization. In accordance, substitution with phosphate, CoA, or other large modifiers considerably lowers the KM values of enzymes utilizing the substituted substrates. It therefore appears that both evolutionary selection pressures and physicochem. constraints shape the kinetic parameters of enzymes. It also seems likely that the catalytic efficiency of some enzymes toward their natural substrates could be increased in many cases by natural or lab. evolution.
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  46. 46
    Chibba, A.; Poloczek, J.; Little, D. J.; Howell, P. L.; Nitz, M. Synthesis and Evaluation of Inhibitors of E. Coli PgaB, a Polysaccharide de-N-Acetylase Involved in Biofilm Formation. Org. Biomol. Chem. 2012, 10 (35), 7103– 7107,  DOI: 10.1039/c2ob26105g
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    46
    Synthesis and evaluation of inhibitors of E. coli PgaB, a polysaccharide de-N-acetylase involved in biofilm formation
    Chibba, Anthony; Poloczek, Joanna; Little, Dustin J.; Howell, P. Lynne; Nitz, Mark
    Organic & Biomolecular Chemistry (2012), 10 (35), 7103-7107CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    Many medically important biofilm forming bacteria produce similar polysaccharide intercellular adhesins (PIA) consisting of partially de-N-acetylated β-(1 6)-N-acetylglucosamine polymers (dPNAG). In Escherichia coli, de-N-acetylation of the β-(16)-N-acetylglucosamine polymer (PNAG) is catalyzed by the carbohydrate esterase family 4 deacetylase PgaB. The de-N-acetylation of PNAG is essential for productive PNAG-dependent biofilm formation. Here, we describe the development of a fluorogenic assay to monitor PgaB activity in vitro and the synthesis of a series of PgaB inhibitors. The synthesized inhibitors consist of a metal chelating functional group on a glucosamine scaffold to target the active site metal ion of PgaB. Optimal inhibition was obsd. with N-thioglycolyl amide (Ki = 480 μM) and N-methyl-N-glycolyl amide (Ki = 320 μM) glucosamine derivs. A chemoenzymic synthesis of an N-thioglycolyl amide PNAG pentasaccharide led to an inhibitor with an improved Ki of 280 μM.
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  47. 47
    Jerga, A.; Raychaudhuri, A.; Tipton, P. A. Pseudomonas Aeruginosa C5-Mannuronan Epimerase: Steady-State Kinetics and Characterization of the Product. Biochemistry 2006, 45 (2), 552– 560,  DOI: 10.1021/bi051862l
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    47
    Pseudomonas aeruginosa C5-Mannuronan Epimerase: Steady-State Kinetics and Characterization of the Product
    Jerga, Agoston; Raychaudhuri, Aniruddha; Tipton, Peter A.
    Biochemistry (2006), 45 (2), 552-560CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Alginate is a major constituent of mature biofilms produced by Pseudomonas aeruginosa. The penultimate step in the biosynthesis of alginate is the conversion of some β-D-mannuronate residues in the polymeric substrate polymannuronan to α-L-guluronate residues in a reaction catalyzed by C5-mannuronan epimerase. Specificity studies conducted with size-fractionated oligomannuronates revealed that the minimal substrate contained nine monosaccharide residues. The max. velocity of the reaction increased from 0.0018 to 0.0218 s-1 as the substrate size increased from 10 to 20 residues, and no addnl. increase in kcat was obsd. for substrates up to 100 residues in length. The Km decreased from 80 μM for a substrate contg. fewer than 15 residues to 4 μM for a substrate contg. more than 100 residues. In contrast to C5-mannuronan epimerases that have been characterized in other bacterial species, P. aeruginosa C5-mannuronan epimerase does not require Ca2+ for activity, and the Ca2+-alginate complex is not a substrate for the enzyme. Anal. of the purified, active enzyme by inductively coupled plasma-emission spectroscopy revealed that no metals were present in the protein. The pH dependence of the kinetic parameters revealed that three residues on the enzyme which all have a pKa of ∼7.6 must be protonated for catalysis to occur. The compn. of the polymeric product of the epimerase reaction was analyzed by 1H NMR spectroscopy, which revealed that tracts of adjacent guluronate residues were readily formed. The reaction reached an apparent equil. when the guluronate compn. of the polymer was 75%.
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    Figure 1

    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)

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    Figure 2

    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].

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    Figure 3

    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].

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    Figure 4

    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.

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    Figure 5

    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.

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    Figure 6

    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.

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    This article references 47 other publications.

    1. 1
      Mah, T.-F. C.; O’Toole, G. A. Mechanisms of Biofilm Resistance to Antimicrobial Agents. Trends Microbiol. 2001, 9 (1), 34– 39,  DOI: 10.1016/S0966-842X(00)01913-2
      1
      Mechanisms of biofilm resistance to antimicrobial agents
      Mah, Thien-Fah C.; O'Toole, George A.
      Trends in Microbiology (2001), 9 (1), 34-39CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Science Ltd.)
      A review with 46 refs. Biofilms are communities of microorganisms attached to a surface. It has become clear that biofilm-grown cells express properties distinct from planktonic cells, one of which is an increased resistance to antimicrobial agents. Recent work has indicated that slow growth and/or induction of an rpoS-mediated stress response could contribute to biocide resistance. The phys. and/or chem. structure of exopolysaccharides or other aspects of biofilm architecture could also confer resistance by exclusion of biocides from the bacterial community. Finally, biofilm-grown bacteria might develop a biofilm-specific biocide-resistant phenotype. Owing to the heterogeneous nature of the biofilm, it is likely that there are multiple resistance mechanisms at work within a single community. Recent research has begun to shed light on how and why surface-attached microbial communities develop resistance to antimicrobial agents.
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    2. 2
      Flemming, H.-C.; Wingender, J.; Szewzyk, U.; Steinberg, P.; Rice, S. A.; Kjelleberg, S. Biofilms: An Emergent Form of Bacterial Life. Nat. Rev. Microbiol. 2016, 14 (9), 563– 575,  DOI: 10.1038/nrmicro.2016.94
      2
      Biofilms: an emergent form of bacterial life
      Flemming, Hans-Curt; Wingender, Jost; Szewzyk, Ulrich; Steinberg, Peter; Rice, Scott A.; Kjelleberg, Staffan
      Nature Reviews Microbiology (2016), 14 (9), 563-575CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
      A review. Bacterial biofilms are formed by communities that are embedded in a self-produced matrix of extracellular polymeric substances (EPS). Importantly, bacteria in biofilms exhibit a set of 'emergent properties' that differ substantially from free-living bacterial cells. In this Review, we consider the fundamental role of the biofilm matrix in establishing the emergent properties of biofilms, describing how the characteristic features of biofilms - such as social cooperation, resource capture and enhanced survival of exposure to antimicrobials - all rely on the structural and functional properties of the matrix. Finally, we highlight the value of an ecol. perspective in the study of the emergent properties of biofilms, which enables an appreciation of the ecol. success of biofilms as habitat formers and, more generally, as a bacterial lifestyle.
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    3. 3
      Marsh, P. Dental Plaque as a Microbial Biofilm Dental Plaque – Existing Perspective. Caries Res. 2004, 38, 204– 211,  DOI: 10.1159/000077756
      3
      Dental plaque as a microbial biofilm
      Marsh, P. D.
      Caries Research (2004), 38 (3), 204-211CODEN: CAREBK; ISSN:0008-6568. (S. Karger AG)
      A review. New technologies have provided novel insights into how dental plaque functions as a biofilm. Confocal microscopy has confirmed that plaque has an open architecture similar to other biofilms, with channels and voids. Gradients develop in areas of dense biomass over short distances in key parameters that influence microbial growth and distribution. Bacteria exhibit an altered pattern of gene expression either as a direct result of being on a surface or indirectly as a response to the local environmental heterogeneity within the biofilm. Bacteria communicate via small diffusible signalling mols. (e.g. competence-stimulating peptide, CSP; autoinducer 2); CSP induces both genetic competence and acid tolerance in recipient sessile cells. Thus, rates of gene transfer increase in biofilm communities, and this is one of several mechanisms (others include: diffusion-reaction, neutralization/inactivation, slow growth rates, novel phenotype) that contribute to the increased antimicrobial resistance exhibited by bacteria in biofilms. Oral bacteria in plaque do not exist as independent entities but function as a coordinated, spatially organized and fully metabolically integrated microbial community, the properties of which are greater than the sum of the component species. A greater understanding of the significance of dental plaque as a mixed culture biofilm will lead to novel control strategies.
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    4. 4
      Flickinger, S. T.; Copeland, M. F.; Downes, E. M.; Braasch, A. T.; Tuson, H. H.; Eun, Y.-J.; Weibel, D. B. Quorum Sensing between Pseudomonas Aeruginosa Biofilms Accelerates Cell Growth. J. Am. Chem. Soc. 2011, 133 (15), 5966– 5975,  DOI: 10.1021/ja111131f
      4
      Quorum sensing between Pseudomonas aeruginosa biofilms accelerates cell growth
      Flickinger, Shane T.; Copeland, Matthew F.; Downes, Eric M.; Braasch, Andrew T.; Tuson, Hannah H.; Eun, Ye-Jin; Weibel, Douglas B.
      Journal of the American Chemical Society (2011), 133 (15), 5966-5975CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      This paper describes the fabrication of arrays of spatially confined chambers embossed in a layer of poly(ethylene glycol) diacrylate (PEGDA) and their application to studying quorum sensing between communities of Pseudomonas aeruginosa. The authors hypothesized that biofilms may produce stable chem. signaling gradients in close proximity to surfaces, which influence the growth and development of nearby microcolonies into biofilms. To test this hypothesis, they embossed a layer of PEGDA with 1.5-mm wide chambers in which P. aeruginosa biofilms grew, secreted homoserine lactones (HSLs, small mol. regulators of quorum sensing), and formed spatial and temporal gradients of these compds. In static growth conditions (i.e., no flow), nascent biofilms secreted N-(3-oxododecanoyl) HSL that formed a gradient in the hydrogel and was detected by P. aeruginosa cells that were ≤8 mm away. Diffusing HSLs increased the growth rate of cells in communities that were < 3 mm away from the biofilm, where the concn. of HSL was > 1 μM, and had little effect on communities farther away. The HSL gradient had no observable influence on biofilm structure. Surprisingly, 0.1-10 μM of N-(3-oxododecanoyl) HSL had no effect on cell growth in liq. culture. The results suggest that the secretion of HSLs from a biofilm enhances the growth of neighboring cells in contact with surfaces into communities and may influence their compn., organization, and diversity.
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    5. 5
      Fux, C. A.; Costerton, J. W.; Stewart, P. S.; Stoodley, P. Survival Strategies of Infectious Biofilms. Trends Microbiol. 2005, 13 (1), 34– 40,  DOI: 10.1016/j.tim.2004.11.010
      5
      Survival strategies of infectious biofilms
      Fux, C. A.; Costerton, J. W.; Stewart, P. S.; Stoodley, P.
      Trends in Microbiology (2005), 13 (1), 34-40CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)
      A review. Modern medicine is facing the spread of biofilm-related infections. Bacterial biofilms are difficult to detect in routine diagnostics and are inherently tolerant to host defenses and antibiotic therapies. In addn., biofilms facilitate the spread of antibiotic resistance by promoting horizontal gene transfer. The authors review current concepts of biofilm tolerance with special emphasis on the role of the biofilm matrix and the physiol. of biofilm-embedded cells. The heterogeneity in metabolic and reproductive activity within a biofilm correlates with a non-uniform susceptibility of enclosed bacteria. Recent studies have documented similar heterogeneity in planktonic cultures. Nutritional starvation and high cell d., 2 key characteristics of biofilm physiol., also mediate antimicrobial tolerance in stationary-phase planktonic cultures. Advances in characterizing the role of stress response genes, quorum sensing and phase variation in stationary-phase planktonic cultures have shed new light on tolerance mechanisms within biofilm communities.
      >> More from SciFinder ®
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    6. 6
      Römling, U.; Bokranz, W.; Rabsch, W.; Zogaj, X.; Nimtz, M.; Tschäpe, H. Occurrence and Regulation of the Multicellular Morphotype in Salmonella Serovars Important in Human Disease. Int. J. Med. Microbiol. 2003, 293 (4), 273– 285,  DOI: 10.1078/1438-4221-00268
      6
      Occurrence and regulation of the multicellular morphotype in Salmonella serovars important in human disease
      Romling Ute; Bokranz Werner; Rabsch Wolfgang; Zogaj Xhavit; Nimtz Manfred; Tschape Helmut
      International journal of medical microbiology : IJMM (2003), 293 (4), 273-85 ISSN:1438-4221.
      Multicellular behavior in Salmonella Typhimurium ATCC14028 called the rdar morphotype is characterized by the expression of the extracellular matrix components cellulose and curli fimbriae. Over 90% of S. Typhimurium and S. Enteritidis strains from human disease, food and animals expressed the rdar morphotype at 28 degrees C. Regulation of the rdar morphotype occurred via the response regulator ompR, which activated transcription of csgD required for production of cellulose and curli fimbriae. Serovar-specific regulation of csgD required rpoS in S. Typhimurium, but was partially independent of rpoS in S. Enteritidis. Rarely, strain-specific temperature-deregulated expression of the rdar morphotype was observed. The host-restricted serovars S. Typhimurium var. Copenhagen phage type DT2 and DT99, Salmonella Typhi and Salmonella Choleraesuis did not express the rdar morphotype, while in Salmonella Gallinarum cellulose expression at 37 degrees C was seen in some strains. Therefore, the expression pattern of the rdar morphotype is serovar specific and correlates with a disease phenotype breaching the intestinal epithelial cell lining.
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    7. 7
      Saldaña, Z.; Xicohtencatl-Cortes, J.; Avelino, F.; Phillips, A. D.; Kaper, J. B.; Puente, J. L.; Girón, J. A. Synergistic Role of Curli and Cellulose in Cell Adherence and Biofilm Formation of Attaching and Effacing Escherichia Coli and Identification of Fis as a Negative Regulator of Curli. Environ. Microbiol. 2009, 11 (4), 992– 1006,  DOI: 10.1111/j.1462-2920.2008.01824.x
      7
      Synergistic role of curli and cellulose in cell adherence and biofilm formation of attaching and effacing Escherichia coli and identification of Fis as a negative regulator of curli
      Saldana, Zeus; Xicohtencatl-Cortes, Juan; Avelino, Fabiola; Phillips, Alan D.; Kaper, James B.; Puente, Jose L.; Giron, Jorge A.
      Environmental Microbiology (2009), 11 (4), 992-1006CODEN: ENMIFM; ISSN:1462-2912. (Wiley-Blackwell)
      Curli are adhesive fimbriae of Escherichia coli and Salmonella enterica. Expression of curli (csgA) and cellulose (bcsA) is co-activated by the transcriptional activator CsgD. In this study, we investigated the contribution of curli and cellulose to the adhesive properties of enterohemorrhagic (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) O127:H6. While single mutations in csgA, csgD or bcsA in EPEC and EHEC had no dramatic effect on cell adherence, double csgAbcsA mutants were significantly less adherent than the single mutants or wild-type strains to human colonic HT-29 epithelial cells or to cow colon tissue in vitro. Overexpression of csgD (carried on plasmid pCP994) in a csgD mutant, but not in the single csgA or bscA mutants, led to significant increase in adherence and biofilm formation in EPEC and EHEC, suggesting that synchronized over-prodn. of curli and cellulose enhances bacterial adherence. In line with this finding, csgD transcription was activated significantly in the presence of cultured epithelial cells as compared with growth in tissue culture medium. Anal. of the influence of virulence and global regulators in the prodn. of curli in EPEC identified Fis (factor for inversion stimulation) as a, heretofore unrecognized, neg. transcriptional regulator of csgA expression. An EPEC E2348/69Δfis produced abundant amts. of curli whereas a double fis/csgD mutant yielded no detectable curli prodn. Our data suggest that curli and cellulose act in concert to favor host colonization, biofilm formation and survival in different environments.
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    8. 8
      Spiers, A. J.; Bohannon, J.; Gehrig, S. M.; Rainey, P. B. Biofilm Formation at the Air-Liquid Interface by the Pseudomonas Fluorescens SBW25 Wrinkly Spreader Requires an Acetylated Form of Cellulose. Mol. Microbiol. 2003, 50 (1), 15– 27,  DOI: 10.1046/j.1365-2958.2003.03670.x
      8
      Biofilm formation at the air-liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose
      Spiers, Andrew J.; Bohannon, John; Gehrig, Stefanie M.; Rainey, Paul B.
      Molecular Microbiology (2003), 50 (1), 15-27CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)
      The wrinkly spreader (WS) genotype of Pseudomonas fluorescens SBW25 colonizes the air-liq. interface of spatially structured microcosms resulting in formation of a thick biofilm. Its ability to colonize this niche is largely due to overprodn. of a cellulosic polymer, the product of the wss operon. Chem. anal. of the biofilm matrix shows that the cellulosic polymer is partially acetylated cellulose, which is consistent with predictions of gene function based on in silico anal. of wss. Both polar and non-polar mutations in the sixth gene of the wss operon (wssF) or adjacent downstream genes (wssGHIJ) generated mutants that overproduce non-acetylated cellulose, thus implicating WssFGHIJ in acetylation of cellulose. WssGHI are homologs of AlgFIJ from P. aeruginosa, which together are necessary and sufficient to acetylate alginate polymer. WssF belongs to a newly established Pfam family and is predicted to provide acyl groups to WssGHI. The role of WssJ is unclear, but its similarity to MinD-like proteins suggests a role in polar localization of the acetylation complex. Fluorescent microscopy of Calcofluor-stained biofilms revealed a matrix structure composed of networks of cellulose fibers, sheets and clumped material. Quant. analyses of biofilm structure showed that acetylation of cellulose is important for effective colonization of the air-liq. interface: mutants identical to WS, but defective in enzymes required for acetylation produced biofilms with altered phys. properties. In addn., mutants producing non-acetylated cellulose were unable to spread rapidly across solid surfaces. Inclusion in these assays of a WS mutant with a defect in the GGDEF regulator (WspR) confirmed the requirement for this protein in expression of both acetylated cellulose polymer and bacterial attachment. These results suggest a model in which WspR regulation of cellulose expression and attachment plays a role in the co-ordination of surface colonization.
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    9. 9
      Hu, L.; Grim, C. J.; Franco, A. A.; Jarvis, K. G.; Sathyamoorthy, V.; Kothary, M. H.; McCardell, B. A.; Tall, B. D. Analysis of the Cellulose Synthase Operon Genes, BcsA, BcsB, and BcsC in Cronobacter Species: Prevalence among Species and Their Roles in Biofilm Formation and Cell-Cell Aggregation. Food Microbiol. 2015, 52, 97– 105,  DOI: 10.1016/j.fm.2015.07.007
      9
      Analysis of the cellulose synthase operon genes, bcsA, bcsB, and bcsC in Cronobacter species: Prevalence among species and their roles in biofilm formation and cell-cell aggregation
      Hu, Lan; Grim, Christopher J.; Franco, Augusto A.; Jarvis, Karen G.; Sathyamoorthy, Vengopal; Kothary, Mahendra H.; McCardell, Barbara A.; Tall, Ben D.
      Food Microbiology (2015), 52 (), 97-105CODEN: FOMIE5; ISSN:0740-0020. (Elsevier Ltd.)
      Cronobacter species are emerging food-borne pathogens that cause severe sepsis, meningitis, and necrotizing entercolitis in neonates and infants. Bacterial pathogens such as Escherichia coli and Salmonella species produce extracellular cellulose which has been shown to be involved in rugosity, biofilm formation, and host colonization. In this study the distribution and prevalence of cellulose synthase operon genes (bcsABZC) were detd. by polymerase chain reaction (PCR) anal. in 231 Cronobacter strains isolated from clin., food, environmental, and unknown sources. Furthermore, bcsA and bcsB isogenic mutants were constructed in Cronobacter sakazakii BAA894 to det. their roles. In calcofluor binding assays bcsA and bcsB mutants did not produce cellulose, and their colonial morphotypes were different to that of the parent strain. Biofilm formation and bacterial cell-cell aggregation were significantly reduced in bcsA and bcsB mutants compared to the parental strain. bcsA or bcsAB PCR-neg. strains of C. sakazakii did not bind calcofluor, and produced less biofilm and cell-cell aggregation compared to strains possessing bcsAB genes. These data indicated that Cronobacter bcsABZC were present in all clin. isolates and most of food and environmental isolates. bcsA and bcsB genes of Cronobacter were necessary to produce cellulose, and were involved in biofilm formation and cell-cell aggregation.
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    10. 10
      Ross, P.; Mayer, R.; Benziman, M. Cellulose Biosynthesis and Function in Bacteria. Microbiol. Rev. 1991, 55 (1), 35– 58,  DOI: 10.1128/mr.55.1.35-58.1991
      10
      Cellulose biosynthesis and function in bacteria
      Ross, Peter; Mayer, Raphael; Benziman, Moshe
      Microbiological Reviews (1991), 55 (1), 35-58CODEN: MBRED3; ISSN:0146-0749.
      A review with >150 refs., including regulation and comparative biochem. of cellulose formation.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXktVCkt7Y%253D&md5=261a5ff345094b27877de240df3c3228
    11. 11
      Canale-Parola, E.; Borasky, R.; Wolfe, R. S. Studies on Sarcina Ventriculi III. Localization of Cellulose. J. Bacteriol. 1961, 81 (2), 311– 318,  DOI: 10.1128/jb.81.2.311-318.1961
      11
      Studies on Sarcina ventriculi. III. Localization of cellulose
      CANALE-PAROLA E; BORASKY R; WOLFE R S
      Journal of bacteriology (1961), 81 (), 311-8 ISSN:0021-9193.
      There is no expanded citation for this reference.
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    12. 12
      Scott, W.; Lowrance, B.; Anderson, A. C.; Weadge, J. T. Identification of the Clostridial Cellulose Synthase and Characterization of the Cognate Glycosyl Hydrolase, CcsZ. PLoS One 2020, 15 (12), e0242686,  DOI: 10.1371/journal.pone.0242686
      12
      Identification of the Clostridial cellulose synthase and characterization of the cognate glycosyl hydrolase, CcsZ
      Scott, William; Lowrance, Brian; Anderson, Alexander C.; Weadge, Joel T.
      PLoS One (2020), 15 (12), e0242686CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Biofilms are community structures of bacteria enmeshed in a self-produced matrix of exopolysaccharides. The biofilm matrix serves numerous roles, including resilience and persistence, making biofilms a subject of research interest among persistent clin. pathogens of global health importance. Our current understanding of the underlying biochem. pathways responsible for biosynthesis of these exopolysaccharides is largely limited to Gram-neg. bacteria. Clostridia are a class of Gram-pos., anaerobic and spore-forming bacteria and include the important human pathogens Clostridium perfringens, Clostridium botulinum and Clostridioides difficile, among numerous others. Several species of Clostridia have been reported to produce a biofilm matrix that contains an acetylated glucan linked to a series of hypothetical genes. Here, we propose a model for the function of these hypothetical genes, which, using homol. modeling, we show plausibly encode a synthase complex responsible for polymn., modification and export of an O-acetylated cellulose exopolysaccharide. Specifically, the cellulose synthase is homologous to that of the known exopolysaccharide synthases in Gram-neg. bacteria. The remaining proteins represent a mosaic of evolutionary lineages that differ from the described Gram-neg. cellulose exopolysaccharide synthases, but their predicted functions satisfy all criteria required for a functional cellulose synthase operon. Accordingly, we named these hypothetical genes ccsZABHI, for the Clostridial cellulose synthase (Ccs), in keeping with naming conventions for exopolysaccharide synthase subunits and to distinguish it from the Gram-neg. Bcs locus with which it shares only a single one-to-one ortholog. To test our model and assess the identity of the exopolysaccharide, we subcloned the putative glycoside hydrolase encoded by ccsZ and solved the X-ray crystal structure of both apo- and product-bound CcsZ, which belongs to glycoside hydrolase family 5 (GH-5). Although not homologous to the Gram-neg. cellulose synthase, which instead encodes the structurally distinct BcsZ belonging to GH-8, we show CcsZ displays specificity for cellulosic materials. This specificity of the synthase-assocd. glycosyl hydrolase validates our proposal that these hypothetical genes are responsible for biosynthesis of a cellulose exopolysaccharide. The data we present here allowed us to propose a model for Clostridial cellulose synthesis and serves as an entry point to an understanding of cellulose biofilm formation among class Clostridia.
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    13. 13
      Whitfield, G. B.; Marmont, L. S.; Howell, P. L. Enzymatic Modifications of Exopolysaccharides Enhance Bacterial Persistence. Front. Microbiol. 2015, 6, 471,  DOI: 10.3389/fmicb.2015.00471
      13
      Enzymatic modifications of exopolysaccharides enhance bacterial persistence
      Whitfield Gregory B; Marmont Lindsey S; Howell P Lynne
      Frontiers in microbiology (2015), 6 (), 471 ISSN:1664-302X.
      Biofilms are surface-attached communities of bacterial cells embedded in a self-produced matrix that are found ubiquitously in nature. The biofilm matrix is composed of various extracellular polymeric substances, which confer advantages to the encapsulated bacteria by protecting them from eradication. The matrix composition varies between species and is dependent on the environmental niche that the bacteria inhabit. Exopolysaccharides (EPS) play a variety of important roles in biofilm formation in numerous bacterial species. The ability of bacteria to thrive in a broad range of environmental settings is reflected in part by the structural diversity of the EPS produced both within individual bacterial strains as well as by different species. This variability is achieved through polymerization of distinct sugar moieties into homo- or hetero-polymers, as well as post-polymerization modification of the polysaccharide. Specific enzymes that are unique to the production of each polymer can transfer or remove non-carbohydrate moieties, or in other cases, epimerize the sugar units. These modifications alter the physicochemical properties of the polymer, which in turn can affect bacterial pathogenicity, virulence, and environmental adaptability. Herein, we review the diversity of modifications that the EPS alginate, the Pel polysaccharide, Vibrio polysaccharide, cepacian, glycosaminoglycans, and poly-N-acetyl-glucosamine undergo during biosynthesis. These are EPS produced by human pathogenic bacteria for which studies have begun to unravel the effect modifications have on their physicochemical and biological properties. The biological advantages these polymer modifications confer to the bacteria that produce them will be discussed. The expanding list of identified modifications will allow future efforts to focus on linking these modifications to specific biosynthetic genes and biofilm phenotypes.
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    14. 14
      Thongsomboon, W.; Serra, D. O.; Possling, A.; Hadjineophytou, C.; Hengge, R.; Cegelski, L. Phosphoethanolamine Cellulose: A Naturally Produced Chemically Modified Cellulose. Science (Washington, DC, U. S.) 2018, 359 (6373), 334– 338,  DOI: 10.1126/science.aao4096
      14
      Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose
      Thongsomboon, Wiriya; Serra, Diego O.; Possling, Alexandra; Hadjineophytou, Chris; Hengge, Regine; Cegelski, Lynette
      Science (Washington, DC, United States) (2018), 359 (6373), 334-338CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Cellulose is a major contributor to the chem. and mech. properties of plants and assumes structural roles in bacterial communities termed biofilms. We find that Escherichia coli produces chem. modified cellulose that is required for extracellular matrix assembly and biofilm architecture. Solid-state NMR spectroscopy of the intact and insol. material elucidates the zwitterionic phosphoethanolamine modification that had evaded detection by conventional methods. Installation of the phosphoethanolamine group requires BcsG, a proposed phosphoethanolamine transferase, with biofilm-promoting cyclic diguanylate monophosphate input through a BcsE-BcsF-BcsG transmembrane signaling pathway. The bcsEFG operon is present in many bacteria, including Salmonella species, that also produce the modified cellulose. The discovery of phosphoethanolamine cellulose and the genetic and mol. basis for its prodn. offers opportunities to modulate its prodn. in bacteria and inspires efforts to biosynthetically engineer alternatively modified cellulosic materials.
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    15. 15
      Whitfield, G. B.; Marmont, L. S.; Bundalovic-Torma, C.; Razvi, E.; Roach, E. J.; Khursigara, C. M.; Parkinson, J.; Howell, P. L. Discovery and Characterization of a Gram- Positive Pel Polysaccharide Biosynthetic Gene Cluster. PLoS Pathog. 2020, 16 (4), e1008281,  DOI: 10.1371/journal.ppat.1008281
      15
      Discovery and characterization of a Gram positive Pel polysaccharide biosynthetic gene cluster
      Whitfield, Gregory B.; Marmont, Lindsey S.; Bundalovic-Torma, Cedoljub; Razvi, Erum; Roach, Elyse J.; Khursigara, Cezar M.; Parkinson, John; Howell, P. Lynne
      PLoS Pathogens (2020), 16 (4), e1008281CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)
      Our understanding of the biofilm matrix components utilized by Gram-pos.bacteria, and the signaling pathways that regulate their prodn.are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the anal. of synthase-dependent exopolysaccharide biosynthetic systems. Here, we explore the finding that many species of Grampos.bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B.cereus PelD ortholog binds cyclic-3',5-dimeric guanosine monophosphate (c-diGMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the prodn. of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signaling in Gram-pos.organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of bacilli, clostridia, streptococci, and actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-pos.bacteria.
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    16. 16
      Römling, U. Molecular Biology of Cellulose Production in Bacteria. Res. Microbiol. 2002, 153 (4), 205– 212,  DOI: 10.1016/S0923-2508(02)01316-5
      16
      Molecular biology of cellulose production in bacteria
      Romling Ute
      Research in microbiology (2002), 153 (4), 205-12 ISSN:0923-2508.
      Cellulose biosynthesis has recently been established for a variety of bacteria of diverse origin at the phenotypic and genetic levels. Novel regulatory pathways, which involve the second messenger bis-(3',5') cyclic diguanylic acid and several proteins with the GGDEF domain, participate in the regulation of cellulose biosynthesis. The biological significance of cellulose production in environmental, commensal and pathogenic bacteria is only punctually resolved. This review summarizes current knowledge on cellulose biosynthesis, its regulation and biological function.
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    17. 17
      Römling, U.; Galperin, M. Y. Bacterial Cellulose Biosynthesis: Diversity of Operons, Subunits, Products, and Functions. Trends Microbiol. 2015, 23 (9), 545– 557,  DOI: 10.1016/j.tim.2015.05.005
      17
      Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions
      Romling Ute; Galperin Michael Y
      Trends in microbiology (2015), 23 (9), 545-57 ISSN:.
      Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits - which differ among various taxa - affect the enzymatic activity and product yield in vivo by modulating (i) the expression of the biosynthesis apparatus, (ii) the export of the nascent β-D-glucan polymer to the cell surface, and (iii) the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of resulting biofilms, which is particularly important for the interactions of bacteria with higher organisms - leading to rhizosphere colonization and modulating the virulence of cellulose-producing bacterial pathogens inside and outside of host cells. We review the organization of four principal types of cellulose synthase operon found in various bacterial genomes, identify additional bcs genes that encode components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms and in the choice between acute infection and persistence in the host.
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    18. 18
      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. U. S. A. 2013, 110 (44), 17856– 17861,  DOI: 10.1073/pnas.1314063110
      18
      BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis
      Omadjela, Okako; Narahari, Adishesh; Strumillo, Joanna; Melida, Hugo; Mazur, Olga; Bulone, Vincent; Zimmer, Jochen
      Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (44), 17856-17861,S17856/1-S17856/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromol. complexes contg. several different enzyme isoforms, prokaryotic synthases assoc. with addnl. subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-assocd. bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochem. studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a d.p. in the range 200-300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation anal. of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-assocd. domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP.
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    19. 19
      Morgan, J. L. W.; McNamara, J. T.; Zimmer, J. Mechanism of Activation of Bacterial Cellulose Synthase by Cyclic Di-GMP. Nat. Struct. Mol. Biol. 2014, 21 (5), 489– 496,  DOI: 10.1038/nsmb.2803
      19
      Mechanism of activation of bacterial cellulose synthase by cyclic di-GMP
      Morgan, Jacob L. W.; McNamara, Joshua T.; Zimmer, Jochen
      Nature Structural & Molecular Biology (2014), 21 (5), 489-496CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
      The bacterial signaling mol. cyclic di-GMP (c-di-GMP) stimulates the synthesis of bacterial cellulose, which is frequently found in biofilms. Bacterial cellulose is synthesized and translocated across the inner membrane by a complex of cellulose synthase BcsA and BcsB subunits. Here we present crystal structures of the c-di-GMP-activated BcsA-BcsB complex. The structures reveal that c-di-GMP releases an autoinhibited state of the enzyme by breaking a salt bridge that otherwise tethers a conserved gating loop that controls access to and substrate coordination at the active site. Disrupting the salt bridge by mutagenesis generates a constitutively active cellulose synthase. Addnl., the c-di-GMP-activated BcsA-BcsB complex contains a nascent cellulose polymer whose terminal glucose unit rests at a new location above BcsA's active site and is positioned for catalysis. Our mechanistic insights indicate how c-di-GMP allosterically modulates enzymic functions.
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    20. 20
      Morgan, J. L. W.; Strumillo, J.; Zimmer, J. Crystallographic Snapshot of Cellulose Synthesis and Membrane Translocation. Nature 2013, 493 (7431), 181– 186,  DOI: 10.1038/nature11744
      20
      Crystallographic snapshot of cellulose synthesis and membrane translocation
      Morgan, Jacob L. W.; Strumillo, Joanna; Zimmer, Jochen
      Nature (London, United Kingdom) (2013), 493 (7431), 181-186CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Cellulose, the most abundant biol. macromol., is an extracellular, linear polymer of glucose mols. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose prodn. frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB subunits of Rhodobacter sphaeroides cellulose synthase (CESA) contg. a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose mol. at a time.
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    21. 21
      Whitfield, C.; Mainprize, I. L. TPR Motifs: Hallmarks of a New Polysaccharide Export Scaffold. Structure 2010, 18 (2), 151– 153,  DOI: 10.1016/j.str.2010.01.006
      21
      TPR Motifs: Hallmarks of a New Polysaccharide Export Scaffold
      Whitfield, Chris; Mainprize, Iain L.
      Structure (Cambridge, MA, United States) (2010), 18 (2), 151-153CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
      A review. Bacteria produce a remarkable range of surface and secreted polysaccharides. Two pathways have been defined for the biosynthesis and export of capsular polysaccharides and exopolysaccharides in Gram-neg. bacteria. A structure of AlgK described in this issue provides structural insight into a third previously unrecognized pathway assocd. with important biopolymers.
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    22. 22
      Low, K. E.; Howell, P. L. Gram-Negative Synthase-Dependent Exopolysaccharide Biosynthetic Machines. Curr. Opin. Struct. Biol. 2018, 53, 32– 44,  DOI: 10.1016/j.sbi.2018.05.001
      22
      Gram-negative synthase-dependent exopolysaccharide biosynthetic machines
      Low, Kristin E.; Howell, P. Lynne
      Current Opinion in Structural Biology (2018), 53 (), 32-44CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)
      A review. Bacteria predominantly exist as matrix embedded communities of cells called biofilms. The biofilm matrix is made up of a variety of self-produced extracellular components including DNA, proteins, and exopolysaccharides. Bacterial exopolysaccharides have been implicated in surface adhesion, resistance to antibiotics, and protection from host immune systems. Herein review the structure and function of the proteins involved in the prodn. of the Gram-neg. synthase-dependent exopolysaccharides: alginate, poly-β(1,6)-N-acetyl-D-glucosamine (PNAG), cellulose, and the Pel polysaccharide. This study highlight the similarities and differences that exist at the mol. level in these synthase systems.
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    23. 23
      Acheson, J. F.; Derewenda, Z. S.; Zimmer, J. Architecture of the Cellulose Synthase Outer Membrane Channel and Its Association with the Periplasmic TPR Domain. Structure 2019, 27 (12), 1855– 1861.e3,  DOI: 10.1016/j.str.2019.09.008
      23
      Architecture of the cellulose synthase outer membrane channel and its association with the periplasmic TPR domain
      Acheson, Justin F.; Derewenda, Zygmunt S.; Zimmer, Jochen
      Structure (Oxford, United Kingdom) (2019), 27 (12), 1855-1861.e3CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
      Extracellular bacterial cellulose contributes to biofilm stability and to the integrity of the bacterial cell envelope. In Gram-neg. bacteria, cellulose is synthesized and secreted by a multi-component cellulose synthase complex. The BcsA subunit synthesizes cellulose and also transports the polymer across the inner membrane. Translocation across the outer membrane occurs through the BcsC porin, which extends into the periplasm via 19 tetra-tricopeptide repeats (TPR). We present the crystal structure of a truncated BcsC, encompassing the last TPR repeat and the complete outer membrane channel domain, revealing a 16-stranded, β barrel pore architecture. The pore is blocked by an extracellular gating loop, while the extended C terminus inserts deeply into the channel and positions a conserved Trp residue near its extracellular exit. The channel is lined with hydrophilic and arom. residues suggesting a mechanism for facilitated cellulose diffusion based on arom. stacking and hydrogen bonding.
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    24. 24
      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 (1), 13018,  DOI: 10.1038/s41598-017-12530-0
      24
      Crystal structure of the flexible tandem repeat domain of bacterial cellulose synthesis subunit C
      Nojima Shingo; Fujishima Ayumi; Kato Koji; Ohuchi Kayoko; Yao Min; Kato Koji; Yao Min; Shimizu Nobutaka; Yonezawa Kento; Tajima Kenji
      Scientific reports (2017), 7 (1), 13018 ISSN:.
      Bacterial cellulose (BC) is synthesized and exported through the cell membrane via a large protein complex (terminal complex) that consists of three or four subunits. BcsC is a little-studied subunit considered to export BC to the extracellular matrix. It is predicted to have two domains: a tetratrico peptide repeat (TPR) domain and a β-barrelled outer membrane domain. Here we report the crystal structure of the N-terminal part of BcsC-TPR domain (Asp24-Arg272) derived from Enterobacter CJF-002. Unlike most TPR-containing proteins which have continuous TPR motifs, this structure has an extra α-helix between two clusters of TPR motifs. Five independent molecules in the crystal had three different conformations that varied at the hinge of the inserted α-helix. Such structural feature indicates that the inserted α-helix confers flexibility to the chain and changes the direction of the TPR super-helix, which was also suggested by structural analysis of BcsC-TPR (Asp24-Leu664) in solution by size exclusion chromatography-small-angle X-ray scattering. The flexibility at the α-helical hinge may play important role for exporting glucan chains.
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    25. 25
      Anderson, A. C.; Burnett, A. J. N.; Hiscock, L.; Maly, K. E.; Weadge, J. T. The Escherichia Coli Cellulose Synthase Subunit G (BcsG) Is a Zn2+-Dependent Phosphoethanolamine Transferase. J. Biol. Chem. 2020, 295 (18), 6225– 6235,  DOI: 10.1074/jbc.RA119.011668
      25
      The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase
      Anderson, Alexander C.; Burnett, Alysha J. N.; Hiscock, Lana; Maly, Kenneth E.; Weadge, Joel T.
      Journal of Biological Chemistry (2020), 295 (18), 6225-6235CODEN: JBCHA3; ISSN:1083-351X. (American Society for Biochemistry and Molecular Biology)
      A review. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrixes has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane-localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alk. phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-neg. bacteria. Informed by our structural studies, we present a functional complementation expt. in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes.
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    26. 26
      Mazur, O.; Zimmer, J. Apo- and Cellopentaose-Bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ. J. Biol. Chem. 2011, 286 (20), 17601– 17606,  DOI: 10.1074/jbc.M111.227660
      26
      Apo- and Cellopentaose-bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ
      Mazur, Olga; Zimmer, Jochen
      Journal of Biological Chemistry (2011), 286 (20), 17601-17606CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Cellulose, a very abundant extracellular polysaccharide, is synthesized in a finely tuned process that involves the activity of glycosyl-transferases and hydrolases. The cellulose microfibril consists of bundles of linear β-1,4-glucan chains that are synthesized inside the cell; however, the mechanism by which these polymers traverse the cell membrane is currently unknown. In Gram-neg. bacteria, the cellulose synthase complex forms a trans-envelope complex consisting of at least four subunits. Although three of these subunits account for the synthesis and translocation of the polysaccharide, the fourth subunit, BcsZ, is a periplasmic protein with endo-β-1,4-glucanase activity. BcsZ belongs to family eight of glycosyl-hydrolases, and its activity is required for optimal synthesis and membrane translocation of cellulose. In this study we report two crystal structures of BcsZ from Escherichia coli. One structure shows the wild-type enzyme in its apo form, and the second structure is for a catalytically inactive mutant of BcsZ in complex with the substrate cellopentaose. The structures demonstrate that BcsZ adopts an (α/α)6-barrel fold and that it binds four glucan moieties of cellopentaose via highly conserved residues exclusively on the nonreducing side of its catalytic center. Thus, the BcsZ-cellopentaose structure most likely represents a posthydrolysis state in which the newly formed nonreducing end has already left the substrate binding pocket while the enzyme remains attached to the truncated polysaccharide chain. We further show that BcsZ efficiently degrades β-1,4-glucans in in vitro cellulase assays with carboxymethyl-cellulose as substrate.
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    27. 27
      Sun, L.; Vella, P.; Schnell, R.; Polyakova, A.; Bourenkov, G.; Li, F.; Cimdins, A.; Schneider, T. R.; Lindqvist, Y.; Galperin, M. Y.; Schneider, G.; Römling, U. Structural and Functional Characterization of the BcsG Subunit of the Cellulose Synthase in Salmonella Typhimurium. J. Mol. Biol. 2018, 430 (18), 3170– 3189,  DOI: 10.1016/j.jmb.2018.07.008
      27
      Structural and functional characterization of the BcsG subunit of the cellulose synthase in Salmonella typhimurium
      Sun, Lei; Vella, Peter; Schnell, Robert; Polyakova, Anna; Bourenkov, Gleb; Li, Fengyang; Cimdins, Annika; Schneider, Thomas R.; Lindqvist, Ylva; Galperin, Michael Y.; Schneider, Gunter; Roemling, Ute
      Journal of Molecular Biology (2018), 430 (18_Part_B), 3170-3189CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Many bacteria secrete cellulose, which forms the structural basis for bacterial multicellular aggregates, termed biofilms. The cellulose synthase complex of S. typhimurium consists of catalytic subunits BcsA and BcsB and several auxiliary subunits that are encoded by 2 divergently transcribed operons, bcsRQABZC and bcsEFG. Expression of the bcsEFG operon is required for full-scale cellulose prodn., but the functions of its products are not fully understood. This work aimed to characterize the BcsG subunit of cellulose synthase, which consists of an N-terminal transmembrane fragment and a C-terminal domain in the periplasm. Deletion of the bcsG gene substantially decreased the total amt. of BcsA and cellulose prodn. BcsA levels were partially restored by the expression of the transmembrane segment, whereas restoration of cellulose prodn. required the presence of the C-terminal periplasmic domain and its characteristic metal-binding residues. The high-resoln. crystal structure of the periplasmic domain characterized BcsG as a member of the alk. phosphatase/sulfatase superfamily of metalloenzymes, contg. a conserved Zn2+-binding site. Sequence and structural comparisons showed that BcsG belongs to a specific family within alk. phosphatase-like enzymes, which includes bacterial Zn2+-dependent lipopolysaccharide phosphoethanolamine transferases such as MCR-1 (colistin resistance protein), EptA, and EptC and Mn2+-dependent lipoteichoic acid synthase (phosphoglycerol transferase) LtaS. These enzymes use the phospholipids, phosphatidylethanolamine and phosphatidylglycerol, resp., as substrates. These data were consistent with the recently discovered phosphoethanolamine modification of cellulose by BcsG and showed that its membrane-bound and periplasmic parts play distinct roles in the assembly of the functional cellulose synthase and cellulose prodn.
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    28. 28
      Fang, X.; Ahmad, I.; Blanka, A.; Schottkowski, M.; Cimdins, A.; Galperin, M. Y.; Römling, U.; Gomelsky, M. GIL, a New C-di-GMP-binding Protein Domain Involved in Regulation of Cellulose Synthesis in Enterobacteria. Mol. Microbiol. 2014, 93 (3), 439– 452,  DOI: 10.1111/mmi.12672
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      GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria
      Fang, Xin; Ahmad, Irfan; Blanka, Andrea; Schottkowski, Marco; Cimdins, Annika; Galperin, Michael Y.; Roemling, Ute; Gomelsky, Mark
      Molecular Microbiology (2014), 93 (3), 439-452CODEN: MOMIEE; ISSN:0950-382X. (Wiley-Blackwell)
      In contrast to numerous enzymes involved in c-di-GMP synthesis and degrdn. in enterobacteria, only a handful of c-di-GMP receptors/effectors have been identified. In search of new c-di-GMP receptors, the authors screened the Escherichia coli ASKA overexpression gene library using the Differential Radial Capillary Action of Ligand Assay (DRaCALA) with fluorescently and radioisotope-labeled c-di-GMP. The authors uncovered three new candidate c-di-GMP receptors in E. coli and characterized one of them, BcsE. The bcsE gene is encoded in cellulose synthase operons in representatives of Gammaproteobacteria and Betaproteobacteria. The purified BcsE proteins from E. coli, Salmonella enterica and Klebsiella pneumoniae bind c-di-GMP via the domain of unknown function, DUF2819, which is hereby designated GIL, GGDEF I-site like domain. The RxGD motif of the GIL domain is required for c-di-GMP binding, similar to the c-di-GMP-binding I-site of the diguanylate cyclase GGDEF domain. Thus, GIL is the second protein domain, after PilZ, dedicated to c-di-GMP-binding. In S. enterica, BcsE is not essential for cellulose synthesis but is required for maximal cellulose prodn., and c-di-GMP binding is crit. for BcsE function. It appears that cellulose prodn. in enterobacteria is controlled by a two-tiered c-di-GMP-dependent system involving BcsE and the PilZ domain contg. glycosyltransferase BcsA.
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    29. 29
      Zouhir, S.; Abidi, W.; Caleechurn, M.; Krasteva, P. V. Structure and Multitasking of the C-Di-GMP-Sensing Cellulose Secretion Regulator BcsE. mBio 2020, 11, 4,  DOI: 10.1128/mBio.01303-20
      There is no corresponding record for this reference.
    30. 30
      Acheson, J. F.; Ho, R.; Goularte, N. F.; Cegelski, L.; Zimmer, J. Molecular Organization of the E. Coli Cellulose Synthase Macrocomplex. Nat. Struct. Mol. Biol. 2021, 28, 310,  DOI: 10.1038/s41594-021-00569-7
      30
      Molecular organization of the E. coli cellulose synthase macrocomplex
      Acheson, Justin F.; Ho, Ruoya; Goularte, Nicolette F.; Cegelski, Lynette; Zimmer, Jochen
      Nature Structural & Molecular Biology (2021), 28 (3), 310-318CODEN: NSMBCU; ISSN:1545-9993. (Nature Research)
      Abstr.: Cellulose is frequently found in communities of sessile bacteria called biofilms. Escherichia coli and other enterobacteriaceae modify cellulose with phosphoethanolamine (pEtN) to promote host tissue adhesion. The E. coli pEtN cellulose biosynthesis machinery contains the catalytic BcsA-B complex that synthesizes and secretes cellulose, in addn. to five other subunits. The membrane-anchored periplasmic BcsG subunit catalyzes pEtN modification. Here we present the structure of the roughly 1 MDa E. coli Bcs complex, consisting of one BcsA enzyme assocd. with six copies of BcsB, detd. by single-particle cryo-electron microscopy. BcsB homo-oligomerizes primarily through interactions between its carbohydrate-binding domains as well as intermol. beta-sheet formation. The BcsB hexamer creates a half spiral whose open side accommodates two BcsG subunits, directly adjacent to BcsA's periplasmic channel exit. The cytosolic BcsE and BcsQ subunits assoc. with BcsA's regulatory PilZ domain. The macrocomplex is a fascinating example of cellulose synthase specification.
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    31. 31
      Abidi, W.; Zouhir, S.; Caleechurn, M.; Roche, S.; Krasteva, P. V. Architecture and Regulation of an Enterobacterial Cellulose Secretion System. Sci. Adv. 2021, 7 (5), 1– 16,  DOI: 10.1126/sciadv.abd8049
      There is no corresponding record for this reference.
    32. 32
      Hinchliffe, P.; Yang, Q. E.; Portal, E.; Young, T.; Li, H.; Tooke, C. L.; Carvalho, M. J.; Paterson, N. G.; Brem, J.; Niumsup, P. R.; Tansawai, U.; Lei, L.; Li, M.; Shen, Z.; Wang, Y.; Schofield, C. J.; Mulholland, A. J.; Shen, J.; Fey, N.; Walsh, T. R.; Spencer, J. Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1. Sci. Rep. 2017, 7, 39392,  DOI: 10.1038/srep39392
      32
      Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1
      Hinchliffe, Philip; Yang, Qiu E.; Portal, Edward; Young, Tom; Li, Hui; Tooke, Catherine L.; Carvalho, Maria J.; Paterson, Neil G.; Brem, Jurgen; Niumsup, Pannika R.; Tansawai, Uttapoln; Lei, Lei; Li, Mei; Shen, Zhangqi; Wang, Yang; Schofield, Christopher J.; Mulholland, Adrian J.; Shen, Jianzhong; Fey, Natalie; Walsh, Timothy R.; Spencer, James
      Scientific Reports (2017), 7 (), 39392CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      The polymixin colistin is a "last line" antibiotic against extensively-resistant Gram-neg. bacteria. Recently, the mcr-1 gene was identified as a plasmid-mediated resistance mechanism in human and animal Enterobacteriaceae, with a wide geog. distribution and many producer strains resistant to multiple other antibiotics. Mcr-1 encodes a membrane-bound enzyme catalyzing phosphoethanolamine transfer onto bacterial lipid A. Here we present crystal structures revealing the MCR-1 periplasmic, catalytic domain to be a zinc metalloprotein with an alk. phosphatase/sulphatase fold contg. three disulfide bonds. One structure captures a phosphorylated form representing the first intermediate in the transfer reaction. Mutation of residues implicated in zinc or phosphoethanolamine binding, or catalytic activity, restores colistin susceptibility of recombinant E. coli. Zinc deprivation reduces colistin MICs in MCR-1-producing lab., environmental, animal and human E. coli. Conversely, over-expression of the disulfide isomerase DsbA increases the colistin MIC of lab. E. coli. Preliminary d. functional theory calcns. on cluster models suggest a single zinc ion may be sufficient to support phosphoethanolamine transfer. These data demonstrate the importance of zinc and disulfide bonds to MCR-1 activity, suggest that assays under zinc-limiting conditions represent a route to phenotypic identification of MCR-1 producing E. coli, and identify key features of the likely catalytic mechanism.
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    33. 33
      McCall, K. A.; Huang, C.; Fierke, C. A. Function and Mechanism of Zinc Metalloenzymes. J. Nutr. 2000, 130 (5), 1437S– 1446S,  DOI: 10.1093/jn/130.5.1437S
      33
      Function and mechanism of zinc metalloenzymes
      McCall, Keith A.; Huang, Chih-Chinx; Fierke, Carol A.
      Journal of Nutrition (2000), 130 (5S), 1437S-1446SCODEN: JONUAI; ISSN:0022-3166. (American Society for Nutritional Sciences)
      A review with 115 refs. Zn is required for the activity of >300 enzymes, covering all 6 classes of enzymes. Zn-binding sites in proteins are often distorted tetrahedral or trigonal bipyramidal geometry, made up of the S atom of Cys, the N atom of His, or the O atom of Asp and Glu residues, or a combination. Zn in proteins can either participate directly in chem. catalysis or be important for maintaining protein structure and stability. In all catalytic sites, the Zn ion functions as a Lewis acid. Researchers in the authors' lab. are dissecting the determinants of mol. recognition and catalysis in the Zn-binding site of carbonic anhydrase. These studies demonstrate that the chem. nature of the direct ligands and the structure of the surrounding H-bond network are crucial for both the activity of carbonic anhydrase and the metal cation affinity of the Zn-binding site. An understanding of naturally occurring Zn-binding sites will aid in creating de novo Zn-binding proteins and in designing new metal sites in existing proteins for novel purposes such as to serve as metal ion biosensors.
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    34. 34
      Fage, C. D.; Brown, D. B.; Boll, J. M.; Keatinge-Clay, A. T.; Trent, M. S. Crystallographic Study of the Phosphoethanolamine Transferase EptC Required for Polymyxin Resistance and Motility in Campylobacter Jejuni. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2014, 70 (10), 2730– 2739,  DOI: 10.1107/S1399004714017623
      34
      Crystallographic study of the phosphoethanolamine transferase EptC required for polymyxin resistance and motility in Campylobacter jejuni
      Fage, Christopher D.; Brown, Dusty B.; Boll, Joseph M.; Keatinge-Clay, Adrian T.; Trent, M. Stephen
      Acta Crystallographica, Section D: Biological Crystallography (2014), 70 (10), 2730-2739CODEN: ABCRE6; ISSN:1399-0047. (International Union of Crystallography)
      The food-borne enteric pathogen Campylobacter jejuni decorates a variety of its cell-surface structures with phosphoethanolamine (pEtN). Modifying lipid A with pEtN promotes cationic antimicrobial peptide resistance, whereas post-translationally modifying the flagellar rod protein FlgG with pEtN promotes flagellar assembly and motility, which are processes that are important for intestinal colonization. EptC, the pEtN transferase required for all known pEtN cell-surface modifications in C. jejuni, is a predicted inner-membrane metalloenzyme with a five-helix N-terminal transmembrane domain followed by a sol. sulfatase-like catalytic domain in the periplasm. The at. structure of the catalytic domain of EptC (cEptC) was crystd. and solved to a resoln. of 2.40 Å. cEptC adopts the α/β/α fold of the sulfatase protein family and harbors a zinc-binding site. A phosphorylated Thr-266 residue was obsd. that was hypothesized to mimic a covalent pEtN-enzyme intermediate. The requirement for Thr-266 as well as the nearby residues Asn-308, Ser-309, His-358 and His-440 was ascertained via in vivo activity assays on mutant strains. The results establish a basis for the design of pEtN transferase inhibitors.
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    35. 35
      Wanty, C.; Anandan, A.; Piek, S.; Walshe, J.; Ganguly, J.; Carlson, R. W.; Stubbs, K. A.; Kahler, C. M.; Vrielink, A. The Structure of the Neisserial Lipooligosaccharide Phosphoethanolamine Transferase A (LptA) Required for Resistance to Polymyxin. J. Mol. Biol. 2013, 425 (18), 3389– 3402,  DOI: 10.1016/j.jmb.2013.06.029
      35
      The Structure of the Neisserial Lipooligosaccharide Phosphoethanolamine Transferase A (LptA) Required for Resistance to Polymyxin
      Wanty, Christopher; Anandan, Anandhi; Piek, Susannah; Walshe, James; Ganguly, Jhuma; Carlson, Russell W.; Stubbs, Keith A.; Kahler, Charlene M.; Vrielink, Alice
      Journal of Molecular Biology (2013), 425 (18), 3389-3402CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Gram-neg. bacteria possess an outer membrane envelope consisting of an outer leaflet of lipopolysaccharides, also called endotoxins, which protect the pathogen from antimicrobial peptides and have multifaceted roles in virulence. Lipopolysaccharide consists of a glycan moiety attached to lipid A, embedded in the outer membrane. Modification of the lipid A headgroups by phosphoethanolamine (PEA) or 4-amino-arabinose residues increases resistance to the cationic cyclic polypeptide antibiotic, polymyxin. Lipid A PEA transferases are members of the YhjW/YjdB/YijP superfamily and usually consist of a transmembrane domain anchoring the enzyme to the periplasmic face of the cytoplasmic membrane attached to a sol. catalytic domain. The crystal structure of the sol. domain of the protein of the lipid A PEA transferase from Neisseria meningitidis has been detd. crystallog. and refined to 1.4 Å resoln. The structure reveals a core hydrolase fold similar to that of alk. phosphatase. Loop regions in the structure differ, presumably to enable interaction with the membrane-localized substrates and to provide substrate specificity. A phosphorylated form of the putative nucleophile, Thr280, is obsd. Metal ions present in the active site are coordinated to Thr280 and to residues conserved among the family of transferases. The structure reveals the protein components needed for the transferase chem.; however, substrate-binding regions are not evident and are likely to reside in the transmembrane domain of the protein.
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    36. 36
      Anandan, A.; Evans, G. L.; Condic-Jurkic, K.; O’Mara, M. L.; John, C. M.; Phillips, N. J.; Jarvis, G. A.; Wills, S. S.; Stubbs, K. A.; Moraes, I.; Kahler, C. M.; Vrielink, A. Structure of a Lipid A Phosphoethanolamine Transferase Suggests How Conformational Changes Govern Substrate Binding. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (9), 2218– 2223,  DOI: 10.1073/pnas.1612927114
      36
      Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding
      Anandan, Anandhi; Evans, Genevieve L.; Condic-Jurkic, Karmen; O'Mara, Megan L.; John, Constance M.; Phillips, Nancy J.; Jarvis, Gary A.; Wills, Siobhan S.; Stubbs, Keith A.; Moraes, Isabel; Kahler, Charlene M.; Vrielink, Alice
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (9), 2218-2223CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Multidrug-resistant (MDR) gram-neg. bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin contg. lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addn. of phosphoethanolamine to the 1 and 4' headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from Neisseria meningitidis, detd. to 2.75-Å resoln. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing sol. domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helixes located in a periplasmic loop between two transmembrane helixes contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and mol. dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different-sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.
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    37. 37
      Moynihan, M. M.; Murkin, A. S. Cysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3-Nitropropionate. Biochemistry 2014, 53 (1), 178– 187,  DOI: 10.1021/bi401432t
      37
      Cysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3-Nitropropionate
      Moynihan, Margaret M.; Murkin, Andrew S.
      Biochemistry (2014), 53 (1), 178-187CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Isocitrate lyase (ICL) catalyzes the reversible cleavage of isocitrate into succinate and glyoxylate. It is the first committed step in the glyoxylate cycle used by some organisms, including Mycobacterium tuberculosis, where it has been shown to be essential for cell survival during chronic infection. The pH-rate and pD-rate profiles measured in the direction of isocitrate synthesis revealed solvent kinetic isotope effects (KIEs) of 1.7 ± 0.4 for D2OV and 0.56 ± 0.07 for D2O(V/Ksuccinate). Whereas the D2OV is consistent with partially rate-limiting proton transfer during formation of the hydroxyl group of isocitrate, the large inverse D2O(V/Ksuccinate) indicates that substantially different kinetic parameters exist when the enzyme is satd. with succinate. Inhibition by 3-nitropropionate (3-NP), a succinate analog, was found to proceed through an unusual double slow-onset process featuring formation of a complex with a Ki of 3.3 ± 0.2 μM during the first minute, followed by formation of a final complex with a Ki* of 44 ± 10 nM over the course of several minutes to hours. Stopped-flow measurements during the first minute revealed an apparent solvent KIE of 0.40 ± 0.03 for assocn. and unity for dissocn. In contrast, itaconate, a succinate analog lacking an acidic α-proton, did not display slow-binding behavior and yielded a D2OKi of 1.0 ± 0.2. These results support a common mechanism for catalysis with succinate and inhibition by 3-NP featuring (1) an unfavorable prebinding isomerization of the active site Cys191-His193 pair to the thiolate-imidazolium form, a process that is favored in D2O, and (2) the transfer of a proton from succinate or 3-NP to Cys191. These findings also indicate that propionate-3-nitronate, which is the conjugate base of 3-NP and the "true inhibitor" of ICL, does not bind directly and must be generated enzymically.
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    38. 38
      Zhao, Y.; Meng, Q.; Lai, Y.; Wang, L.; Zhou, D.; Dou, C.; Gu, Y.; Nie, C.; Wei, Y.; Cheng, W. Structural and Mechanistic Insights into Polymyxin Resistance Mediated by EptC Originating from Escherichia Coli. FEBS J. 2019, 286 (4), 750– 764,  DOI: 10.1111/febs.14719
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      Structural and mechanistic insights into polymyxin resistance mediated by EptC originating from Escherichia coli
      Zhao, Yanqun; Meng, Qiang; Lai, Yujie; Wang, Li; Zhou, Dan; Dou, Chao; Gu, Yijun; Nie, Chunlai; Wei, Yuquan; Cheng, Wei
      FEBS Journal (2019), 286 (4), 750-764CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
      Gram-neg. bacteria defend against the toxicity of polymyxins by modifying their outer membrane lipopolysaccharide (LPS). This modification mainly occurs through the addn. of cationic mols. such as phosphoethanolamine (PEA). EcEptC is a PEA transferase from Escherichia coli (E. coli). However, unlike its homologs CjEptC (Campylobacter jejuni) and MCR-1, EcEptC is unable to mediate polymyxin resistance when overexpressed in E. coli. Here, we report crystal structures of the C-terminal putative catalytic domain (EcEptCΔN, 205-577 aa) of EcEptC in apo and Zn2+-bound states at 2.10 and 2.60 Å, resp. EcEptCΔN is arranged into an α-β-α fold and equipped with the zinc ion in a conserved mode. Coupled with isothermal titrn. calorimetry (ITC) data, we provide insights into the mechanism by which EcEptC recognizes Zn2+. Furthermore, structure comparison anal. indicated that disulfide bonds, which play a key role in polymyxin resistance, were absent in EcEptCΔN. Supported by structural and biochem. evidence, we reveal mechanistic implications for disulfide bonds in PEA transferase-mediated polymyxin resistance. Significantly, because the structural effects exhibited by disulfide bonds are absent in EcEptC, it is impossible for this protein to participate in polymyxin resistance in E. coli.
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    39. 39
      Arciola, C. R.; Campoccia, D.; Ravaioli, S.; Montanaro, L. Polysaccharide Intercellular Adhesin in Biofilm: Structural and Regulatory Aspects. Front. Cell. Infect. Microbiol. 2015, 5, 7,  DOI: 10.3389/fcimb.2015.00007
      39
      Polysaccharideintercellularadhesininbiofilm:structuralandregulatoryaspects
      Arciola, Carla Renata; Campoccia, Davide; Ravaioli, Stefano; Montanaro, Lucio
      Frontiers in Cellular and Infection Microbiology (2015), 5 (), 7/1-7/10CODEN: FCIMAB; ISSN:2235-2988. (Frontiers Media S.A.)
      Staphylococcus aureus and Staphylococcus epidermidis are the leading etiol. agents of implant-related infections. Biofilm formation is the main pathogenetic mechanism leading to the chronicity and irreducibility of infections. The extracellular polymeric substances of staphylococcal biofilms are the polysaccharide intercellular adhesin (PIA), extracellular-DNA, proteins, and amyloid fibrils. PIA is a poly-β(1-6)-N-acetylglucosamine (PNAG), partially deacetylated, pos. charged, whose synthesis is mediated by the icaADBC locus. DNA sequences homologous to icaADBC locus are present in many coagulase-neg. staphylococcal species, among which S. lugdunensis, however, produces a biofilm prevalently consisting of proteins. The product of icaA is an N-acetylglucosaminyltransferase that synthesizes PIA oligomers from UDP-N-acetylglucosamine. The product of icaD gives optimal efficiency to IcaA. The product of icaC is involved in the externalization of the nascent polysaccharide. The product of icaB is an N-deacetylase responsible for the partial deacetylation of PIA. The expression of ica locus is affected by environmental conditions. In S. aureus and S. epidermidis ica-independent alternative mechanisms of biofilm prodn. have been described. S.epidermidis and S. aureus undergo to a phase variation for the biofilm prodn. that has been ascribed, in turn, to the transposition of an insertion sequence in the icaC gene or to the expansion/contraction of a tandem repeat naturally harbored within icaC. A role is played by the quorum sensing system, which neg. regulates biofilm formation, favoring the dispersal phase that disseminates bacteria to new infection sites. Interfering with the QS system is a much debated strategy to combat biofilm-related infections. In the search of vaccines against staphylococcal infections deacetylated PNAG retained on the surface of S. aureus favors opsonophagocytosis and is a potential candidate for immune-protection.
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    40. 40
      Little, D. J.; Milek, S.; Bamford, N. C.; Ganguly, T.; Difrancesco, B. R.; Nitz, M.; Deora, R.; Howell, P. L. The Protein BpsB Is a Poly-β-1,6-N-Acetyl-D-Glucosamine Deacetylase Required for Biofilm Formation in Bordetella Bronchiseptica. J. Biol. Chem. 2015, 290 (37), 22827– 22840,  DOI: 10.1074/jbc.M115.672469
      40
      The protein BpsB is a poly-β-1,6-N-acetyl-D-glucosamine deacetylase required for biofilm formation in Bordetella bronchiseptica
      Little, Dustin J.; Milek, Sonja; Bamford, Natalie C.; Ganguly, Tridib; Di Francesco, Benjamin R.; Nitz, Mark; Deora, Rajendar; Howell, P. Lynne
      Journal of Biological Chemistry (2015), 290 (37), 22827-22840CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Bordetella pertussis and Bordetella bronchiseptica are the causative agents of whooping cough in humans and a variety of respiratory diseases in animals, resp. Bordetella species produce an exopolysaccharide, known as the Bordetella polysaccharide (Bps), which is encoded by the bpsABCD operon. Bps is required for Bordetella biofilm formation, colonization of the respiratory tract, and confers protection from complement-mediated killing. Here, the authors investigated the role of BpsB in the biosynthesis of Bps and biofilm formation by B. bronchiseptica. BpsB is a two-domain protein that localizes to the periplasm and outer membrane. BpsB displays metal- and length-dependent deacetylation on poly-β-1,6-N-acetyl-D-glucosamine (PNAG) oligomers, supporting previous immunogenic data that suggests Bps is a PNAG polymer. BpsB can use a variety of divalent metal cations for deacetylase activity and showed highest activity in the presence of Ni2+ and Co2+. The structure of the BpsB deacetylase domain is similar to the PNAG deacetylases PgaB and IcaB and contains the same circularly permuted family four carbohydrate esterase motifs. Unlike PgaB from Escherichia coli, BpsB is not required for polymer export and has unique structural differences that allow the N-terminal deacetylase domain to be active when purified in isolation from the C-terminal domain. The enzymic characterizations here highlight the importance of conserved active site residues in PNAG deacetylation and demonstrate that the C-terminal domain is required for maximal deacetylation of longer PNAG oligomers. Furthermore, the authors show that BpsB is crit. for the formation and complex architecture of B. bronchiseptica biofilms.
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    41. 41
      Little, D. J.; Poloczek, J.; Whitney, J. C.; Robinson, H.; Nitz, M.; Howell, P. L. The Structure- and Metal-Dependent Activity of Escherichia Coli PgaB Provides Insight into the Partial de-N-Acetylation of Poly-β-1,6-N-Acetyl- D-Glucosamine. J. Biol. Chem. 2012, 287 (37), 31126– 31137,  DOI: 10.1074/jbc.M112.390005
      41
      The structure- and metal-dependent activity of Escherichia coli PgaB provides insight into the partial de-N-acetylation of poly-β-1,6-N-acetyl-D-glucosamine
      Little, Dustin J.; Poloczek, Joanna; Whitney, John C.; Robinson, Howard; Nitz, Mark; Howell, P. Lynne
      Journal of Biological Chemistry (2012), 287 (37), 31126-31137CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In Escherichia coli, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-D-glucosamine (PNAG) by the periplasmic protein PgaB is required for polysaccharide intercellular adhesin-dependent biofilm formation. To understand the mol. basis for PNAG de-N-acetylation, the structure of PgaB in complex with Ni2+ and Fe3+ have been detd. to 1.9 and 2.1 Å resoln., resp., and its activity on β-1,6-GlcNAc oligomers has been characterized. The structure of PgaB reveals two (β/α)x barrel domains: a metal-binding de-N-acetylase that is a member of the family 4 carbohydrate esterases (CE4s) and a domain structurally similar to glycoside hydrolases. PgaB displays de-N-acetylase activity on β-1,6-GlcNAc oligomers but not on the β-1,4-(GlcNAc)4 oligomer chitotetraose and is the first CE4 member to exhibit this substrate specificity. De-N-acetylation occurs in a length-dependent manner, and specificity is obsd. for the position of de-N-acetylation. A key aspartic acid involved in de-N-acetylation, normally seen in other CE4s, is missing in PgaB, suggesting that the activity of PgaB is attenuated to maintain the low levels of de-N-acetylation of PNAG obsd. in vivo. The metal dependence of PgaB is different from most CE4s, because PgaB shows increased rates of de-N-acetylation with Co2+ and Ni2+ under aerobic conditions, and Co2+, Ni2+ and Fe2+ under anaerobic conditions, but decreased activity with Zn2+. The work presented herein will guide inhibitor design to combat biofilm formation by E. coli and potentially a wide range of medically relevant bacteria producing polysaccharide intercellular adhesin-dependent biofilms.
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    42. 42
      Little, D. J.; Li, G.; Ing, C.; DiFrancesco, B. R.; Bamford, N. C.; Robinson, H.; Nitz, M.; Pomes, R.; Howell, P. L. Modification and Periplasmic Translocation of the Biofilm Exopolysaccharide Poly- −1,6-N-Acetyl-D-Glucosamine. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (30), 11013– 11018,  DOI: 10.1073/pnas.1406388111
      42
      Modification and periplasmic translocation of the biofilm exopolysaccharide poly-β-1,6-N-acetyl-D-glucosamine
      Little, Dustin J.; Li, Grace; Ing, Christopher; Di Francesco, Benjamin R.; Bamford, Natalie C.; Robinson, Howard; Nitz, Mark; Pomes, Regis; Howell, P. Lynne
      Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (30), 11013-11018CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Poly-β-1,6-N-acetyl-D-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of medically important bacteria. Polyglucosamine subunit B (PgaB) is responsible for the de-N-acetylation of PNAG, a process required for polymer export and biofilm formation. PgaB is located in the periplasm and likely bridges the inner membrane synthesis and outer membrane export machinery. Here, we present structural, functional, and mol. simulation data that suggest PgaB assocs. with PNAG continuously during periplasmic transport. We show that the assocn. of PgaB's N- and C-terminal domains forms a cleft required for the binding and de-N-acetylation of PNAG. Mol. dynamics (MD) simulations of PgaB show a binding preference for N-acetylglucosamine (GlcNAc) to the N-terminal domain and glucosammonium to the C-terminal domain. Continuous ligand binding d. is obsd. that extends around PgaB from the N-terminal domain active site to an electroneg. groove on the C-terminal domain that would allow for a processive mechanism. PgaB's C-terminal domain (PgaB310-672) directly binds PNAG oligomers with dissocn. consts. of ∼1-3 mM, and the structures of PgaB310-672 in complex with β-1,6-(GlcNAc)6, GlcNAc, and glucosamine reveal a unique binding mode suitable for interaction with de-N-acetylated PNAG (dPNAG). Furthermore, PgaB310-672 contains a β-hairpin loop (βHL) important for binding PNAG that was disordered in previous PgaB32-655 structures and is highly dynamic in the MD simulations. We propose that conformational changes in PgaB32-655 mediated by the βHL on binding of PNAG/dPNAG play an important role in the targeting of the polymer for export and its release.
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    43. 43
      Pokrovskaya, V.; Poloczek, J.; Little, D. J.; Griffiths, H.; Howell, P. L.; Nitz, M. Functional Characterization of Staphylococcus Epidermidis IcaB, a De-N-Acetylase Important for Biofilm Formation. Biochemistry 2013, 52 (32), 5463– 5471,  DOI: 10.1021/bi400836g
      43
      Functional characterization of Staphylococcus epidermidis IcaB, a de-N-acetylase important for biofilm formation
      Pokrovskaya, Varvara; Poloczek, Joanna; Little, Dustin J.; Griffiths, Heather; Howell, P. Lynne; Nitz, Mark
      Biochemistry (2013), 52 (32), 5463-5471CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      A polymer of partially de-N-acetylated β-1,6-linked N-acetylglucosamine (dPNAG), also known as the polysaccharide intercellular adhesin (PIA), is an important component of many bacterial biofilm matrixes. In S. epidermidis, the poly-N-acetylglucosamine polymer is partially de-N-acetylated by the extracellular protein, polysaccharide deacetylase IcaB. To understand the mechanism of action of IcaB, the enzyme was overexpressed and purified. IcaB demonstrated metal-dependent de-N-acetylase activity on β-1,6-linked N-acetylglucosamine oligomers with a broad preference for divalent metals. Steady-state kinetic anal. revealed the low catalytic efficiency (pentasaccharide kcat/Km = 0.03 M-1 s-1) of the enzyme toward the oligomeric substrates. While IcaB displays similar rates of de-N-acetylation with tri- through hexasaccharide PNAG oligomers, position-specific de-N-acetylation was only obsd. with penta- and hexasaccharides. The enzyme preferentially de-N-acetylated the 2nd residue from the reducing terminus in the pentasaccharide and 2nd and 3rd residues from the reducing terminus in the hexasaccharide. The data described here represent an important step toward a detailed understanding of dPNAG biosynthesis in S. epidermidis, an important nosocomial pathogen, as well as in other Gram-pos. bacteria. The low catalytic activity of IcaB was consistent with reports of other enzymes which act on biofilm-related polysaccharides, and this emerging trend may indicate a common feature among this group of polysaccharide-processing enzymes.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFams7nE&md5=f23003884b480873e1c46a90b8b30584
    44. 44
      Suardíaz, R.; Lythell, E.; Hinchliffe, P.; Van Der Kamp, M.; Spencer, J.; Fey, N.; Mulholland, A. J. Catalytic Mechanism of the Colistin Resistance Protein MCR-1. Org. Biomol. Chem. 2021, 19, 3813– 3819,  DOI: 10.1039/D0OB02566F
      44
      Catalytic mechanism of the colistin resistance protein MCR-1
      Suardiaz, Reynier; Lythell, Emily; Hinchliffe, Philip; van der Kamp, Marc; Spencer, James; Fey, Natalie; Mulholland, Adrian J.
      Organic & Biomolecular Chemistry (2021), 19 (17), 3813-3819CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      The mcr-1 gene encodes a membrane-bound Zn2+-metalloenzyme, MCR-1, which catalyzes phosphoethanolamine transfer onto bacterial lipid A, making bacteria resistant to colistin, a last-resort antibiotic. Mechanistic understanding of this process remains incomplete. Here, we investigate possible catalytic pathways using DFT and ab initio calcns. on cluster models and identify a complete two-step reaction mechanism. The first step, formation of a covalent phosphointermediate via transfer of phosphoethanolamine from a membrane phospholipid donor to the acceptor Thr285, is rate-limiting and proceeds with a single Zn2+ ion. The second step, transfer of the phosphoethanolamine group to lipid A, requires an addnl. Zn2+. The calcns. suggest the involvement of the Zn2+ orbitals directly in the reaction is limited, with the second Zn2+ acting to bind incoming lipid A and direct phosphoethanolamine addn. The new level of mechanistic detail obtained here, which distinguishes these enzymes from other phosphotransferases, will aid in the development of inhibitors specific to MCR-1 and related bacterial phosphoethanolamine transferases.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXktVOmtL4%253D&md5=ae9da798795700edcf4ef7d68ded2482
    45. 45
      Bar-Even, A.; Noor, E.; Savir, Y.; Liebermeister, W.; Davidi, D.; Tawfik, D. S.; Milo, R. The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters. Biochemistry 2011, 50 (21), 4402– 4410,  DOI: 10.1021/bi2002289
      45
      The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters
      Bar-Even, Arren; Noor, Elad; Savir, Yonatan; Liebermeister, Wolfram; Davidi, Dan; Tawfik, Dan S.; Milo, Ron
      Biochemistry (2011), 50 (21), 4402-4410CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The kinetic parameters of enzymes are key to understanding the rate and specificity of most biol. processes. Although specific trends are frequently studied for individual enzymes, global trends are rarely addressed. We performed an anal. of kcat and KM values of several thousand enzymes collected from the literature. We found that the "av. enzyme" exhibits a kcat of ∼10 s-1 and a kcat/KM of ∼ 105 s-1 M-1, much below the diffusion limit and the characteristic textbook portrayal of kinetically superior enzymes. Why do most enzymes exhibit moderate catalytic efficiencies Maximal rates may not evolve in cases where weaker selection pressures are expected. We find, for example, that enzymes operating in secondary metab. are, on av., ∼ 30-fold slower than those of central metab. We also find indications that the physicochem. properties of substrates affect the kinetic parameters. Specifically, low mol. mass and hydrophobicity appear to limit KM optimization. In accordance, substitution with phosphate, CoA, or other large modifiers considerably lowers the KM values of enzymes utilizing the substituted substrates. It therefore appears that both evolutionary selection pressures and physicochem. constraints shape the kinetic parameters of enzymes. It also seems likely that the catalytic efficiency of some enzymes toward their natural substrates could be increased in many cases by natural or lab. evolution.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXlsFWnur8%253D&md5=6cca5d0e98fe4f835de63adfe4059a56
    46. 46
      Chibba, A.; Poloczek, J.; Little, D. J.; Howell, P. L.; Nitz, M. Synthesis and Evaluation of Inhibitors of E. Coli PgaB, a Polysaccharide de-N-Acetylase Involved in Biofilm Formation. Org. Biomol. Chem. 2012, 10 (35), 7103– 7107,  DOI: 10.1039/c2ob26105g
      46
      Synthesis and evaluation of inhibitors of E. coli PgaB, a polysaccharide de-N-acetylase involved in biofilm formation
      Chibba, Anthony; Poloczek, Joanna; Little, Dustin J.; Howell, P. Lynne; Nitz, Mark
      Organic & Biomolecular Chemistry (2012), 10 (35), 7103-7107CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Many medically important biofilm forming bacteria produce similar polysaccharide intercellular adhesins (PIA) consisting of partially de-N-acetylated β-(1 6)-N-acetylglucosamine polymers (dPNAG). In Escherichia coli, de-N-acetylation of the β-(16)-N-acetylglucosamine polymer (PNAG) is catalyzed by the carbohydrate esterase family 4 deacetylase PgaB. The de-N-acetylation of PNAG is essential for productive PNAG-dependent biofilm formation. Here, we describe the development of a fluorogenic assay to monitor PgaB activity in vitro and the synthesis of a series of PgaB inhibitors. The synthesized inhibitors consist of a metal chelating functional group on a glucosamine scaffold to target the active site metal ion of PgaB. Optimal inhibition was obsd. with N-thioglycolyl amide (Ki = 480 μM) and N-methyl-N-glycolyl amide (Ki = 320 μM) glucosamine derivs. A chemoenzymic synthesis of an N-thioglycolyl amide PNAG pentasaccharide led to an inhibitor with an improved Ki of 280 μM.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1aitb3I&md5=2a93ce23411b8d2596c10e5aaee145f7
    47. 47
      Jerga, A.; Raychaudhuri, A.; Tipton, P. A. Pseudomonas Aeruginosa C5-Mannuronan Epimerase: Steady-State Kinetics and Characterization of the Product. Biochemistry 2006, 45 (2), 552– 560,  DOI: 10.1021/bi051862l
      47
      Pseudomonas aeruginosa C5-Mannuronan Epimerase: Steady-State Kinetics and Characterization of the Product
      Jerga, Agoston; Raychaudhuri, Aniruddha; Tipton, Peter A.
      Biochemistry (2006), 45 (2), 552-560CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Alginate is a major constituent of mature biofilms produced by Pseudomonas aeruginosa. The penultimate step in the biosynthesis of alginate is the conversion of some β-D-mannuronate residues in the polymeric substrate polymannuronan to α-L-guluronate residues in a reaction catalyzed by C5-mannuronan epimerase. Specificity studies conducted with size-fractionated oligomannuronates revealed that the minimal substrate contained nine monosaccharide residues. The max. velocity of the reaction increased from 0.0018 to 0.0218 s-1 as the substrate size increased from 10 to 20 residues, and no addnl. increase in kcat was obsd. for substrates up to 100 residues in length. The Km decreased from 80 μM for a substrate contg. fewer than 15 residues to 4 μM for a substrate contg. more than 100 residues. In contrast to C5-mannuronan epimerases that have been characterized in other bacterial species, P. aeruginosa C5-mannuronan epimerase does not require Ca2+ for activity, and the Ca2+-alginate complex is not a substrate for the enzyme. Anal. of the purified, active enzyme by inductively coupled plasma-emission spectroscopy revealed that no metals were present in the protein. The pH dependence of the kinetic parameters revealed that three residues on the enzyme which all have a pKa of ∼7.6 must be protonated for catalysis to occur. The compn. of the polymeric product of the epimerase reaction was analyzed by 1H NMR spectroscopy, which revealed that tracts of adjacent guluronate residues were readily formed. The reaction reached an apparent equil. when the guluronate compn. of the polymer was 75%.
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