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Originally published In Press as doi:10.1074/jbc.M401366200 on June 23, 2004

J. Biol. Chem., Vol. 279, Issue 35, 36470-36480, August 27, 2004
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Investigation of the Structural Requirements in the Lipopolysaccharide Core Acceptor for Ligation of O Antigens in the Genus Salmonella

WAAL "LIGASE" IS NOT THE SOLE DETERMINANT OF ACCEPTOR SPECIFICITY*

Natalia A. Kaniuk{ddagger}§, Evgeny Vinogradov¶, and Chris Whitfield{ddagger}||

From the {ddagger}Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1 and Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A 0R6, Canada

Received for publication, February 6, 2004 , and in revised form, June 2, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ligation of O antigen polysaccharide to lipid A-core oligosaccharide is a late step in the formation of the complex glycolipid known as lipopolysaccharide. Although the process has been localized to the periplasmic face of the inner membrane, details of the ligation mechanism have not been resolved. To date, there is only one gene product (WaaL, often referred to as "ligase") known to be required. There exists a requirement for a specific lipid A-core oligosaccharide acceptor structure for ligation activity, and it has been proposed that the WaaL protein imparts this acceptor specificity. Here the structural requirements in the core oligosaccharide acceptor for O antigen ligation are investigated in prototype serovars of Salmonella enterica. Complementation experiments in mutants with defined core oligosaccharide structure indicate that the specificity of the ligation reaction for a particular core oligosaccharide structure is not dependent on the WaaL protein alone. The data provide the first indication of a more complicated recognition process involving additional cellular components.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipopolysaccharide (LPS)1 is a complex glycolipid located in the outer membrane of Gram-negative bacteria. LPS contributes to permeability-barrier properties and stability of the outer membrane (1). LPS molecules can be subdivided into three structurally distinct regions. The hydrophobic lipid A forms the outer leaflet of the outer membrane and is responsible for the endotoxic properties of LPS. The core oligosaccharide (core OS) serves as a link between lipid A and the distal repeating unit polysaccharide known as O antigen. O antigen plays a direct role in virulence, contributing to the resistance of the bacterium to complement-mediated serum killing (2).

LPS O antigens provide important elements in serological distinction among isolates from a genus or species. The genus Salmonella is subdivided into two species, Salmonella enterica (including the subspecies I, II, IIIA, IIIB, IV, VI, and VII) and Salmonella bongori (subspecies V) (3). Assigned to these eight groups are over 2500 recognized serovars (4). Currently, there are ~60 characterized O antigens in S. enterica (5). Some serovars have a broad animal host range. For example, S. enterica serovar Typhimurium, (a member of subspecies I) causes enteric illness in livestock, rodents, birds, and humans (6). Other serovars, such as Arizonae (subspecies IIIA) and Diarizonae (subspecies IIIB), have a more limited range and are most commonly isolated from reptiles (7, 8).

S. enterica serovar Typhimurium has served as a prototype for many studies on LPS structure and biosynthesis. During core OS assembly, glycosyl residues are transferred from nucleotide sugar precursors onto preformed lipid A by the action of glycosyltransferases. These enzymes are membrane-associated proteins found on the cytoplasmic side of the inner membrane (reviewed in Ref. 1). Two Salmonella core OS structures have been reported, represented by the prototypes from subspecies 1 (serovar Typhimurium) and subspecies IIIA (serovar Arizonae IIIA) (9). The structures are highly conserved, with an identical inner core region and the same outer core backbone. They differ in the terminal glycose attached to the GlcII residue, i.e. GlcNAc in serovar Typhimurium and GlcIII in serovar Arizonae IIIA (Fig. 1A). The genetic basis for these differences has been established (10). Once complete, the lipid A-core OS molecule is transferred across the inner membrane by the ABC transporter, MsbA (11, 12). The O antigen is synthesized separately in one of three known pathways that are each believed to lead to the formation of undecaprenol pyrophosphate (und-PP)-linked polymer at the periplasmic face of the inner membrane (1). A ligation reaction transfers the nascent O antigen to lipid A-core prior to translocation of the completed LPS molecule to the outer membrane. The GlcII residue is the site of O antigen ligation in both Salmonella core OS structures (Fig. 1A).



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  		FIG. 1.
Structures and biosynthesis of the LPS outer core OS from S. enterica sv. Typhimurium (subspecies I) and sv. Arizonae (subspecies IIIA). A shows the outer core OS structures and identifies the glycosyltransferases (Waa* proteins) involved in the formation of each linkage. HepII is the first residue of the inner core OS in these structures. All linkages are in the {alpha}-configuration. Glc, D-glucose; Gal, D-galactose, Hep, L-glycero-D-manno-heptose. B shows the organization of the large central waa* operon responsible for outer core OS biosynthesis (adapted from Ref. 10). Serovars Typhimurium and Arizonae IIIA are distinguished by the presence of either waaK or waaH; only one of these genes is present in each serovar, but remnants of the waaK sequence are identifiable in serovar Arizonae IIIA. The SARC 8 (subspecies IIIB) isolate contains complete copies of both waaK and waaH. The critical genes for this study, waaK, waaH (both gray shaded), and waaL (black shaded), are highlighted.

 
The mechanism of ligation of O antigen to lipid A-core OS remains one of the most important unresolved issues in LPS assembly. WaaL proteins (or "ligases") are currently the only protein known to be involved in ligation, but despite the importance of the ligation reaction, little information is available concerning the process or the requirement for additional cellular factors. WaaL proteins in Escherichia coli and Salmonella share highly conserved predicted membrane topology and overall secondary structure (13). waaL mutants produce rough LPS (lacking O antigen repeat units) and accumulate intracellular und-PP-linked O antigen (14). E. coli K12 can ligate structurally diverse O antigens from cloned O antigen gene clusters; these O antigens can be formed by any of the three known biosynthesis pathways (1). It is presumed that the observed promiscuity in ligation substrates reflects recognition by the ligation machinery of the conserved und-PP-linked glycan carrier, rather than the specific O antigen structure. To the extent it has been studied directly, the structure of the lipid A-core acceptor is important in ligation. In the serovar Typhimurium, for example, the activity of the waaK gene product is required for ligation (15). WaaK is the {alpha}-1,2-GlcNAc transferase that forms the terminal GlcNAc-1,2-GlcII motif in the outer core OS. Similar results were obtained with the structurally identical ligation site in the R2 core OS of E. coli (13). However, structural analysis of the core OS of serovar Arizonae IIIA (9) showed that some LPS molecules possessed linked O antigen but lacked the corresponding {alpha}-1,2-linked Glc-1,2-GlcII motif formed by the {alpha}-1,2-Glc transferase, WaaH (10) (Fig. 1A). Therefore, either the ligation machinery in serovar Arizonae IIIA has a different specificity or, as speculated by Olsthoorn et al. (9), some molecules are processed (post-ligation) to remove the substituent. To address these open questions regarding ligase specificity, we have investigated the requirement for a specific core OS acceptor of O antigen for prototype WaaL proteins in Salmonella.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Plasmids, and Growth Conditions—Bacterial strains and plasmids used in this study are listed in Table I. The Salmonella Reference Collection (SARC) contains different strains that exemplify different subspecies that make up the genus Salmonella (16). In this collection, there are two isolates representing each of the subspecies, and these isolates have been categorized based on multilocus enzyme electrophoresis analysis. For example, SARC 1 and SARC 2 represent subspecies I, SARC 5 and SARC 6 represent subspecies IIIA (Arizonae), and SARC 7 and SARC 8 represent subspecies IIIB (Diarizonae). All strains were grown at 37 °C in Luria Bertani (LB) medium, supplemented as appropriate with ampicillin (100 µg/ml), chloramphenicol (30 µg/ml), gentamicin (30 µg/ml), kanamycin (50 µg/ml), streptomycin (100 µg/ml), or tetracycline (100 µg/ml) (Sigma). Gene expression from pBAD-derivative plasmids was induced with 0.02% L-arabinose. For selection in allelic exchange experiments with the suicide-delivery vector, pWQ173 (17), bacteria were grown at 45 °C on YEG-Cl media containing 2 g/liter of the analogue p-chlorophenylalanine (17, 18).


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  		TABLE I
Properties of strains and plasmids used in this study

 
DNA Manipulation—DNA fragments cloned in pBAD24, pBAD18, pWQ173, and pRE112 were obtained by PCR amplification, using primers incorporating appropriate restriction sites to facilitate cloning. Reaction conditions were optimized for each primer pair. Chromosomal DNA PCR templates were prepared using the Insta-gene matrix (Bio-Rad), and PwoI polymerase from Roche Diagnostics was used in all PCRs. Transformation with plasmid DNA was done by electroporation (19). All plasmids were maintained in E. coli DH5{alpha}, except pWQ320 and pWQ321 which were maintained in E. coli TOP10. Primers used for construction of pWQ307 (containing waaH) were 5'-GCATGAATTCTGTATGTTCCTTTTCATATG-3' (the EcoRI site incorporated in the primer is underlined) and 5'-CCCAATCTAGACTATATTTATACTGAGACC-3' (XbaI site is underlined). Primers used for construction of pWQ308 and pWQ309 (containing waaK) were 5'-CTGGATTGAATTCATAATGATTAAAAAAT-3' (EcoRI) and 5'-CCTAGGCAGGATCCAATCACTTCTCAAAC-3' (BamHI). Primers used for the construction of pWQ310, pWQ311, pWQ312, and pWQ313 containing cloned waaL genes were 5'-CCTATTGTGAATTCAAGATGCTAACC-3' (EcoRI) and 5'-AAGGTATCTAGACGTTTTTTTATTTATTTC-3' (XbaI). The plasmids pWQ314 and pWQ315 were constructed by digestion of pWQ307 and pWQ308 with NheI and XbaI, and the fragment containing the waaH (pWQ307) or waaK (pWQ308) gene was purified and cloned into pBAD18-Km (20). The waaK gene was also cloned into pBADHisB, which incorporated an N-terminal hexahistidine (His6) tag. This was done by digesting pWQ315 with EcoRI and HindIII and cloning into the multiple cloning site of the pBADHisB vector (Invitrogen). Site-directed mutagenesis of the waaK gene was carried out using the QuikChange site-directed mutagenesis kit from Stratagene. The template for PCR was pWQ315, and the primers used were 5'-TCAGGTTGAAGCAGCATTTTGCATGGTGGCTGTAGCAGCGATGGCAGCAGG-3' and 5'-TGCCATCGCTGCTACAGCCACCATGCAAAATGCTGCTTCAACCTGAGATGGC-3'. The various constructs were verified by sequencing the complete open reading frame. From here, the resulting plasmid was digested with EcoRI and HindIII, and the fragment carrying waaK was cloned into the pBADHisB vector, adding an N-terminal His6 tag to the gene product. Plasmid pWQ322 was constructed by using the forward primer used to construct pWQ310 (see above) and the primer 5'-TGATCTCTAGATTAGTGGTGGTGGTGGTGGTGTTTATTTCTTAGCGCCAGCAGAAAACCGGTAATG-3' (XbaI) thus incorporating a C-terminal His6 tag in the waaLSARC1 gene product. Plasmid pWQ310 was used as template to amplify the waaL gene, and the fragment was digested with EcoRI and XbaI to facilitate cloning in pBAD24.

In Vitro Mutagenesis and Gene Replacement—The cloned SARC 5, SARC 6, and CWG620 waaH genes were mutated by insertion of a gentamicin resistance cassette (aacC1) within the open reading frame. When cloned in the same transcriptional orientation as the target gene, the aacC1 cassette is nonpolar. Briefly, the waaH gene was cloned into the pBAD24 expression vector (pWQ307) and subsequently digested with BbsI, a restriction enzyme that cuts approximately in the middle of the waaH gene. The resulting sticky ends were filled in by using the Klenow fragment (Invitrogen). A SmaI fragment (containing the aacC1 gentamicin-resistance cassette) from the pUCGm plasmid was ligated to the linearized pWQ307. The resulting plasmid was digested with BamHI and XbaI, and the waaH::aacC1 fragment was isolated and cloned into the temperature-sensitive suicide-delivery vector, pWQ173 (17). The pWQ173-derived knockout construct containing waaH::aacC1 was transformed into SARC 5, SARC 6, and CWG620. The strains were grown at the permissive temperature (30 °C) overnight. Cells were then collected, washed with saline, and plated on the selective media YEG-Cl at 45 °C (18). Colonies were screened for chloramphenicol sensitivity and gentamicin resistance to select allelic exchange mutants. Chromosomal insertions were confirmed by PCR and sequencing.

Chromosomal waaK mutations were constructed in SARC 8 (CWG619) and SL3749 (CWG621) by using the plasmid pWQ306, a sucrose-sensitive pRE112 derivative (21). Plasmid pWQ306 carries an ~700-bp fragment from the middle of the SARC 8 waaK gene (the waaK gene in SARC 8 is 95% homologous to the waaK gene in serovar Typhimurium). Plasmid pWQ306 was transferred by conjugation from E. coli SM10{lambda}pir into streptomycin-resistant derivatives of SARC 8 and SL3749. Chromosomal insertions resulted from a single crossover event and the integration of the plasmid through homologous recombination, mediated by the internal gene fragment. The mutants were selected on media containing streptomycin and chloramphenicol. A waaL chromosomal mutation also was made in SARC 6 (CWG620), using the pRE112 derivative pWQ305 and the method above. Briefly, a fragment of ~500 bp from the middle of the SARC 6 waaL gene was amplified by PCR and cloned into pRE112. Plasmid pWQ305 was transferred by conjugation from E. coli SM10{lambda}pir into a streptomycin-resistant strain of SARC 6. A chromosomal insertion event was selected by plating on streptomycin and chloramphenicol. All mutations were confirmed by sequence analysis of a PCR product spanning the insertion site.

LPS Analysis by PAGE and Western Immunoblotting—Small scale LPS preparations were made from SDS-proteinase K whole cell lysates by the method of Hitchcock and Brown (22). LPS was separated on 4–12% gradient NuPAGE gels that were obtained from NOVEX (San Diego, CA). PAGE conditions were those recommended by the manufacturer, and silver staining was performed as described elsewhere (23). For immunoblotting, samples were transferred to nitrocellulose membranes using conventional methods (24). The samples were probed using T6 monoclonal antibody (25). The secondary antibody was a goat anti-mouse alkaline phosphatase conjugate, and the detection system was 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium.

Purification and Structural Analysis of CWG619 Core Oligosaccharide—Large scale LPS preparations were made using the phenol, chloroform, and petroleum ether method outlined elsewhere (26). The LPS backbone oligosaccharides were isolated by N,O-deacylation of 120 mg of LPS in 4 ml of 4 M KOH at 120 °C for 16 h (27). The mixture was cooled and neutralized with 2 M HCl, and the precipitate was removed by centrifugation. The supernatant was desalted by gel chromatography on Sephadex G-50. Individual compounds from the N,O-deacylated LPS were isolated by high performance anion-exchange chromatography on a column (250 x 9 mm) of Carbopac PA1 that was eluted with a linear gradient of 10–80% 1 M sodium acetate in 0.1 M NaOH at a flow rate of 3 ml/min over 60 min. After desalting, oligosaccharides were isolated as single compounds in yields of 2–10 mg. Purified oligosaccharides were then analyzed by mass spectrometry and NMR. Electrospray mass spectroscopy was carried out as described previously (28). NMR spectra were recorded at 25 °C in D2O on a Varian UNITY INOVA 600 instrument using acetone as reference (1H, 2.225 ppm, 13C, 31.45 ppm). Varian standard programs COSY, NOESY (mixing time of 300 ms), TOCSY (spinlock time of 120 ms), HSQC, and gHMBC (evolution delay of 100 ms) were used with digital resolution in F2 dimension <2 Hz/point. Spectra were assigned using the computer program Pronto (29).

SDS-PAGE and Western Immunoblotting of Proteins—Bacteria were lysed by sonication, and unbroken cells and large debris were removed by centrifugation at 20,000 x g for 20 min. Membranes were isolated from the cell-free lysates by centrifugation at 100,000 x g for 60 min and were solubilized in SDS-containing sample buffer (30). In order to visualize the WaaL-His6 protein, it was essential to incubate the protein samples at 45 °C for 30 min. Higher temperatures resulted in no detection of the expressed protein, a phenomenon seen for some other bacterial integral membrane proteins (31–33). Proteins were separated using SDS-PAGE gels containing 12% acrylamide, and the gels were stained with Coomassie Brilliant Blue. For Western immunoblotting, samples were transferred to nitrocellulose membranes, using conventional procedures (24). The anti-His6-antibody (H-3): sc-8036 (Santa Cruz Biotechnology Inc.), was used to detect the His6-WaaK derivatives, and the Qiagen anti-His5 antibody was used to detect WaaL-His6. The second antibody was a goat anti-mouse alkaline phosphatase conjugate, and the detection system used was 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
S. enterica sv. Typhimurium Requires a Terminal GlcNAc-1,2-GlcII Motif in the Core OS for O Antigen Ligation—In S. enterica sv. Typhimurium and E. coli R2, mutation of the waaK gene abolishes ligation of the O antigen (13, 15). The loss of the O antigen may result from the GlcNAc-1,2-GlcII structure being essential for a ligation competent core OS. Alternatively, the WaaK protein itself may be required for essential protein-protein interactions in a functional ligation complex. To resolve this question, site-directed WaaK mutants were constructed, rendering the resulting GlcNAc transferase catalytically inactive without preventing synthesis of the WaaK protein.

The WaaK glycosyltransferase is a member of family 4 in the CAZY glycosyltransferase classification system (afmb.cnrsmrs.fr/CAZY/index.html). Unfortunately, no solved structures are available to provide information regarding catalysis that could serve to guide the site-directed mutagenesis. However, family 4 glycosyltransferases do contain a conserved motif that includes a signature sequence (E(X7)E) shared by a subset of {alpha}-glycosyltransferases (34). Examples with known activity include the outer core OS glycosyltransferase WaaB (UDP-galactose:(glucosyl) LPS {alpha}1,6-galactosyltransferase) (Fig. 1A) in S. enterica (35) and inner core OS {alpha}-heptosyltransferases (WaaC and WaaF) (13). The E(X7)E motif is also found in some {beta}-glycosyltransferases, including WaaX and WaaV involved in biosynthesis of the type R1 and R4 outer core OS in E. coli (36). Although the precise role of the motif is unknown, its conservation suggests an important role in glycosyltransferase structure-function (37). In an attempt to eliminate catalytic activity in WaaK, the two conserved glutamate amino acids 288E (AFCMVAV)E296 were replaced with alanine residues. Plasmid pWQ320 (expressing His6-WaaK from the SARC 1 isolate) and plasmid pWQ321 (expressing the mutant His6-WaaKSARC1 E288/296A) were transformed into Salmonella SL733 (waaK953). Addition of the N-terminal His6 tag to the native WaaK protein did not influence its function in complementation experiments, but His6-WaaKSARC1 E288/296A was unable to restore O antigen ligation (Fig. 2A). To confirm expression of His6-WaaKSARC1 E288/296A, membrane proteins of E. coli TOP10 (pWQ320), TOP10 (pWQ321), and the control TOP10 (pBADHisB) were separated by PAGE and examined by Western blot analysis using anti-His6 antibodies (Fig. 2C). Although WaaK-related protein bands were difficult to visualize in Coomassie Blue-stained gels, despite the use of an expression vector, they were readily detected in the corresponding Western blot. Polypeptides migrating with an apparent molecular mass consistent with that predicted for His6-WaaKSARC1 (48,082 Da) were evident in the membrane fraction (Fig. 2C). These results are consistent with the ligation machinery in S. enterica sv. Typhimurium requiring a terminal GlcNAc-1,2-GlcII motif.



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  		FIG. 2.
O antigen ligation in serovar Typhimurium is absolutely dependent on a GlcNAc-1,2-GlcII motif in the core OS. A shows silver-stained PAGE profiles of LPS from serovar Typhimurium SL733 (waaK) expressing His6-WaaKSARC1 from plasmid pWQ320 and its catalytically inactive derivative, His6-WaaKSARC1 E288/296A. Only the wild type WaaK derivative restores O antigen ligation. B illustrates that the waaK deficiency cannot be overcome by expression of the waaH gene product from SARC 5. Note the altered PAGE migration of the lipid A-core modified by WaaH, consistent with the predicted addition of a single Glc residue. C shows expression of the WaaK protein derivatives. The Coomassie Brilliant Blue-stained SDS-PAGE (left) shows expression of the wild type His6-WaaK protein (pWQ320) and the mutated version His6-WaaKE288/296A (pWQ321) in membrane fractions of E. coli TOP10. The corresponding Western immunoblot (right) shows single bands of each WaaK derivative located at the predicted molecular mass of 48,082 Da in membrane fractions.

 
To investigate the possibility that another glycose substitution could functionally replace the terminal GlcNAc residue, plasmid pWQ307 expressing the functional waaH gene from S. enterica sv. Arizonae IIIA was transformed into SL733. The waaH gene product has been established as the Glc transferase that assembles the GlcIII-1,2-GlcII terminal structure in serovar Arizonae IIIA core OS (10) (Fig. 1A). Expression of waaH in SL733 results in a shift of the migration of the lipid A-core in PAGE, reflecting the expected addition of a single Glc residue (Fig. 2B). However, this core modification did not restore proficiency for the O antigen ligation, indicating that in serovar Typhimurium the ligation of O antigen is dependent on a specific terminal structure (GlcNAc-1,2-GlcII).

S. enterica sv. Arizonae IIIA SARC 6 Does Not Require WaaH Activity for O Antigen Ligation—Given the essential requirement for GlcNAc substitution of GlcII for ligation in serovar Typhimurium, the structural results (9) that indicated only partial substitution of GlcII in serovar Arizonae IIIA LPS molecules carrying O antigen were intriguing. One possible explanation is that WaaH activity is essential for ligation, but its core OS product is subsequently processed in some molecules. To test this hypothesis, a chromosomal waaH::aacC1 mutation (strain CWG618) was constructed in the SARC 6 representative of serovar Arizonae IIIA. As shown in the LPS PAGE profiles in Fig. 3, the mutation in waaH has no effect on ligation of the O antigen. The migration of the lipid A-core OS of CWG618 in PAGE is consistent with a serovar Typhimurium standard lacking substitution at GlcII (data not shown), ruling out any unexpected activity compensating for loss of WaaH. The comigration of the major lipid A-core from SARC 6 and CWG618 indicates that the level of GlcII substitution in the parent is low. The ligation machinery therefore can attach O antigen to GlcII irrespective of the presence or absence of a residue linked at the 2-position of GlcII. Similar experiments could not be performed with the prototype serovar Arizonae IIIA SARC 5 isolate because of an uncharacterized mutation in the O antigen biosynthesis genes that leaves the SARC 5 isolate with rough LPS. However, a SARC 5 (waaH) mutant could ligate a reporter O antigen from Klebsiella pneumoniae when the O antigen biosynthesis locus was supplied on a plasmid (data not shown), indicating that here too the ligation is independent of modification of GlcII. Notably, the sequences of the waa gene clusters from SARC 5 and SARC 6 have the same organization, and the corresponding waaH gene products are highly conserved (83% identity and 90% similarity) (10).



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  		FIG. 3.
Modification of the outer core OS by WaaH is not essential for O antigen ligation in S. enterica sv. Arizonae IIIA (SARC 6). The figure shows silver-stained PAGE profiles of LPS from serovar Arizonae IIIA SARC 6 and CWG618. CWG618 is a waaH mutant of SARC 6, and ligation of O antigen is still evident in this strain.

 
S. enterica Subspecies IIIB Isolate SARC 8 Requires a Terminal GlcNAc for O Antigen Ligation—The SARC 8 isolate (a representative of subspecies IIIB) contains both the waaK and waaH genes in the waa cluster, and sequence data predict that both potentially encode complete and functional proteins (10). However, it is not known if both proteins are expressed or if both enzymes are active. In terms of waa locus structure, SARC 8 is a hybrid of serovar Typhimurium and serovar Arizonae IIIA, but it is unclear why this isolate has retained genes for two glycosyltransferases that potentially compete to add different glycoses to the same site in the acceptor. The waaK gene in SARC 8 and the waaK gene in serovar Typhimurium share 94% identity at the nucleotide level, and this translates into protein sequences with 91% identity (93% similarity). The waaH gene in SARC 8 shares 95% nucleotide sequence similarity with the waaH gene from SARC 5 (serovar Arizonae IIIA), and the two predicted proteins share 93% identity (97% similarity). In order to determine the required core OS structure for ligation in the SARC 8 strain, a waaK chromosomal mutation was constructed (CWG619). The PAGE profile of CWG619 LPS revealed loss of O antigen ligation (Fig. 4A). To confirm that the ligation-deficient phenotype was due only to the waaK mutation, plasmids expressing WaaKSARC8 (pWQ309) and WaaKSARC1 (pWQ308) were transformed into CWG619. The resulting LPS profiles were identical to wild type SARC 8 (Fig. 4A). Plasmid pWQ307 (expressing WaaHSARC5) was also transformed into CWG619, but no O antigen ligation was restored by WaaHSARC5 expression. These results indicate that the terminal GlcNAc-1,2-GlcII motif formed by WaaK is essential for O antigen ligation in the SARC 8 isolate, and WaaH cannot replace WaaK, as seen in serovar Typhimurium.



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  		FIG. 4.
A GlcNAc-1,2-GlcII structural motif is essential for O antigen ligation in S. enterica subsp. IIIB (SARC 8). A shows silver-stained PAGE profiles of LPS from derivatives of S. enterica subspecies IIIB CWG619, a waaK mutant of SARC 8. O antigen ligation is restored by expression of plasmid-encoded copies of either WaaKSARC1 or WaaKSARC8 but not by expression of WaaH. B shows PAGE and Western immunoblotting analysis of LPS samples from SARC 1, SARC 5, SARC 6, SARC 8, the waaK mutant in SARC 8 (CWG619), and CWG619 complemented with waaK genes from SARC 1 (pWQ308) and SARC 8 (pWQ309). The upper frame of B shows the silver-stained PAGE, and the lower frame is the corresponding Western immunoblot probed with monoclonal antibody T6, which recognizes the GlcNAc-1,2-GlcII motif. The basis for the differential antibody reactivity with the core components of O antigen-substituted LPS species from SARC 1 and SARC 8 is unknown but could reflect steric differences influencing exposure of the epitope.

 
To verify endogenous WaaK activity in the SARC 8 isolate, the LPS profiles of SARC 1, SARC 5, SARC 6, and SARC 8 were probed with the T6 monoclonal antibody (Fig. 4B). The T6 epitope has been determined to be the GlcNAc-1,2-GlcII disaccharide of the outer core OS (25, 38, 39). As expected, LPS of SARC 5 and SARC 6, both representatives of subspecies IIIA, did not react with T6. However, SARC 1 and SARC 8 LPS both showed T6 reactivity, affirming presence of the terminal Glc-NAc-1,2-GlcII motif. T6 reactivity in SARC 8 was eliminated by the waaK mutation in CWG619 and was restored by complementation with the waaK gene from either SARC 8 or SARC 1 (Fig. 4B).

These data do not address the possible simultaneous biosynthesis of a core OS modified by WaaH, which would presumably no longer serve as an acceptor for WaaK and, as a result, would not be a suitable ligation acceptor. The structure of the core OS from CWG619 (SARC 8 waaK) was therefore examined with the expectation that elimination of a potentially competing GlcNAc transferase would increase the probability of detecting any products formed by the WaaH Glc transferase. The LPS was N,O-deacylated by treatment with aqueous KOH, and the single major oligosaccharide was isolated, and its structure was determined by NMR. A set of two-dimensional spectra was recorded and fully interpreted (Table II; Fig. 5A). The oligosaccharide contains the characteristic Salmonella core OS backbone with a single side branch 1,6-Gal (residue I) substitution on the first sugar of the outer core OS (GlcI, residue H; Fig. 5B). No substitution was detected on GlcII (residue K; Fig. 5B), and the mass of the oligosaccharide (2244.2 experimental, 2245.7 predicted; data not shown) is entirely consistent with the deduced structure (Fig. 5B). The CWG619 (SARC 8 waaK) core OS therefore lacks the GlcNAc-1,2-GlcII motif as predicted from the waaK defect but also contains no Glc-1,2-GlcII. Although we cannot rule out the possibility of minor fractions (too small for purification and detailed analysis) of SARC 8 LPS containing Glc-1,2-GlcII, there is currently no evidence of any significant WaaH activity in this strain background.


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  		TABLE II
NMR data for the major product of the N,O-deacylated LPS backbone of Salmonella CWG619 (SARC 8 waaK)

 



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  		FIG. 5.
Structural analysis of the major core OS fraction isolated from N,O-deacylated LPS from CWG619 (SARC 8 waaK). A shows the 1H-13C HSQC correlation spectrum of the oligosaccharide, and the established structure is given in B.

 
The WaaL Enzymes in sv. Typhimurium and sv. Arizonae IIIA Are Functionally Interchangeable—It has been hypothesized that differences in waaL sequences of E. coli and Salmonella may reflect the difference in outer core OS structures that serve as acceptors for O antigen (13, 40). Despite their different outer core structural requirements for O antigen ligation, the WaaL protein sequences of serovar Typhimurium (SARC 1), serovar Arizonae IIIA (SARC 6), and subspecies IIIB (SARC 8) share considerable homology (Fig. 6), and their hydrophilicity plots (Kyte-Doolittle) are indistinguishable (data not shown). To investigate whether the minor differences in WaaL proteins could confer the observed acceptor specificities, cloned waaL ligase genes from SARC 1, 5, 6, and 8 were used in complementation studies with waaL chromosomal mutants in both serovar Typhimurium (SL3749 waaL446) and SARC 6 (CWG620).



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  		FIG. 6.
Comparison of the sequences of WaaL proteins from serovar Typhimurium (subspecies I, SARC 1; GenBankTM accession number NP 462613), serovar Arizonae IIIA (subspecies IIIA, SARC 6; GenBankTM accession number AY533856 [GenBank] ),,and subspecies IIIB (SARC 8; GenBankTM accession number AY533857 [GenBank] ). Sequences were aligned by Clustal3 (www.ebi.ac.uk/clustalw/), and the lines below sequences reflect the positions of predicted transmembrane helices identified by TMPred2 (www.ch.embnet.org/software/TMPRED_form.html).

 
Introduction of plasmids pWQ310 (expressing WaaLSARC1), pWQ311 (WaaLSARC5), pWQ312 (expressing WaaLSARC6), and pWQ313 (WaaLSARC8) into SL3749 (serovar Typhimurium waaL) restored the ability to ligate the O antigen to lipid A-core in all cases (Fig. 7A). The positive complementation results with WaaLSARC1 and WaaLSARC8 were anticipated because these ligases require the GlcNAc-1,2-GlcII. The complementation by WaaLSARC5 and WaaLSARC6 indicates that these ligases not only operate independently of the presence of a substituting glycose-attached GlcII but are also apparently unaffected by the nature (Glc or GlcNAc) of the substitution.



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  		FIG. 7.
Function of heterologous WaaL proteins in waaL mutants of serovar Typhimurium (SL3749) and serovar Arizonae IIIA (CWG620). Shown are silver-stained PAGE profiles of LPS that indicate that all of the tested ligases are fully functional in both genetic backgrounds. A shows strain SL3749 (serovar Typhimurium waaL). The waaL genes from SARC 1 (pWQ310), SARC 5 (pWQ311), SARC 6 (pWQ312), and SARC 8 (pWQ313) were transformed into SL3749, and all constructs complemented the waaL mutation and restored O antigen ligation. B shows strain CWG620, the waaL mutant of serovar Arizonae IIIA SARC 6. O antigen was also ligated to lipid A-core when this strain was transformed with plasmids pWQ310, pWQ311, pWQ312, and pWQ313.

 
When the same cloned ligase genes were expressed in the waaL mutant CWG620 SARC 6 (serovar Arizonae IIIA), O antigen ligation was restored (Fig. 7B). The activity of WaaLSARC5 and WaaLSARC6 was expected, because they are unaffected by the substitution status of GlcII. However, the positive complementation by WaaLSARC1 and WaaLSARC8 was entirely unanticipated. The SARC 1 and SARC 8 ligases require GlcNAc-1,2-GlcII in their natural genetic background, but CWG620 contains only remnants of the waaK gene necessary for formation of this structure (10), and the genetic data are entirely consistent with the structure of the complete core OS from serovar Arizonae IIIA (9) (Fig. 1). The waaL complementation results indicate that the recognition of a lipid A-core acceptor for ligation is not simply dependent only on WaaL and points to a much more complex process that must involve additional factors in the genetic background.

Further Evidence for a Multifactorial Core OS Recognition Process in Ligation by Reconstruction of Hybrid Core OS Structures in sv. Typhimurium and sv. Arizonae IIIA—In order to investigate the contribution of genetic background to the core OS acceptor specificity for ligation, CWG621 (a waaK mutant of SL3749 waaL446) was constructed. The lack of WaaK-mediated GlcNAc substitution of GlcII in CWG621 was confirmed by separately introducing plasmids pWQ314 (expressing WaaHSARC5) and pWQ315 (WaaKSARC1). Whereas the transformants were unable to ligate O antigen because of the waaL defect, both showed lipid A-core molecules with reduced mobility in PAGE, reflecting the addition of GlcIII or GlcNAc, respectively, to the GlcII residue (data not shown). To determine the impact of these changes on WaaL activity, plasmids pWQ310 (expressing WaaLSARC1), pWQ311 (WaaLSARC5), pWQ312 (expressing WaaLSARC6), and pWQ313 (WaaLSARC8) were introduced. In the presence of WaaKSARC1 (Fig. 8C), all of the WaaL proteins were active. In contrast, WaaHSARC5 expression was unable to restore detectable ligation activity for any of the WaaL proteins (Fig. 8B). To ensure proper expression of WaaL was occurring in the double mutant (CWG621), independent of the presence or absence of waaK and waaH, the waaL gene product from SARC 1 was expressed as a derivative containing a C-terminal His6 tag (plasmid pWQ322) to facilitate its detection in Western immunoblots. The presence of the His6 tag did not influence ligation activity of the protein, and the LPS of CWG621 showed identical PAGE profiles when expressing either WaaL or WaaL-His6 (data not shown). An immunoreactive band with an apparent molecular mass of ~47 kDa was present in all three transformants (Fig. 9), reflecting the expression of WaaL-His6 whose predicted molecular mass is 46,854 Da.



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  		FIG. 8.
Function of WaaL proteins expressed in strains with specific core OS structures. The figure shows silver-stained PAGE profiles of LPS from S. enterica sv. Typhimurium CWG621 (waaK waaL). A shows the inability of any of the WaaL proteins to function in the absence of the waaK gene product. B shows the same mutants expressing plasmid-encoded WaaH and demonstrates that WaaH cannot functionally replace WaaK to restore O antigen ligation. C shows the ability of WaaK to restore ligation capacity in the presence of each of the WaaL proteins.

 



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  		FIG. 9.
Expression of the SARC 1 WaaL-His6 protein derivatives in membrane fractions. A shows a Coomassie Brilliant Blue-stained SDS-PAGE of membrane proteins from CWG621 (serovar Typhimurium waaK waaL) expressing WaaL-His6 from SARC 1 (pWQ322). WaaL-His6 was detectable in this background in the presence or absence of WaaK (pWQ315) and WaaH (pWQ314). CWG621 containing pBAD24 vector provides the negative control. B shows the same samples in a Western immunoblot probed with a monoclonal antibody recognizing the His6 tag. The antibody recognizes a 47-kDa protein species, and the predicted size of WaaL-His6 is 46,854 Da.

 
In a corresponding experimental series, a waaH mutation was constructed in CWG620 (SARC 6 waaL). The LPS profile of the resulting double mutant (CWG622) showed no O antigen ligation (Fig. 10). All of the WaaL proteins were functional in this background. This includes WaaLSARC1 and WaaLSARC8 which, in their natural backgrounds, act only with core OS structures containing GlcNAc-1,2-GlcII.



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  		FIG. 10.
Function of heterologous WaaL proteins in Salmonella CWG622 (serovar Arizonae IIIA, SARC 6 waaH waaL) is not dependent on WaaH activity. The figure shows silver-stained PAGE profiles of LPS from the mutant transformed with plasmids encoding each ligase. All restored ligation activity.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The molecular process of O antigen ligation has not yet been elucidated, and the only enzyme currently known to be required for the process is WaaL. In the prototype system, S. enterica sv. Typhimurium, the site of ligation has been established as the periplasmic face of the inner membrane, and the substrate is und-P-P-linked glycan (41). Because WaaL proteins from Salmonella and other bacteria can function with diverse O antigens (1), specificity is apparently confined to recognition of the core OS acceptor. However, because assays with purified substrates and acceptors are not available for the ligase enzyme, and purification of this integral membrane protein is not practical, it has been impossible to assess the exact role WaaL plays in either O antigen ligation or acceptor specificity. The experiments reported here address the role of WaaL in acceptor specificity by using an in vivo approach in which core OS structure is modulated by the mutation or expression of glycosyltransferase genes of established function. The use of varied genetic backgrounds and possible differences in the extent of expression of cloned genes can influence LPS biosynthesis activities and the subtle details of LPS structure. This rules out any interpretation of quantitative differences in ligation efficiency but does not preclude conclusions about plus or minus results for ligation competence.

Some lipid A-core acceptors require specific glycose residues for O antigen ligation (13, 15), and the ligase enzyme has been proposed to play a part in recognizing those residues (13). It is clear from these studies using core OSs from serovar Typhimurium and serovar Arizonae IIIA with defined ligation sites that the WaaL ligases are in fact functionally interchangeable and are not themselves responsible for discrimination between acceptors. This explains the highly conserved WaaL sequences in strains where the structural constraints in the core OS acceptors differ. Although the data still support a central role for WaaL in ligation, it also clearly points to a more complex acceptor-recognition process requiring additional strain-specific factors.

The additional components in a more sophisticated ligation specificity could potentially involve components (and, potentially, additional protein-protein interactions) from the O antigen biosynthesis system. It has been speculated that the WaaL enzyme in E. coli and Salmonella functions as part of a complex that may involve precise interactions with O antigen intermediates on an und-PP carrier and specific lipid A-core acceptor. Feldman et al. (42) have suggested an O antigen processing model for the Wzy-dependent O antigen biosynthesis pathway (e.g. serovar Typhimurium), where a complex consisting of Wzx (O antigen translocase), Wzy (O antigen polymerase), and WaaL are all involved in recognizing the und-PP-linked sugars. The O antigen of serovar Typhimurium has been a critical model for the Wzy-dependent pathway (1). The serotype O62 antigen found in the prototype serovar Arizonae IIIA isolate has a branched hexasaccharide structure (9, 43), indicative of a Wzy-dependent pathway (44). Thus differences in the acceptor specificity of the respective WaaL proteins cannot be explained by interactions with fundamentally different O antigen assembly mechanisms. This is consistent with the finding that O antigens formed by any of the three known biosynthesis pathways (with different biosynthetic components) can be expressed in the same heterologous host with a single waaL gene (reviewed in Refs. 1 and 45). However, one element that may differ in the assembly of the Salmonella O antigens is the identity of the initiating sugar (9, 46). In serovar Typhimurium, this is a Gal residue, and it is added to the und-PP-linked O antigen intermediate by the Gal-1-P transferase, WbaP (47). In serovar Arizonae IIIA, the initial residue is GlcNAc and must be added by a different enzyme, most likely the well characterized GlcNAc-1-P transferase known as WecA (1). WbaP and WecA represent the only known O antigen initiation enzymes in the Enterobacteriaceae. Although beyond the scope of the current work, a possible recognition between WaaL and initiating transferases/initiating sugars represents a logical question for subsequent study.

Wzz is another component of the Wzy-dependent O antigen biosynthesis mechanism thought to be complexed with WaaL. The Wzz protein is responsible for the strain-dependent modal distribution of O antigen chain lengths reflected in the banding pattern LPS forms on polyacrylamide gels (48, 49). It is hypothesized that Wzz may interact with WaaL, Wzy, and und-PP-linked sugars to form a complex, and specific ratios of each protein determine O antigen chain elongation or termination (50, 51). However, there is currently no conclusive evidence of protein-protein interactions involving WaaL. The PAGE profiles of the O antigen-substituted LPS molecules in the different isolates reflect subtle serotype-specific features in O antigen biosynthesis systems, as well as the structure of the O antigens. These profiles were not influenced by expression of heterologous waaL variants from different serovars.

The core OS assembly and ligation situation in isolates such as SARC 8 was particularly intriguing because the waa sequence data suggested that both WaaK and WaaH might be expressed (10). Only WaaK generates a GlcNAc-1,2-GlcII structure that is competent for ligation. It is clear from the work reported here that SARC 8 ligation requires GlcNAc-1,2-GlcII, and therefore any SARC 8 core OS molecules that are modified by WaaH activity would no longer provide viable acceptors for O antigen ligation. Furthermore, LPS analyses performed with a SARC 8 waaK mutant showed no evidence of WaaH activity. Because waaK and waaH are located in the same operon (10, 52), there is no obvious way to regulate differentially these genes at the transcriptional level, and there is currently no precedent for post-translational regulation of these enzymes. It is therefore likely that the sequence variations distinguishing SARC 5 and SARC 8 WaaH proteins (93% identical; 97% similar) lead to an inactive WaaHSARC8. Unfortunately, there is no detailed structure-function information for this family of glycosyltransferases to facilitate interpretation of the sequence differences. The importance of smooth LPS in the biology of Salmonella (and the critical role of WaaK-modified core OS for ligation competence in some isolates) may provide the driving force for selection of the waaH deletions and frameshift mutations frequently identified in isolates that also have functional waaK genes (10). These observations underscore the importance of the ligation process in bacterial survival within the host and reinforce the possibility that this complex reaction could be exploited for novel therapeutic approaches.


   FOOTNOTES
 
* This work was supported in part through funding by the Canadian Bacterial Disease Network (to C. W.) and the National Sciences and Engineering Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of a National Sciences and Engineering Research Council PGS-B graduate scholarship. Back

|| To whom correspondence should be addressed. Tel.: 519-824-4120 (ext. 53361); Fax: 519-837-1802; E-mail: cwhitfie{at}uoguelph.ca.

1 The abbreviations used are: LPS, lipopolysaccharide; core OS, core oligosaccharide; und-PP, undecaprenol pyrophosphate. Back


   ACKNOWLEDGMENTS
 
K. E. Sanderson (University of Calgary) generously provided many of the bacterial strains used in this work. The T6 monoclonal antibody was provided by Dr. R. Tsang (Health Canada, Winnipeg, Manitoba, Canada).



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Raetz, C. R. H., and Whitfield, C. (2002) Annu. Rev. Biochem. 71, 635–700[CrossRef][Medline] [Order article via Infotrieve]
  2. Joiner, K. A. (1985) Curr. Top. Microbiol. Immunol. 121, 99–133[Medline] [Order article via Infotrieve]
  3. Brenner, F. W., Villar, R. G., Angulo, F. J., Tauxe, R., and Swaminathan, B. (2000) J. Clin. Microbiol. 38, 2465–2467[Free Full Text]
  4. Popoff, M. Y., Bockemuhl, J., and Gheesling, L. L. (2003) Res. Microbiol. 154, 173–174[Medline] [Order article via Infotrieve]
  5. Reeves, P. R. (1993) Trends Genet. 9, 17–22[CrossRef][Medline] [Order article via Infotrieve]
  6. Rabsch, W., Andrews, H. L., Kingsley, R. A., Prager, R., Tschape, H., Adams, L. G., and Baumler, A. J. (2002) Infect. Immun. 70, 2249–2255[Free Full Text]
  7. Kingsley, R. A., and Baumler, A. J. (2000) Mol. Microbiol. 36, 1006–1014[CrossRef][Medline] [Order article via Infotrieve]
  8. Geue, L., and Loschner, U. (2002) Vet. Microbiol. 84, 79–91[CrossRef][Medline] [Order article via Infotrieve]
  9. Olsthoorn, M. M. A., Petersen, B. O., Schlecht, S., Haverkamp, J., Bock, K., Thomas-Oates, J. E., and Holst, O. (1998) J. Biol. Chem. 273, 3817–3829[Abstract/Free Full Text]
  10. Kaniuk, N. A., Monteiro, M. A., Parker, C. T., and Whitfield, C. (2002) Mol. Microbiol. 46, 1305–1318[CrossRef][Medline] [Order article via Infotrieve]
  11. Zhou, Z., White, K. A., Polissi, A., Georgopoulos, C., and Raetz, C. R. (1998) J. Biol. Chem. 273, 12466–12475[Abstract/Free Full Text]
  12. Chang, G., and Roth, C. B. (2001) Science 293, 1793–1800[Abstract/Free Full Text]
  13. Heinrichs, D. E., Monteiro, M. A., Perry, M. B., and Whitfield, C. (1998) J. Biol. Chem. 273, 8849–8859[Abstract/Free Full Text]
  14. Mulford, C. A., and Osborn, M. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1159–1163[Abstract/Free Full Text]
  15. MacLachlan, P. R., Kadam, S. K., and Sanderson, K. E. (1991) J. Bacteriol. 173, 7151–7163[Abstract/Free Full Text]
  16. Boyd, E. F., Wang, F. S., Whittam, T. S., and Selander, R. K. (1996) Appl. Environ. Microbiol. 62, 804–808[Abstract]
  17. Rahn, A., and Whitfield, C. (2003) Mol. Microbiol. 47, 1045–1060[CrossRef][Medline] [Order article via Infotrieve]
  18. Kast, P. (1994) Gene (Amst.) 138, 109–114[CrossRef][Medline] [Order article via Infotrieve]
  19. Binotto, J., MacLachlan, P. R., and Sanderson, P. R. (1991) Can. J. Microbiol. 37, 474–477[Medline] [Order article via Infotrieve]
  20. Guzman, L.-M., Belin, D., Carson, M. J., and Beckwith, J. (1995) J. Bacteriol. 177, 4121–4130[Abstract/Free Full Text]
  21. Edwards, R. A., Keller, L. H., and Schifferli, D. M. (1998) Gene (Amst.) 207, 149–157[CrossRef][Medline] [Order article via Infotrieve]
  22. Hitchcock, P. J., and Brown, T. M. (1983) J. Bacteriol. 154, 269–277[Abstract/Free Full Text]
  23. Tsai, G. M., and Frasch, C. E. (1982) Anal. Biochem. 119, 115–119[CrossRef][Medline] [Order article via Infotrieve]
  24. Towbin, M. T., Staehelin, T., and Gordon, G. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350–4354[Abstract/Free Full Text]
  25. Tsang, R. S., Chan, K. H., Chau, P. Y., Wan, K. C., Ng, M. H., and Schlecht, S. (1987) Infect. Immun. 55, 211–216[Abstract/Free Full Text]
  26. Galanos, C., Luderitz, O., and Westphal, O. (1969) Eur. J. Biochem. 9, 245–249[Medline] [Order article via Infotrieve]
  27. Holst, O., Thomas-Oates, J. E., and Brade, H. (1994) Eur. J. Biochem. 222, 183–194[Medline] [Order article via Infotrieve]
  28. Vinogradov, E., Korenevsky, A., and Beveridge, T. J. (2002) Carbohydr. Res. 337, 1285–1289[CrossRef][Medline] [Order article via Infotrieve]
  29. Kjaer, M., Andersen, K. V., and Poulsen, F. M. (1994) Methods Enzymol. 239, 288–307[Medline] [Order article via Infotrieve]
  30. Laemmli, U. K. (1970) Nature 227, 680–685[CrossRef][Medline] [Order article via Infotrieve]
  31. Amer, A. O., and Valvano, M. A. (2000) J. Bacteriol. 182, 498–503[Abstract/Free Full Text]
  32. Dan, N., Middleton, R. B., and Lehrman, M. A. (1996) J. Biol. Chem. 271, 30717–30724[Abstract/Free Full Text]
  33. Seol, W., and Shatkin, A. J. (1993) J. Bacteriol. 175, 565–567[Abstract/Free Full Text]
  34. Geremia, R. A., Petroni, E. A., Ielpi, L., and Henrissat, B. (1996) Biochem. J. 318, 133–138
  35. Kadam, S. K., Rehemtulla, A., and Sanderson, K. E. (1985) J. Bacteriol. 161, 277–284[Abstract/Free Full Text]
  36. Heinrichs, D. E., Yethon, J. A., Amor, P. A., and Whitfield, C. (1998) J. Biol. Chem. 273, 29497–29505[Abstract/Free Full Text]
  37. Kostova, Z., Yan, B. C., Vainauskas, S., Schwartz, R., Menon, A. K., and Orlean, P. (2003) Eur. J. Biochem. 270, 4507–4514[Medline] [Order article via Infotrieve]
  38. Tsang, R. S., Schlecht, S., Aleksic, S., Chan, K. H., and Chau, P. Y. (1991) Res. Microbiol. 142, 521–533[Medline] [Order article via Infotrieve]
  39. Tsang, R. S. W., Schlecht, S., and Mayer, H. (1992) Zentbl. Bakteriol. 276, 330–339
  40. Heinrichs, D. E., Yethon, J. A., and Whitfield, C. (1998) Mol. Microbiol. 30, 221–232[CrossRef][Medline] [Order article via Infotrieve]
  41. Osborn, M. J., Rick, P. D., and Rasmussen, N. S. (1980) J. Biol. Chem. 255, 4246–4251[Abstract/Free Full Text]
  42. Feldman, M. F., Marolda, C. L., Monteiro, M. A., Perry, M. B., Parodi, A. J., and Valvano, M. A. (1999) J. Biol. Chem. 274, 35129–35138[Abstract/Free Full Text]
  43. Vinogradov, E. V., Knirel, Y. A., Kochetkov, N. K., Schlecht, S., and Mayer, H. (1994) Carbohydr. Res. 253, 101–110[CrossRef][Medline] [Order article via Infotrieve]
  44. Whitfield, C. (1995) Trends Microbiol. 3, 178–185[CrossRef][Medline] [Order article via Infotrieve]
  45. Whitfield, C., Amor, P. A., and Köplin, R. (1997) Mol. Microbiol. 23, 629–638[CrossRef][Medline] [Order article via Infotrieve]
  46. Olsthoorn, M. M., Petersen, B. O., Duus, J., Haverkamp, J., Thomas-Oates, J. E., Bock, K., and Holst, O. (2000) Eur. J. Biochem. 267, 2014–2027[Medline] [Order article via Infotrieve]
  47. Wang, L., and Reeves, P. R. (1994) J. Bacteriol. 176, 4348–4356[Abstract/Free Full Text]
  48. Batchelor, R. A., Haraguchi, G. E., Hull, R. A., and Hull, S. I. (1991) J. Bacteriol. 173, 5699–5704[Abstract/Free Full Text]
  49. Bastin, D. A., Stevenson, G., Brown, P. K., Haase, A., and Reeves, P. R. (1993) Mol. Microbiol. 7, 725–734[Medline] [Order article via Infotrieve]
  50. Morona, R., Van Den Bosch, L., and Manning, P. A. (1995) J. Bacteriol. 177, 1059–1068[Abstract/Free Full Text]
  51. Daniels, C., and Morona, R. (1999) Mol. Microbiol. 34, 181–194[CrossRef][Medline] [Order article via Infotrieve]
  52. Roncero, C., and Casadaban, M. J. (1992) J. Bacteriol. 174, 3250–3260[Abstract/Free Full Text]
  53. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  54. Miller, V. L., and Mekalanos, J. J. (1988) J. Bacteriol. 170, 2575–2583[Abstract/Free Full Text]
  55. Schweizer, H. P. (1993) BioTechniques 15, 831–833[Medline] [Order article via Infotrieve]

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