Analysis of the Topology and Active-Site Residues of WbbF, aPutative O-Polysaccharide Synthase from Salmonella entericaSerovar Borreze

Samantha S. Wear,a Brittany A. Hunt,a Bradley R. Clarke,a Chris Whitfielda

aDepartment of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada

ABSTRACT Bacterial lipopolysaccharides are major components and contributorsto the integrity of Gram-negative outer membranes. The more conserved lipidA-core part of this complex glycolipid is synthesized separately from the hyper-variable O-antigenic polysaccharide (OPS) part, and they are joined in the periplasm priorto translocation to the outer membrane. Three different biosynthesis strategies arerecognized for OPS biosynthesis, and one, the synthase-dependent pathway, is cur-rently confined to a single example: the O:54 antigen from Salmonella enterica sero-var Borreze. Synthases are complex enzymes that have the capacity to both polym-erize and export bacterial polysaccharides. Although synthases like cellulose synthaseare widespread, they typically polymerize a glycan without employing a lipid-linkedintermediate, unlike the O:54 synthase (WbbF), which produces an undecaprenoldiphosphate-linked product. This raises questions about the overall similarity be-tween WbbF and conventional synthases. In this study, we examine the topology ofWbbF, revealing four membrane-spanning helices, compared to the eight in cellu-lose synthase. Molecular modeling of the glycosyltransferase domain of WbbF indi-cates a similar architecture, and site-directed mutagenesis confirmed that residuesimportant for catalysis and processivity in cellulose synthase are conserved in WbbFand required for its activity. These findings indicate that the glycosyltransferasemechanism of WbbF and classic synthases are likely conserved despite the use of alipid acceptor for chain extension by WbbF.

IMPORTANCE Glycosyltransferases play a critical role in the synthesis of a wide vari-ety of bacterial polysaccharides. These include O-antigenic polysaccharides, whichform the distal component of lipopolysaccharides and provide a protective barrierimportant for survival and host-pathogen interactions. Synthases are a subset of gly-cosyltransferases capable of coupled synthesis and export of glycans. Currently, theO:54 antigen of Salmonella enterica serovar Borreze involves the only example of anO-polysaccharide synthase, and its generation of a lipid-linked product differentiatesit from classical synthases. Here, we explore features conserved in the O:54 enzymeand classical synthases to shed light on the structure and function of the unusualO:54 enzyme.

KEYWORDS O antigens, glycosyltransferase, lipopolysaccharide, polysaccharidebiosynthesis, polysaccharide export, synthase

The O-antigenic polysaccharides (OPS) of Gram-negative bacteria are part of aglycolipid known as lipopolysaccharide (LPS), which represents a major component

of the outer membranes (OM) of most Gram-negative bacteria (1). The OPS extendsoutwards from the cell surface, forming a barrier that protects the bacteria from factorsin the external environment, including elements of the host immune response (2). OPSis a hypervariable structure, and the repeat-unit structures define the O-antigen sero-

Citation Wear SS, Hunt BA, Clarke BR, Whitfield C.2020. Analysis of the topology and active-siteresidues of WbbF, a putative O-polysaccharidesynthase from Salmonella enterica serovarBorreze. J Bacteriol 202:e00625-19. https://doi.org/10.1128/JB.00625-19.

Editor Thomas J. Silhavy, Princeton University

Copyright © 2020 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Chris Whitfield,cwhitfie@uoguelph.ca.

Received 30 September 2019Accepted 22 November 2019

Accepted manuscript posted online 2December 2019Published

RESEARCH ARTICLE

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type in bacterial serological typing systems. In Salmonella, there are 46 recognizedO-antigens (3). Three different strategies are used by bacteria to synthesize OPS, andthese are distinguished by the process used for their export across the inner membrane(IM). All three pathways assemble OPS glycans using the lipid carrier C55 undecaprenyl-phosphate (und-P) and ligate the finished product to LPS lipid A-core at the periplasmicface of the IM (2, 4). In the Wzy-dependent process, individual lipid-linked repeat unitsare delivered to a periplasmic polymerase by a representative of the multidrug/oligosaccharyl-lipid/polysaccharide (MOP) flippase (5). Some OPS are polymerized inthe cytoplasm and are exported by a pathway-defining member of the ATP-bindingcassette (ABC) transporter superfamily (4, 6). In contrast to these well-studied mecha-nisms, the synthase-dependent pathway is poorly understood, and the O:54 antigen ofSalmonella enterica serovar Borreze is the only known OPS that follows this process.

The O:54 antigen is a homopolymer of N-acetylmannosamine (ManNAc) with alter-nating �-(1¡3) and �-(1¡4) linkages. The enzymes involved in its production areencoded by genes carried on a naturally occurring 6.9-kb ColE1-based mobilizableplasmid (7, 8). The plasmid is unique in possessing a functional O-antigen gene clustercomprising three genes: wbbE, wbbF, and mnaA (formerly rfbA, rfbB, and rfbC, respec-tively). Assembly of the O:54 repeating unit (Fig. 1) begins on the cytoplasmic face ofthe IM, with the transfer of N-acetylglucosamine-1-phosphate (GlcNAc-1-P) to und-P byWecA. WecA is a phosphoglycosyltransferase possessing UDP-GlcNAc:undecaprenyl-phosphate GlcNAc-1-P transferase activity and is encoded by the locus directingbiosynthesis of enterobacterial common antigen (ECA) (9, 10). The WecA reaction isemployed by examples from all three OPS assembly pathways (2). MnaA, a UDP-GlcNAc2-epimerase, converts UDP-GlcNAc to UDP-ManNAc, the precursor needed for synthesisof the O:54 glycan, but the plasmid-borne mnaA is not essential for O:54 biosynthesisin members of the Enterobacteriaceae due to the existence of a functional homolog(wecB) in the ECA locus. Following precursor synthesis, a monofunctional glycosyltrans-ferase (GT), WbbE, transfers the first ManNAc residue to und-diphosphate (PP)-GlcNAc,committing the intermediate to O:54 biosynthesis and creating an adaptor on whichchain extension can occur (7). Next, WbbF is believed to polymerize the polysaccharideby forming the repeat domain of the OPS, with growth occurring at the nonreducingend (7). The absence of a dedicated transporter for the O:54 antigen led to the proposalthat WbbF is sufficient for both polymerization and export of the nascent OPS. Such

repeat-unit domain

→β-D-ManpNAc-(1→4)-β-D-ManpNAc-(1→3)-β-D-ManpNAc-(1→?)-β-D-ManpNAc-(1→?)-β-D-GlcpNAc→PP-und

adaptor primer

Periplasm

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Cytoplasm

UDPUDP UDP

WbbF

P PP

UDP-

UDP- UDP-

UDP- UDP-

WbbE

2.1. 3.

ligation tolipid A-core

GlcNAc

ManNAc

PP

PP

PP

FIG 1 Synthase-dependent pathway for O:54 antigen biosynthesis. (1) WecA primes und-P for synthesis through the transfer of GlcNAc-1-P. MnaA,an epimerase, converts UDP-GlcNAc to UDP-ManNAc, the precursor needed for synthesis. (2) WbbE transfers the initial ManNAc residue tound-PP-GlcNAc, creating the adaptor region. (3) WbbF is the proposed synthase, which polymerizes the O:54 polysaccharide by sequentialaddition of ManNAc residues and exports the product to the periplasmic face of the IM, where ligation to the lipid A-core occurs.

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dual properties are found in a class of enzymes called synthases, leading to the nameof the pathway.

Members of the synthase family are found in bacteria and eukaryotes and arecapable of the coordinated synthesis and export of a variety of important biopolymers,such as cellulose, chitin, and hyaluronan (11). The GT modules of synthases are typicallyassigned to the GT2 family of inverting GTs in the CAZy database (http://www.cazy.org/) and share sequence motifs which correlate with the processivity of these en-zymes (12–14). Cellulose synthase from Rhodobacter sphaeroides is the only examplewith a solved X-ray crystal structure and offers an influential prototype (12). Whilecellulose synthesis involves several proteins in a heterocomplex, the BcsAB subcomplexis sufficient for in vitro synthesis (15). BcsB is a periplasmic protein possessing aC-terminal transmembrane helix (TMH) that anchors the protein to the IM, while BcsAis an integral membrane protein possessing the catalytic site. The GT module in BcsAadopts a GT-A fold, characteristic of GT2 enzymes, and it lies between four amino-terminal and four carboxy-terminal TMHs, which form the narrow cellulose transloca-tion channel that is occupied by a nascent glucan chain in the solved structure (12).Subsequent structures revealed a stepwise synthetic system, with a ratcheting mech-anism to extrude the translocating cellulose chain (16).

Biosynthesis of cellulose, chitin, and hyaluronan occurs without the involvement oflipid-linked intermediates (12, 13, 17). In contrast, WecA-derived und-PP-linked inter-mediates initiate O:54 biosynthesis, and the final translocated product is transferred tolipid A-core at the periplasmic face of the IM by a ligase that requires an undecaprenyl-linked glycan as the glycosylation donor (8). This suggests fundamental differencesbetween the activity of WbbF and that of other characterized synthases, yet WbbF doesshare some sequence features with the known synthases. To begin to address thestructural and functional relationships between these enzymes, we investigated themembrane topology of WbbF and the functional importance of sequence motifs sharedby WbbF and the characterized synthases.

RESULTS AND DISCUSSIONWbbF topology. WbbF shares 40% similarity with the prototypic synthase, BcsA,

and possesses conserved synthase motifs (Fig. 2). The conserved motifs and catalyticresidues are discussed in detail below. To gain insight into the organization of theconserved motifs and the overall structure of WbbF, a structural model of WbbF wasgenerated using the BcsA structure (PDB ID 4HG6). Despite having a low sequenceidentity with BcsA (24%), the model for WbbF produced by the Phyre2 server (18)showed 87% coverage of the full-length protein (Fig. 3). The model predicts a largecytoplasmic GT domain (see below), as expected for an enzyme whose donor substrateis a nucleotide diphosphosugar. Based on this prediction, the two proteins differsubstantially in the number of TMHs: eight from the BcsA crystal structure, and threepredicted in WbbF. Chitin synthase (CHS), its ortholog NodC, and hyaluronan synthase(HAS) from Saccharomyces cerevisiae, Sinorhizobium meliloti, and Streptococcus pyo-genes, respectively, possess only three or four TMHs compared to the eight in BcsA (12,13, 19). An earlier hydrophobic cluster analysis predicted four TMHs located betweenresidues 11 and 40, 325 and 340, 385 and 406, and 416 and 438 and a PhoA activefusion at residue 368 (7). The Phyre2 (18) model contains one less TMH becauseresidues 367 and 409, which are predicted to contain a TMH, were not modeled due tosequence variation between WbbF and BcsA. However, both predictions differ fromresults obtained with the Consensus Constrained TOPology (CCTOP) server, whichintegrates the output of 11 different topology servers to generate a reliable model (20,21). CCTOP predicted a five-TMH topology with a reliability of 88% with a periplasmicN terminus, a cytoplasmic C terminus, and TMHs at positions 12 to 36, 290 to 305, 316to 340, 385 to 405, and 418 to 438. To resolve the contradictory outputs from differentmethods, the topology was investigated experimentally using a cysteine-labelingstrategy.

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TMH1 TMH2

TMH3 TMH4

TMH1

TMH2 TMH2 TMH3

TMH3TMH5 TMH6

TMH7

TMH7 TMH8

FIG 2 Multiple sequence and secondary structure alignment of WbbF (GenBank accession no. AAC98402), NodC(GenBank accession no. WP_014531649), and BcsA. The predicted WbbF secondary structure (modeled byPhyre2 [18]) and that determined from the solved BcsA crystal structure (PDB ID 4HG6) are displayed above and

(Continued on next page)

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The native WbbF protein contains seven cysteine residues, so these were allreplaced with alanine residues to facilitate the reintroduction of individual cysteineresidues at specific reporter sites. In total, eight residues were replaced with cysteine toprobe the number of TMHs. The changed residues are N2, A102, S285, V312, I348, S368,L413, and V447, and their positions are shown in the context of the four-TMH model inFig. 4A. Each WbbF derivative contained a C-terminal FLAG epitope for protein detec-tion, to ensure comparable levels of protein expression in whole-cell lysates (Fig. 4B).The wild-type (WT) and variant proteins consistently migrated as a doublet on SDS-PAGE. The predicted molecular weight of WbbF-FLAG is 54.5 kDa. The upper band(apparent molecular weight of �55.6 kDa calculated using Image Lab [Bio-Rad]) cor-responds to the fully unfolded version. The lower band (�47.3 kDa) may represent apartially unfolded form of WbbF or a degradation product. The denaturation step ofWbbF sample preparation was performed at 37°C, because incubation at highertemperatures results in protein aggregation, rendering WbbF undetectable. A similarphenomenon has been experienced with other membrane proteins (22–24). To confirmthat the numerous variant proteins retained function in O:54 biosynthesis and trans-port, cells expressing them were tested for surface-exposed immunofluorescence usingrabbit polyclonal anti-O:54 antibodies (Fig. 4C). WbbFC¡A (cysteine-free) retainedactivity, as did each variant; as anticipated, control cells transformed with the vectoralone gave no signal.

FIG 2 Legend (Continued)below the alignment, respectively. �-Helices, �-strands, strict �-turns, and strict �-turns are represented bysquiggles, arrows, TT, and TTT, respectively. Proposed catalytic residues, which were replaced with alanine, areindicated by a triangle, while the conserved DXD, (E/D)DX, and Q(Q/R)XRW motifs are highlighted in red. Thealignment was generated with ClustalW (46) and displayed using ESPript (47).

BcsA

Y149

D246D343

(E/D)DX

W383

Q(Q/R)XRW

Periplasm

Cytoplasm

H62

D244D151

W284

Q(Q/R)XRW

(E/D)DX

WbbF

TMH1

TMH2

TMH3

TMH1

TMH3

TMH4

TMH5

TMH2

TMH6

TMH7

TMH8

FIG 3 Structural comparison of BcsA and WbbF. The Phyre2 (18) model of WbbF was generated using thestructure of BcsA (PDB ID 4HG6) as a template. Residues 9 to 368 and 410 to 459 were modeled. Thehorizontal lines represent the approximate location of the membrane bilayer that divides the cytoplasmicand periplasmic compartments. �-Helices are colored red, �-strands are yellow, and loops are green. Theactive sites of each protein are shown below the model. Known and putative active-site residues areshown as cyan sticks, with the conserved Q(Q/R)XRW (top helix) and T(E/D)DX (left helix) motifs areshown on the orange helices as sticks.

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The eight single-cysteine WbbFX¡C FLAG variants were examined for reactivity witha membrane-impermeant fluorescent reagent, Oregon Green 488 maleimide carboxylicacid (OGM) (25, 26). WbbFC¡A-FLAG (pWQ1026) provided the negative control inlabeling experiments. The topology determination strategy was based on the reactivityof the single cysteine residues, which is dependent on whether they are located withinthe periplasm or cytoplasm. Cysteines in the periplasm (Fig. 5, lanes P) are expected to

Periplasm

Cytoplasm

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Wbb

FL4

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Wbb

FV

447C

-FLA

G

Wbb

FN

2C-F

LAG

Wbb

FI3

48C-F

LAG

wb*O:54

cysteine-free WbbF

S. enterica sv. Borreze vector

S285C V312CN2C A102C S368C L413C V447CI348C

C

FIG 4 (A) Topology model of WbbFC¡A (7). The conserved DXD, (E/D)DX, and Q(Q/R)XRW motifs are highlightedin orange. The residues which were replaced with cysteine and probed for OGM reactivity are highlighted in green.The initial model output was generated using Protter (48). (B) Western immunoblots of whole-cell lysates of E. coliTop10 harboring plasmid-encoded WbbF-FLAG and variants, within the O:54 cluster. Immunoblots were probedwith the FLAG epitope-specific antibody to confirm that all proteins were expressed at comparable levels. (C)Immunofluorescence microscopy of S. enterica serovar Borreze and E. coli Top10 cells. Cells were fixed and probedwith anti-O:54 antigen antibody and a fluorescent secondary antibody for detection.

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be labeled in intact cells, as OGM can penetrate the OM. In contrast, those within thelipid bilayer or cytoplasm remain inaccessible in the absence of cellular lysis to disruptthe IM barrier. The addition of OGM pre- and postlysis therefore distinguishes betweenperiplasmic and cytoplasmic cysteines, respectively. Following OGM labeling, the cellmembranes were collected, and WbbF-FLAG was extracted. Solubilized protein sampleswere enriched for WbbF-FLAG using magnetic beads specific for the FLAG epitope toremove as many contaminants as possible. However, attempts to purify WbbF usingalternative methods were not successful due to low protein yields, typical of an integralmembrane protein. The amounts of detergent-extracted WbbF variant proteins werecomparable, as shown by protein staining in SDS-PAGE (Fig. 5, middle panel) andWestern immunoblotting (Fig. 5, bottom panel), consistent with the data for whole-celllysates (Fig. 4B).

Exposure of an SDS-polyacrylamide gel using UV light revealed a fluorescent WbbF-FLAG doublet in samples corresponding to OGM labeling in intact cells of N2C, I348,S368C, and V447C variants (Fig. 5, top panel). In contrast, the WbbF-FLAG doublet inA102C, S285C, V312C, and L413C was labeled only following lysis. These data areconsistent with the originally proposed topology model (7), placing the N and C terminiin the periplasm, the GT domain in the cytoplasm, and four domains spanning themembrane (Fig. 4A). Cysteine-labeling analysis was confined to determining the num-ber of TMHs. No attempt was made to precisely define the borders of the TMHs byintroducing additional cysteine residues, since this was not central to the goals, nor didit influence the conclusions of this study.

WbbF possesses catalytic core residues resembling known synthases. ThePhyre2 (18) model predicts a large cytoplasmic GT domain with a GT-A fold containingtwo neighboring �/�/� domains (Fig. 3). Although WbbF shares a limited identity withthe other synthases (BcsA, 24%; HAS, 21%; CHS, 19%; and its ortholog, NodC, 23%), itpossesses conserved motifs shared by those enzymes in a comparable (predicted)structural context (Fig. 2). All known synthases contain a signature Q(Q/R)XRW motif inthe GT domain, which correlates with the processivity of these enzymes (27). This motiflines the active site, with the Trp residue acting to stabilize the growing end of thepolymer (the acceptor) via CH-� stacking interactions (11). Two additional catalytic GTmotifs are also conserved: DXD and (E/D)DX. The Asp residues of the DXD motif areinvolved in coordination of Mg2� (or Mn2�), which is crucial for the activity of some GTs

50

64

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kDa P P/CN2C

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P P/C P P/CA102C S285C

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αF

LAG

Sim

plyBlue

UV

light

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* * * *

FIG 5 Site-directed fluorescent labeling of WbbF. The panels show SDS-PAGE and Western immunoblotting ofDDM-solubilized WbbF-FLAG variants labeled with OGM. Top panel, SDS-polyacrylamide gel after exposure to UVlight for detection of OGM-labeled residues. Asterisks indicate the location of WbbF-FLAG on the gel. Middle panel,the same gel as shown in the top panel, stained with SimplyBlue to confirm comparable protein loading. Bottompanel, Western immunoblot probed with anti-FLAG epitope antibodies, illustrating comparable expression of WbbFvariants. Addition of OGM-labeled periplasmic (P) cysteine residues in unlysed cells, while addition postlysis labeledboth periplasmic and cytoplasmic (P/C) cysteines.

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(28). BcsA and WbbF also possess the TED motif, which overlaps with the (E/D)D fromthe (E/D)DX motif. These motifs are located on what is known as a “finger helix” and areoriented toward the nonreducing end of the glycan (28). In BcsA, the Thr formshydrogen bonds with the 2=- or 3=-OH of the acceptor, while the Asp is proximal to the4=-OH of the acceptor and therefore acts as a general base to catalyze its deprotonation(11). Additionally, an aromatic residue (Y149 in BcsA) at the entrance of the active siteis speculated to interact with the uracil moiety of the UDP-sugar. This residue is criticalfor NodC and CHS activity (13). Aligning the sequences of WbbF, BcsA, and NodCidentified four putative catalytic residues in WbbF, i.e., H62, D151, D244, and W284,which correspond to established catalytic residues in BcsA (Y149, D246, D343, andW383) and NodC (F58, D140, D241, and W281) (13, 28).

To validate the importance of these putative active-site residues in WbbF, each wasreplaced with alanine and the effect on O:54 antigen biosynthesis was examined. EachWbbF variant was expressed at levels comparable to that of wild-type WbbF (Fig. 6A).The activity of each WbbF variant was assessed by PAGE of whole-cell lysates andWestern immunoblotting using O:54 antibodies (Fig. 6B), as well as immunofluores-cence (Fig. 6C). Silver-stained gels label only the LPS-linked form of OPS (29), and O:54LPS typically stains poorly due to (unknown) aspects of the OPS chemistry. In contrast,the Western blot reveals LPS-linked OPS as well as any unlinked (or unexported)biosynthetic intermediates. The D151A, D244A, and W284A variants were unable tosupport O:54 biosynthesis, confirming the importance of the DXD, TED, and Q(Q/R)XRWmotifs in WbbF (Fig. 6B and C). Examination of permeabilized cells and the correspond-ing immunoblot revealed no evidence of internal O:54 antigen, ruling out unexpecteduncoupled biosynthesis in the absence of export in any of the variants. The cells ofthese variants were typically smaller than those of the control. To rule out anysecond-site mutations in the other cloned O:54 genes (or elsewhere on the chromo-some) that might confer an O:54-deficient phenotype, plasmids encoding each inactivevariant were cotransformed with a plasmid carrying wild-type wbbF. As anticipated, thewild-type copy of wbbF restored both O:54 production and WT cell size. The cell sizedefects in these variants may result from sequestering initiated (but incomplete andunexported) und-PP-linked intermediates resulting from WbbE activity. Various cellsize/shape defects have been observed in Escherichia coli cells accumulating pathwayintermediates for other OPS and ECA (30). In contrast to the variants described above,the H62A variant still supported O:54 production. It is possible that the function of thisresidue is fulfilled by another nearby residue or that the interactions it makes with thedonor are not essential for activity of WbbF. In summary, while some mutationseliminated O:54 biosynthesis, as predicted, none of the mutations examined hereresulted in significant changes of OPS chain length distribution.

Implications for WbbF function. The O:54 antigen is atypical, as it is the onlyknown OPS in which synthesis is encoded by a naturally occurring ColE1-basedmobilizable plasmid (7, 31). ColE1 plasmids can be transferred between bacteria in thepresence of conjugative transfer functions provided by another plasmid (32, 33). Thesecharacteristics explain why the O:54 factor can be coexpressed with Salmonella isolatespossessing additional chromosomally encoded OPS, which use a Wzx/Wzy-dependentpathway (31, 34, 35). The maintenance of two separate OPSs may be helped by thebiosynthetic separation, but in one example, the isolate expresses only the chromo-somally encoded OPS in the absence of the O:54 antigen. The expression of O:54antigen can also be lost (presumably due to loss of the plasmid), while expression ofchromosomally encoded OPS is retained. The coexpression of O:54 with other OPStypes in Salmonella differs from other situations involving lateral transfer of OPS clustersin this species. There are examples of Salmonella isolates with two chromosomal OPSgene clusters, but in these cases, one cluster is no longer functional and only a singleOPS is produced (3).

Although synthases are rare in the context of OPS synthesis (and currently confinedto the O:54 antigen), a variety of other polymers, such as cellulose, are constructed

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FIG 6 Expression of WbbF variants and their effect on O:54 antigen production. (A) Western immunoblots ofwhole-cell lysates of E. coli Top10 harboring plasmid-encoded WbbF and variants. Immunoblots were probed withFLAG epitope-specific antibodies to confirm comparable expression of WbbF variants. WbbF-His6 was utilized forcomplementation. (B) Silver-stained SDS-polyacrylamide gel (upper) and corresponding representative Westernimmunoblot (lower) of proteinase K-digested whole-cell lysates of S. enterica serovar Borreze and E. coli Top10transformants harboring plasmid-encoded WbbF alone (WT WbbF) or the entire wb*O:54 operon. The immunoblotwas probed with anti-O:54 serum. The gel and immunoblot are representative of three biological replicates. (C)Fixed-cell immunofluorescence microscopy of nonpermeabilized and permeabilized S. enterica serovar Borreze andE. coli cells harboring the same plasmids as used in panel B. Cells were probed with anti-O:54 antigen antibody.Fluorescence observed in whole cells is due to the presence of O:54 antigen on the surface, while fluorescentpermeabilized cells indicate total (including intracellular) polymer.

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using these systems. The synthesis and translocation of cellulose have been studied indepth and have provided insight into how these processes are coupled. BcsA extendsthe cellulose polymer one glucose molecule at a time to the nonreducing terminus,with the newly added glucose acting as the acceptor in the reaction that follows (12).While the same direction of growth is shared in the biosynthesis of chitin and the O:54antigen (7, 12, 13), only the O:54 product is assembled as an und-PP-linked interme-diate, and this must be retained through export to provide a viable donor to glycosylatelipid A-core in the periplasm. As a result, some differences between the translocationpathways in cellulose synthase and WbbF are predicted.

Translocation of cellulose through the translocation channel is a result of confor-mational changes in BcsA. These changes are produced by a ratcheting mechanism,which involves movements of the finger helix and the “gating loop” (16). The fingerhelix interacts with the terminal glucose of the cellulose polymer, while the gating loophouses the FXVTXK motif and spans the entrance of the active site, controllingsubstrate access (28). Upon substrate binding, the finger helix shifts upwards and thegating loop transitions into the active site, pushing the polymer into the channel. Oncethe active site is empty, the gating loop retracts, and the finger helix shifts downwards.These movements are repeated for each subsequent binding of substrate and elonga-tion. The finger helix and its motifs are conserved in WbbF. It is believed that movementof the finger helix is reliant on the gating loop. BcsA possesses an Ile upstream of theTED motif, which is speculated to cause steric clashes with the gating loop, in theabsence of their coupled movements (16). This Ile is conserved among bacterialcellulose synthases, but in eukaryotic cellulose synthases and WbbF, the correspondingposition is occupied by a Leu residue, which may play a similar role. Consistent with thispossibility, an Ile¡Leu replacement in BcsA retained 50% of wild-type activity (16). It isunclear whether the same gating loop structure exists in WbbF. The initial residue ofthe gating loop (Phe) aids in positioning the uracil moiety of the donor sugar (28) andis conserved in WbbF, but the portion of the sequence which aligns with the gatingloop in BcsA could not be modeled in WbbF.

The cellulose synthase translocation channel is packed tightly against the cytoplas-mic GT2 domain, and the Phyre2 model of WbbF reveals a similar structural arrange-ment (Fig. 3). Despite having fewer TMHs than cellulose synthase, and a portion of thesequence not being modeled, the model of WbbF reveals a putative translocationchannel. It is feasible that these synthases exist as homodimers to facilitate formationof an adequate translocation channel with eight TMHs. Dimerization has been shownto occur in mammalian HAS, as well as HAS from Streptococcus equisimilis (SeHAS) (17,36). The HAS enzymes from different Streptococcus species are highly conserved (�70%sequence identity) (37). The homolog from S. pyogenes (SpHAS) also possesses fourTMHs and a GT2 module, with conserved DXD, (E/D)DX, and Q(Q/R)XRW motifs (19).Nascent hyaluronan produced by SeHAS reconstituted in liposomes was inaccessible toa hyaluronan-degrading enzyme, indicating that polymer synthesis and translocationare spatially coupled events, resembling cellulose synthesis (17). Other bacterial trans-porters which export und-PP-linked oligo- and polysaccharides have been described.These include the MOP transporter (MurJ) for lipid II (38) and ABC transporters for bothO-antigen (Wzm-Wzt) (39, 40) and N-linked glycan export (PglK) (41). In the ABCtransporters, lateral gates allow the glycan part of the substrate to access the trans-porter lumen, while the undecaprenol lipid remains in the lipid bilayer, and this mayalso apply to MurJ. Whether dimerization offers an avenue to a similar strategy in WbbFawaits a solved structure. Unfortunately, we have been unable to produce sufficientamounts of purified WbbF to facilitate crystallization trials, and there are currently nohomologs that can be pursued as alternatives. To date, WbbF remains the sole synthasewhich requires a lipid-linked acceptor.

MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains used in this study were E. coli Top10

[F� mcrA Δ(mrr-hsdRMS-mcrBC) �80 lacZΔM15 ΔlacX74 deoR nupG recA1 araD139 Δ(ara-leu)7697 galU

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galK rpsL(Strr) endA1] (Invitrogen) and S. enterica serovar Borreze (54:f,g,s:-1; contains plasmids of 96, 4.5and 2.3 MDa) (31). Bacteria were grown at 37°C in lysogeny broth (LB). When appropriate, media weresupplemented with ampicillin (100 �g/ml), chloramphenicol (34 �g/ml), anhydrotetracycline (2.5 ng/ml),or L-arabinose (0.01% [wt/vol]).

Plasmids and DNA methods. The plasmids used are listed in Table 1. A synthetic wb*O54 operon wasdesigned to include the three biosynthetic genes (wbbE, wbbF, and mnaA) in their native arrangement.For detection of WbbF, the nucleotide sequence encoding a FLAG tag epitope was introduced at the 3=terminus of wbbF, and EagI and SacI restriction sites were added to either end of wbbF to facilitate itsexcision and cloning. The operon was synthesized by DNA 2.0 (Atum). Following synthesis, the syntheticwb*O54 operon was amplified by PCR and cloned into pBAD24 using Gibson assembly as described by themanufacturer (New England Biolabs). Custom oligonucleotide primers used in this study were purchasedfrom Sigma and are listed in Table 2. Plasmid DNA was purified using the PureLink quick plasmidminiprep kit (Invitrogen). Either KOD hot start (EMD Millipore) or PfuUltra high-fidelity (Agilent) DNApolymerase was used for amplification of DNA fragments and site-directed mutagenesis. Site-directedmutagenesis was performed using the QuikChange protocol (Agilent). DNA fragments from PCRs andrestriction enzyme digests were purified using the PureLink PCR purification kit (Invitrogen). Restrictionendonucleases and T4 DNA ligase (New England Biolabs) were used according to the manufacturer’sinstructions. All constructs were verified by DNA sequencing (Advanced Analysis Center, University ofGuelph).

Detection of WbbF-FLAG and WbbF-His6 in whole-cell lysates. Overnight cultures were diluted1:50 in 5 ml of LB medium and grown to an optical density at 600 nm (OD600) of 0.5, at which point theculture was transferred to 18°C and allowed to grow until an OD600 of 0.6 was reached. Protein expressionwas then induced using L-arabinose (0.01% [wt/vol]), and the culture was grown for an additional 16 h.The OD600 of the culture was measured, and cells from a volume corresponding to 2 OD600 units werecollected by centrifugation (13,000 � g for 1 min). Ten microliters of cOmplete Mini EDTA-free proteaseinhibitor (Roche Applied Science) solution (1 tablet per 1 ml of water) and 25 �l of B-PER II (ThermoFisher)were added to the pellet. The pellet was then resuspended in 63 �l of water, and 1 �l of both rLysozyme(EMD Millipore) (diluted 1:10 in water) and Benzonase (Novagen) (diluted 1:5 in 50 mM Tris-HCl, 20 mMNaCl, 2 mM MgCl2, pH 8) were added. The mixture was rocked at room temperature for 30 min before100 �l of 2� Laemmli sample buffer (42) was added. Samples were incubated in Laemmli sample bufferat 37°C for 30 min prior to separation by SDS-PAGE using 10% resolving gels in Tris-glycine buffer.Proteins were visualized using SimplyBlue SafeStain (Life Technologies). Western immunoblotting wasperformed with FLAG and His6 epitope-tagged proteins, which were transferred to nitrocellulosemembranes and probed with anti-FLAG (Qiagen; diluted 1:1,000) or anti-His5 (Qiagen; diluted 1:2,000)antibodies. The secondary antibody was horseradish peroxidase-conjugated goat anti-mouse antibody(Jackson ImmunoResearch; diluted 1:3,000), and chemiluminescence detection was accomplished usingLuminata Crescendo substrate (EMD Millipore).

Site-directed fluorescence labeling. Labeling of WbbF variants with Oregon Green 488 maleimidecarboxylic acid (OGM; Fisher Scientific) was based on a protocol described elsewhere (26) and modifiedby Larue et al. (25). Overnight cultures were used to inoculate 200 ml of LB medium (1:100) supple-mented with ampicillin. WbbF variants were expressed as described above. Cells were collected bycentrifugation at 5,000 � g for 10 min at 4°C and resuspended in 10 ml of 25 mM sodium phosphate

TABLE 1 Plasmid summary

Plasmid Description Reference or source

pBAD24 Plasmid vector with L-arabinose-inducible promoter; Apr 49pWQ572 pBAD24 derivative containing a chloramphenicol-resistance cassette and Ptet promoter; Cmr 25pWQ799 Naturally occurring plasmid containing wb*O54 operon 8pWQ203 pWQ572 derivative containing wb*O54 operon from pWQ799; Cmr This studypWQ1015 pWQ203 containing WbbFH62A This studypWQ1016 pWQ203 containing WbbFD151A This studypWQ1017 pWQ203 containing WbbFD244A This studypWQ1018 pWQ203 containing WbbFW284A This studypWQ1019 pBAD24 derivative encoding WbbF-His6; Apr This studypWQ1020 pBAD24 derivative containing synthetic wb*O54 operon encoding WbbF-FLAG This studypWQ1021 pSW1020 containing WbbFH62A-FLAG This studypWQ1022 pSW1020 containing WbbFD151A-FLAG This studypWQ1023 pSW1020 containing WbbFD244A-FLAG This studypWQ1024 pSW1020 containing WbbFW284A-FLAG This studypWQ1025 pWQ203 containing WbbFC¡A This studypWQ1026 pSW1020 containing WbbFC¡A -FLAG from pWQ1025 This studypWQ1027 pSW1026 containing WbbFN2C-FLAG This studypWQ1028 pSW1026 containing WbbFA102C-FLAG This studypWQ1029 pSW1026 containing WbbFS285C-FLAG This studypWQ1030 pSW1026 containing WbbFV312C-FLAG This studypWQ1031 pSW1026 containing WbbFS368C-FLAG This studypWQ1032 pSW1026 containing WbbFL413C-FLAG This studypWQ1033 pSW1026 containing WbbFV447C-FLAG This study

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buffer containing 250 mM NaCl, pH 7.5. Two 3-ml aliquots of the cell suspension were removed. Inaliquot 1, periplasmic cysteine residues were modified with 40 �M OGM during a 15-min incubation atroom temperature. Following labeling, the reaction was quenched with 1 mM �-mercaptoethanol.Aliquot 2 remained untreated. Aliquots 1 and 2 were centrifuged at 5,000 � g for 10 min at 4°C andwashed twice. The pellets were then resuspended in 2 ml of buffer containing 5 mM EDTA, 100 �g/mllysozyme, and 20% (wt/vol) sucrose and incubated for 15 min at room temperature. Cells were disrupted

TABLE 2 Sequences of oligonucleotide primers

Primer Sequence (5=¡3=)a Features

O54fwd gatcggatccGCTGTATAGGAAATTAGGAAATTAG Forward primer to amplify wb*O54 operon; BamHIrestriction site (underlined)

O54rev gatcctgcagTCACTTAATATCCTCCAAGGATAC Reverse primer to amplify wb*O54 operon; PstIrestriction site (underlined)

SW13 CTAATTCTTGTGGCTGCGGCTAATGAAGAGGCTGTGATTGGCTCAACAC Forward primer used to generate WbbFH62A variantSW14 CAATCACAGCCTCTTCATTAGCCGCAGCCACAAGAATTAGAAAGCGG Reverse primer used to generate WbbFH62A variantSW9 TATGATTTGGTCATGGTGTTGGCTGCCGACAATTTTGTTGATGCGAATATCCTTACT Forward primer used to generate WbbFD151A variantSW10 ATCAACAAAATTGTCGGCAGCCAACACCATGACCAAATCATAATTTTCTTTAACAGT Reverse primer used to generate WbbFD151A variantSW7 GTTTTAAATCTCTGACCGAGGCCATTGAACTGGAAATTGAAATTG Forward primer used to generate WbbFD244A variantSW8 CAATTTCAATTTCCAGTTCAATGGCCTCGGTCAGAGATTTAAAAC Reverse primer used to generate WbbFD244A variantSW11 CTCAAACAACGCTATCGCGCGTCAAAGGGACACTGGTATGT Forward primer used to generate WbbFW284A variantSW12 ACCAGTGTCCCTTTGACGCGCGATAGCGTTGTTTGAGGCTTATTC Reverse primer used to generate WbbFW284A variantWbbFf gatcgaattcaccATGAATGATTATATAATTGACATAG Forward primer for the amplification of wbbF; EcoRI

restriction site (underlined)WbbFr gatcctgcagttaatgatgatgatgatgatgTTTGTGCTCTTCCTTTATTTTATTATGC Reverse primer for the amplification of wbbF and

introduction of a his6-tag; PstI restriction site(underlined)

SW35 cccgtttttttgggctagcaggaggaattcaccATGGGGCATCAGTTTACGGTTTGC Forward primer to amplify synthetic wb*O54 operonadding homologous pBAD24 region for Gibsonassembly; EcoRI restriction site (underlined)

SW36 ctcatccgccaaaacacccaagcttctgcagTCACTTAATATCCTCCA Reverse primer to amplify synthetic wb*O54 operonadding homologous pBAD24 region for Gibsonassembly; HindIII restriction site (underlined)

SW17 gatccggccgATGAATGATTATATAATTGACATAGTGG Forward primer for the amplification of wbbF; EagIrestriction site (underlined)

SW18 gatcgagctcTTTGTGCTCTTCCTTTATTTTATTATGC Reverse primer for the amplification of wbbF; SacIrestriction site (underlined)

KB1 GCTAAAAATAAAAAAGACTATCCTGACGCTCCTCCTGAAGCCCG Forward primer used to generate WbbFC49A variantKB2 CAAGAATTAGAAAGCGGGCTTCAGGAGGAGCGTCAGGATAGTC Reverse primer used to generate WbbFC49A variantKB3 CCACTGATCGGACAGGACTTATCGCTGATAGTCATGAAGTAAAG Forward primer used to generate WbbFC102A variantKB4 CCACATGCTTTACTTCATGACTATCAGCGATAAGTCCTGTCCG Reverse primer used to generate WbbFC102A variantKB5 GCCGGAAGCTATACAGGCGTATCTGGATGCTAAAAACTCAACAT Forward primer used to generate WbbFC183A variantKB6 GCCAAAAGAGAGAAGAGATGTTGAGTTTTTAGCATCCAGATACG Reverse primer used to generate WbbFC183A variantKB7 CTCAACATCTCTTCTCTCTTTTGGCTACGCTACATCATACTGGAT Forward primer used to generate WbbFC195A variantKB8 GGAAAAATCGATTCATCATCCAGTATGATGTAGCGTAGCCAAAAG Reverse primer used to generate WbbFC195A variantKB9 CTGATAAATACTGGAGGATTTGCTTTTAAATCTCTGACCGAGGA Forward primer used to generate WbbFC237A variantK10 CCAGTTCAATGTCCTCGGTCAGAGATTTAAAAGCAAATCCTCCA Reverse primer used to generate WbbFC237A variantc328af GGGCCGTGCTTTGCAGGTTGCTATTATTTTCATCAATATCTTTC Forward primer used to generate WbbFC328A variantc328ar GAAAGATATTGATGAAAATAATAGCAACCTGCAAAGCACGGCCC Reverse primer used to generate WbbFC328A variantc394af CGTCACATTAATATCCATCGCTTATGGTATGCTGATTTTACC Forward primer used to generate WbbFC394A variantc394ar GGTAAAATCAGCATACCATAAGCGATGGATATTAATGTGACG Reverse primer used to generate WbbFC394A variantSW115 GTGTTAACGGCCGATGTGTGATTATATAATTGACATAGTGGAATATGTTTTATATG Forward primer used to generate cfWbbFN2C variantSW116 CACTATGTCAATTATATAATCACACATCGGCCGTTAACACCTTAAAATA Reverse primer used to generate cfWbbFN2C variantSW41 GATCGGACAGGACTTATCTGTGATAGTCATGAAGTAAAGCATGTGGATAC Forward primer used to generate cfWbbFA102C variantSW42 GCTTTACTTCATGACTATCACAGATAAGTCCTGTCCGATCAGTGGA Reverse primer used to generate cfWbbFA102C variantSW25 ACAACGCTATCGCTGGTGTAAGGGACACTGGTATGTGGCTTTTAC Forward primer used to generate cfWbbFS285C variantSW26 CACATACCAGTGTCCCTTACACCAGCGATAGCGTTGTTTGAGGCTTA Reverse primer used to generate cfWbbFS285C variantSW27 TTGAGCGTAAGTGGAAATATTGTGATCAATTGTTATATCTGTTCTCTATGGGC Forward primer used to generate cfWbbFV312C variantSW28 GAGAACAGATATAACAATTGATCACAATATTTCCACTTACGCTCAACAAAGGTCAAC Reverse primer used to generate cfWbbFV312C variantSW110 GAAAATTACCATCCAGAGTGTGGAAATATTTCTACGGCGATAAAAGATC Forward primer used to generate cfWbbFI348C variantSW111 CGCCGTAGAAATATTTCCACACTCTGGATGGTAATTTTCTTTAAGAAGAC Reverse primer used to generate cfWbbFI348C variantSW29 CAATATGAGTTTTGCCGACTGTGTGAGTGCGCAGTTTAGCTCAATAAATTG Forward primer used to generate cfWbbFS368C variantSW30 CTAAACTGCGCACTCACACAGTCGGCAAAACTCATATTGGTCACAGTA Reverse primer used to generate cfWbbFS368C variantSW31 GGATGGATAAAGGTATTTTCTGTAATCCATTCAGGGTATTTTTTTCCGGTC Forward primer used to generate cfWbbFL413C variantSW32 GAAAAAAATACCCTGAATGGATTACAGAAAATACCTTTATCCATCCATGCACC Reverse primer used to generate cfWbbFL413C variantSW33 GGAAAAAACAGCATAAATGGTGTGTTACGCCGCATAATAAAATAAAGGAGG Forward primer used to generate cfWbbFV447C variantSW34 ATTTTATTATGCGGCGTAACACACCATTTATGCTGTTTTTTCCAACGAAAAAG Reverse primer used to generate cfWbbFV447C variantaBoldface indicates a point mutation.

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with 18 ml of cold water containing cOmplete Mini EDTA-free protease inhibitor (Roche Applied Science).A 10 �M concentration of OGM was added to aliquot 2, and both aliquots were sonicated for 30 s(10 s on, 10 s off) prior to a 15-min incubation at room temperature. In aliquot 2, all available cysteinesare labeled with OGM. The reaction was again quenched with 1 mM �-mercaptoethanol. Unbroken cellsand debris were removed from both aliquots by centrifugation at 15,000 � g for 15 min at 4°C. Cell-freelysates were centrifuged at 100,000 � g for 1 h at 4°C to collect membranes, which were solubilizedovernight at 4°C in 1 ml of buffer containing 1% N-dodecyl-�-D-maltopyranoside (DDM) (Sigma). Theremaining insoluble material was removed by centrifugation at 100,000 � g for 1 h at 4°C, andWbbF-FLAG was purified from 500 �l solubilized membranes using 25 �l of anti-FLAG M2 magneticbeads (Sigma) in accordance with the manufacturer’s batch protocol. Proteins were eluted using 20 �lof 1� Laemmli sample buffer. Samples were warmed for 30 min at 37°C before 10-�l samples wereanalyzed by SDS-PAGE using 10% resolving gels. OGM was visualized by exposing the gels to UV lightusing a Bio-Rad Gel Doc. Protein visualization and Western immunoblotting were carried out as describedabove.

Immunofluorescence microscopy. To assess O:54 production and export, immunofluorescencemicroscopy was performed with fixed cells, in accordance with the protocol described by Clarke et al.(43), to assess O:54 production and export. In brief, overnight cultures were diluted 1:50 in 5 ml of LBmedium supplemented with the appropriate antibiotics, anhydrotetracycline and/or L-arabinose. Cul-tures were grown at 37°C until an OD600 of 0.5 was reached. One OD600 unit of cells was collected bycentrifugation, resuspended in 5% (vol/vol) formaldehyde, and incubated at 4°C for 16 h. Fixed cells werecollected, washed twice with 1 ml of phosphate-buffered saline (PBS), and finally resuspended in 100 �lof PBS. Ten-microliter samples were added in duplicate to the wells of a poly-L-lysine-coated glass slideand incubated for 10 min at room temperature. Cells in one well for each sample were permeabilizedusing 10 �l of 0.5 mg/ml lysozyme (in 25 mM Tris-HCl, 10 mM EDTA, pH 8) and 0.1% Triton X-100 (in PBS)solutions. Following the addition of each solution, the slide was incubated for 15 min. The wells werethen blocked for 15 min with 1% (wt/vol; in PBS) bovine serum albumin (BSA). After washing, the slideswere incubated with anti-O:54 antiserum (Statens Serum Institut, Denmark; diluted 1:100 in 1% BSA) atroom temperature for 30 min and washed. The slides were then treated with rhodamine red-conjugatedgoat anti-rabbit antibody (Jackson Immunoresearch; diluted 1:50 in 1% BSA) and washed again. Theslides were mounted in Vectashield (Vector Laboratories) and viewed using a Zeiss Axiovert 200microscope with a 100� lens objective. The images were processed using Volocity software (PerkinEl-mer).

LPS analysis. Cells and transformants were grown as described above. Cells from one OD600 unitwere collected by centrifugation (13,000 � g for 1 min), and whole-cell lysates were digested by usingproteinase K (44). Samples (10 �l) were separated on 12% resolving gels in Tris-glycine buffer (42). Silverstaining was then performed by following the protocol described by Tsai and Frasch to visualize LPS (45).O:54 antigen was also detected by immunoblotting separated samples, which were transferred to anitrocellulose membrane (Protran; GE Healthcare) at 200 mA for 60 min in 25 mM Tris, 150 mM glycine,20% (vol/vol) methanol. The membrane was probed with anti-O:54 serum (Statens Serum Institut,Denmark; diluted 1:1,000), followed by alkaline phosphatase-conjugated goat anti-rabbit secondaryantibody (Cedarlane Laboratories; diluted 1:3,000). OPS was detected using nitroblue tetrazolium and5-bromo-4-chloro-3-indolyl phosphate (Roche Applied Science).

ACKNOWLEDGMENTSWe thank Lauren Hampton for generating pWQ1019.This worked was supported by funding from the Natural Sciences and Engineering

Research Council of Canada (to C.W.). C.W. gratefully acknowledges a Canada ResearchChair Award.

REFERENCES1. Whitfield C, Trent MS. 2014. Biosynthesis and export of bacterial lipo-

polysaccharides. Annu Rev Biochem 83:99 –128. https://doi.org/10.1146/annurev-biochem-060713-035600.

2. Raetz CRH, Whitfield C. 2002. Lipopolysaccharide endotoxins. Annu RevBiochem 71:635–700. https://doi.org/10.1146/annurev.biochem.71.110601.135414.

3. Liu B, Knirel YA, Feng L, Perepelov AV, Senchenkova SN, Reeves PR,Wang L. 2014. Structural diversity in Salmonella O antigens and itsgenetic basis. FEMS Microbiol Rev 38:56 – 89. https://doi.org/10.1111/1574-6976.12034.

4. Greenfield LK, Whitfield C. 2012. Synthesis of lipopolysaccharideO-antigens by ABC transporter-dependent pathways. Carbohydr Res356:12–24. https://doi.org/10.1016/j.carres.2012.02.027.

5. Liu MA, Morris P, Reeves PR. 2019. Wzx flippases exhibiting complexO-unit preferences require a new model for Wzx-substrate interactions.Microbiologyopen 8:e00655. https://doi.org/10.1002/mbo3.655.

6. Liston SD, Mann E, Whitfield C. 2017. Glycolipid substrates for ABCtransporters required for the assembly of bacterial cell-envelope and

cell-surface glycoconjugates. Biochim Biophys Acta Mol Cell Biol Lipids1862:1394 –1403. https://doi.org/10.1016/j.bbalip.2016.10.008.

7. Keenleyside WJ, Whitfield C. 1996. A novel pathway for O-polysaccharidebiosynthesis in Salmonella enterica serovar Borreze. J Biol Chem 271:28581–28592. https://doi.org/10.1074/jbc.271.45.28581.

8. Keenleyside WJ, Perry M, Maclean L, Poppe C, Whitfield C. 1994. Aplasmid-encoded rfbO:54 gene cluster is required for biosynthesis of theO:54 antigen in Salmonella enterica serovar Borreze. Mol Microbiol 11:437– 448. https://doi.org/10.1111/j.1365-2958.1994.tb00325.x.

9. Meier-Dieter U, Barr K, Starman R, Hatch L, Rick PD. 1992. Nucleotidesequence of the Escherichia coli rfe gene involved in the synthesis ofenterobacterial common antigen. J Biol Chem 267:746 –753.

10. Lukose V, Walvoort MTC, Imperiali B. 2017. Bacterial phosphoglycosyltransferases: initiators of glycan biosynthesis at the membrane interface.Glycobiology 27:820 – 823. https://doi.org/10.1093/glycob/cwx064.

11. Zimmer J. 2019. Structural features underlying recognition and translo-cation of extracellular polysaccharides. Interface Focus 9:20180060.https://doi.org/10.1098/rsfs.2018.0060.

O:54 Antigen Synthase Journal of Bacteriology

March 2020 Volume 202 Issue 5 e00625-19 jb.asm.org 13

on April 20, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

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

13. Dorfmueller HC, Ferenbach AT, Borodkin VS, van Aalten D. 2014. Astructural and biochemical model of processive chitin synthesis. J BiolChem 289:23020 –23028. https://doi.org/10.1074/jbc.M114.563353.

14. Saxena IM, Brown RM, Dandekar T. 2001. Structure-function characterizationof cellulose synthase: relationship to other glycosyltransferases. Phytochem-istry 57:1135–1148. https://doi.org/10.1016/s0031-9422(01)00048-6.

15. Omadjela O, Narahari A, Strumillo J, Mélida H, Mazur O, Bulone V,Zimmer J. 2013. BcsA and BcsB form the catalytically active core ofbacterial cellulose synthase sufficient for in vitro cellulose synthesis. ProcNatl Acad Sci U S A 110:17856 –17861. https://doi.org/10.1073/pnas.1314063110.

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

17. Hubbard C, McNamara JT, Azumaya C, Patel MS, Zimmer J. 2012. Thehyaluronan synthase catalyzes the synthesis and membrane transloca-tion of hyaluronan. J Mol Biol 418:21–31. https://doi.org/10.1016/j.jmb.2012.01.053.

18. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg M. 2015. The Phyre2web portal for protein modeling, prediction and analysis. Nat Protoc10:845– 858. https://doi.org/10.1038/nprot.2015.053.

19. Heldermon C, DeAngelis PL, Weigel PH. 2001. Topological organizationof the hyaluronan synthase from Streptococcus pyogenes. J Biol Chem276:2037–2046. https://doi.org/10.1074/jbc.M002276200.

20. Dobson L, Reményi I, Tusnády GE. 2015. CCTOP: a Consensus Con-strained TOPology prediction web server. Nucleic Acids Res 43:W408 –W412. https://doi.org/10.1093/nar/gkv451.

21. Dobson L, Reményi I, Tusnády GE. 2015. The human transmembrane pro-teome. Biol Direct 10:1–18. https://doi.org/10.1186/s13062-015-0061-x.

22. Seol W, Shatkin AJ. 1992. Escherichia coli �-ketoglutarate permease is aconstitutively expressed proton symporter. J Biol Chem 267:6409 – 6413.

23. Zhu X, Zeng Y, Lehrman MA. 1992. Evidence that the hamster tunicamycinresistance gene encodes UDP-GlcNAc:dolichol phosphate N-acetyl-glucosamine-1-phosphate transferase. J Biol Chem 267:8895–8902.

24. Amer A, Valvano M. 2000. The N-terminal region of the Escherichia coliWecA (Rfe) protein, containing three predicted transmembrane helices,is required for function but not for membrane insertion. J Bacteriol182:498 –503. https://doi.org/10.1128/jb.182.2.498-503.2000.

25. Larue K, Ford RC, Willis LM, Whitfield C. 2011. Functional and structuralcharacterization of polysaccharide co-polymerase proteins required forpolymer export in ATP-binding cassette transporter-dependent capsulebiosynthesis pathways. J Biol Chem 286:16658 –16668. https://doi.org/10.1074/jbc.M111.228221.

26. Culham DE, Hillar A, Henderson J, Ly A, Vernikovska YI, Racher KI, Boggs JM,Wood JM. 2003. Creation of a fully functional cysteine-less variant of osmo-sensor and proton-osmoprotectant symporter ProP from Escherichia coliand its application to assess the transporter’s membrane orientation. Bio-chemistry 42:11815–11823. https://doi.org/10.1021/bi034939j.

27. Saxena IM, Brown RM. 1997. Identification of cellulose synthase(s) inhigher plants: sequence analysis of processive �-glycosyltransferaseswith the common motif “D, D, D35Q(R,Q)XRW.” Cellulose 4:33– 49.https://doi.org/10.1023/A:1018411101036.

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

29. Mann E, Mallette E, Clarke BR, Kimber MS, Whitfield C. 2016. The Kleb-siella pneumoniae O12 ATP-binding cassette (ABC) transporter recog-nizes the terminal residue of its O-antigen polysaccharide substrate. JBiol Chem 291:9748 –9761. https://doi.org/10.1074/jbc.M116.719344.

30. Jorgenson MA, Young KD. 2016. Interrupting biosynthesis of O antigenor the lipopolysaccharide core produces morphological defects in Esch-erichia coli by sequestering undecaprenyl phosphate. J Bacteriol 198:3070 –3079. https://doi.org/10.1128/JB.00550-16.

31. Popoff MY, Le Minor L. 1985. Expression of antigenic factor O:54 isassociated with the presence of a plasmid in Salmonella. Ann L’InstitutPasteur Microbiol 136:169–179. https://doi.org/10.1016/S0769-2609(85)80042-9.

32. Boyd AC, Archer JA, Sherratt DJ. 1989. Characterization of the ColE1mobilization region and its protein products. Mol Gen Genet 217:488 – 498. https://doi.org/10.1007/bf02464922.

33. Varsaki A, Lucas M, Afendra AS, Drainas C, De La Cruz F. 2003. Geneticand biochemical characterization of MbeA, the relaxase involved inplasmid ColE1 conjugative mobilization. Mol Microbiol 48:481– 493.https://doi.org/10.1046/j.1365-2958.2003.03441.x.

34. Le Minor L, Rohde R, Charié-Marsaines C, Coynault C. 1973. Nouvellesobservations sur les parentés antigéniques de certains sérotypes deSalmonella du groupe O:54 avec d’autres groupes O du schémadeKauffman-White. Ann l’Institut Pasteur Microbiol 124:451– 461.

35. Le Minor L, Rohde R, Charié-Marsaines C, Coynault C. 1971. Étude sur lesrapports antigéniques entre le groupe O:54 et d’autres groupes O deSalmonella. Ann L’Institut Pasteur Microbiol 121:447– 463.

36. Karousou E, Kamiryo M, Skandalis SS, Ruusala A, Asteriou T, Passi A,Yamashita H, Hellman U, Heldin C-H, Heldin P. 2010. The activity ofhyaluronan synthase 2 is regulated by dimerization and ubiquitination.J Biol Chem 285:23647–23654. https://doi.org/10.1074/jbc.M110.127050.

37. Weigel PH, DeAngelis PL. 2007. Hyaluronan synthases: a decade-plus ofnovel glycosyltransferases. J Biol Chem 282:36777–36781. https://doi.org/10.1074/jbc.R700036200.

38. Kuk ACY, Hao A, Guan Z, Lee S-Y. 2019. Visualizing conformation tran-sitions of the lipid II flippase MurJ. Nat Commun 10:1736. https://doi.org/10.1038/s41467-019-09658-0.

39. Caffalette CA, Corey RA, Sansom MSP, Stansfeld PJ, Zimmer J. 2019. A lipidgating mechanism for the channel-forming O antigen ABC transporter. NatCommun 10:824. https://doi.org/10.1038/s41467-019-08646-8.

40. Bi Y, Mann E, Whitfield C, Zimmer J. 2018. Architecture of a channel-forming O-antigen polysaccharide ABC transporter. Nature 553:361–365.https://doi.org/10.1038/nature25190.

41. Perez C, Gerber S, Boilevin J, Bucher M, Darbre T, Aebi M, Reymond J-L,Locher KP. 2015. Structure and mechanism of an active lipid-linkedoligosaccharide flippase. Nature 524:433– 438. https://doi.org/10.1038/nature14953.

42. Laemmli UK. 1970. Cleavage of structural proteins during the assemblyof the head of bacteriophage T4. Nature 227:680 – 685. https://doi.org/10.1038/227680a0.

43. Clarke BR, Cuthbertson L, Whitfield C. 2004. Nonreducing terminal mod-ifications determine the chain length of polymannose O antigens ofEscherichia coli and couple chain termination to polymer export via anATP-binding cassette transporter. J Biol Chem 279:35709 –35718. https://doi.org/10.1074/jbc.M404738200.

44. Hitchcock PJ, Brown TM. 1983. Morphological heterogeneity amongSalmonella lipopolysaccharide chemotypes in silver-stained polyacryl-amide gels. J Bacteriol 154:269 –277.

45. Tsai CM, Frasch CE. 1982. A sensitive silver stain for detecting lipopoly-saccharides in polyacrylamide gels. Anal Biochem 119:115–119. https://doi.org/10.1016/0003-2697(82)90673-x.

46. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWil-liam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ,Higgins DG. 2007. Clustal W and Clustal X version 2.0. Bioinformatics23:2947–2948. https://doi.org/10.1093/bioinformatics/btm404.

47. Robert X, Gouet P. 2014. Deciphering key features in protein structureswith the new ENDscript server. Nucleic Acids Res 42:320 –324. https://doi.org/10.1093/nar/gku316.

48. Omasits U, Ahrens CH, Müller S, Wollscheid B. 2014. Protter: interactiveprotein feature visualization and integration with experimental proteomicdata. Bioinformatics 30:884–886. https://doi.org/10.1093/bioinformatics/btt607.

49. Guzman LLM, Belin D, Carson MJ, Beckwith J. 1995. Tight regulation,modulation, and high-level expression by vectors containing the arabi-nose PBAD promoter. J Bacteriol 177:4121– 4130. https://doi.org/10.1128/jb.177.14.4121-4130.1995.

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