From the Department of Microbiology, University of
Guelph, Guelph, Ontario, Canada N1G 2W1 and ¶ Institute
for Biological Sciences, National Research Council,
Ottawa, Ontario, Canada K1A OR6
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ABSTRACT |
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In Escherichia coli F632, the
14-kilobase pair chromosomal region located between waaC
(formerly rfaC) and waaA (kdtA)
contains genes encoding enzymes required for the synthesis of the type R2 core oligosaccharide portion of lipopolysaccharide. Ten of the 13 open reading frames encode predicted products sharing greater than 90%
total similarity with homologs in E. coli K-12. However, the products of waaK (rfaK) and
waaL (rfaL) each resemble homologs in
Salmonella enterica serovar Typhimurium but
share little similarity with E. coli K-12. The F632 WaaK
and WaaL proteins therefore define differences between the type R2 and
K-12 outer core oligosaccharides of E. coli
lipopolysaccharides. Based on the chemical structure of the core
oligosaccharide of an E. coli F632
waaK::aacC1 mutant and in
vitro glycosyltransferase analyses, waaK encodes
UDP-N-acetylglucosamine:(glucose) lipopolysaccharide
1,2-N-acetylglucosaminyltransferase. The WaaK enzyme
adds a terminal GlcNAc side branch substituent that is crucial for the
recognition of core oligosaccharide acceptor by the O-polysaccharide
ligase, WaaL. Results of complementation analyses of E. coli K-12 and F632 waaL mutants suggest that
structural differences between the WaaL proteins play a role in
recognition of, and interaction with, terminal lipopolysaccharide core
moieties.
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INTRODUCTION |
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Lipopolysaccharides (LPS)1 are major and characteristic components of the outer membrane of Gram-negative bacteria. The hydrophobic lipid component (lipid A) anchors the LPS molecule in the outer membrane. Lipid A is linked to a core oligosaccharide (core OS) of 10-15 sugars; the core OS is often phosphorylated. The resulting basic structure is known as rough or R-LPS. In the Enterobacteriaceae, R-LPS is capped by an O antigen side chain polysaccharide (O-PS) to form LPS molecules termed smooth (or S-LPS). In contrast, some organisms, like Hemophilus influenzae or Neisseria gonorrhoeae, lack O-PS but modify their R-LPS by addition of a few glycosyl residues to produce lipo-oligosaccharide. In the Enterobacteriaceae, the core OS is divided into two structural regions, an inner core containing Kdo and heptose and an outer core region consisting primarily of hexose and acetamido sugars. Whereas the inner core is highly conserved among members of the Enterobacteriaceae, the outer core region exhibits variation in its components and structure. Indeed, although there is only one wild-type core structure currently described in Salmonella spp.2 (Ra core), there are five different core OS structures in Escherichia coli (designated K-12, R1, R2, R3, and R4) which are differentiated based on their outer core OS structures. The structures of the outer core OSs of Salmonella enterica, E. coli K-12, and E. coli R2 are shown in Fig. 1A.
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Lipid A-core and O-PS are formed by independent assembly pathways (1-3). The core OS biosynthesis region of the chromosome (formerly known as the rfa region) contains genes that define unique core OS structures. Many of the genes at this locus code for glycosyltransferases which sequentially elongate the core OS on a lipid A acceptor. The chromosomal core OS biosynthesis region of E. coli K-12 has been entirely sequenced, and the majority of the equivalent region has been completed in S. enterica (Fig. 1B). Most of the known core OS biosynthesis genes in S. enterica have predicted products that are highly similar (greater than 70% total similarity) to E. coli K-12 counterparts. Striking exceptions are WaaK and WaaL, where the similarity is less than 35% (4). The WaaL protein is the only gene product known to be involved in the ligation of pre-assembled O-PS to lipid A-core. This occurs at the periplasmic face of the plasma membrane, prior to translocation of completed S-LPS to the outer membrane (reviewed in 1). WaaL mutants of both E. coli K-12 and S. enterica are unable to "cap" the lipid A-core molecule with an O-PS. The WaaL enzyme of E. coli K-12 has relaxed specificity for the polymer it attaches to lipid A-core since it can effectively ligate a number of "native" E. coli polymers, as well as an ever increasing range of O-PS structures resulting from expression of cloned O-PS-biosynthesis genes in E. coli K-12 (1). From the limited available data, it appears that the S. enterica WaaL protein shows similar relaxed specificity for polymer structure. Ligase enzymes from different bacteria are therefore expected to share a common mechanism of action. Although there is little similarity between the primary sequence of the WaaL homologs of E. coli K-12 and S. enterica, both are integral membrane proteins and have similar hydropathy profiles (4).
Ligation is a crucial step in the assembly of S-LPS. Since the O-PSs of
pathogenic bacteria are usually required for resistance to
complement-mediated killing (5), the ligation step is important for
survival in the host and could potentially be exploited for novel
therapeutic approaches. However, the mechanism of ligation is unknown.
Differences in WaaL sequences of E. coli K-12 and S. enterica most likely reflect the varying structures in the outer
core OSs (see Fig. 1A) which serve as acceptors for O-PS, but the structural requirements for a functional core OS acceptor have
not been addressed in a systematic manner. Attempts to relate structure
and function in WaaL homologs from E. coli K-12 and S. enterica are hampered by differences in both backbone glycan sequence as well as side chain substituents in their respective core
OSs. The E. coli R2 core OS has a backbone identical to
E. coli K-12 but contains a terminal
1,2-GlcpNAc side branch, as is found in S. enterica (Fig. 1A). Analysis of WaaL activity in this
strain therefore allows distinction between structural requirements for
ligation imposed by features of the core OS backbone and terminal side
branch substitutions. The waaK gene of S. enterica has been implicated in the addition of the
1,2-linked
GlcNAc residue (6). Available evidence suggests that this terminal core
OS side branch is important for O-PS ligation activity (7), but the
data are limited by the lack of precisely defined mutations and
individually cloned genes for functional complementation
experiments.
To resolve these ambiguities, the waaK and waaL genes were characterized in E. coli F632, a prototype strain with an E. coli R2 core OS. Structural and biochemical analyses of defined insertions in the E. coli R2 chromosomal genes, together with complementation experiments using single open reading frames and E. coli R2, K-12 and S. enterica core OS acceptors were used to precisely define the effects of the terminal GlcNAc side branch of the E. coli R2 core OS on ligation activity.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and Plasmids-- The bacterial strains and plasmids used in this study are listed in Table I. The R2 prototype strain used in this study, F632, is an O-PS-deficient derivative of E. coli O100, and although it does not produce an O-PS, it does contain a complete core OS (this study and Refs. 8 and 9).
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Media and Growth Conditions-- Bacterial strains were routinely grown in Luria-Bertani (LB) broth (10) at 37 °C, unless otherwise stated. Growth medium was supplemented with ampicillin (100 µg/ml), chloramphenicol (30 µg/ml), gentamicin (15 µg/ml), kanamycin (30 µg/ml), or tetracycline (10 µg/ml) as necessary. L-Arabinose was used at a final concentration of 0.02% for growth and induction of strains containing pBAD18 derivatives.
DNA Methods-- Restriction endonuclease digestion and ligation was performed essentially as described by Sambrook et al. (11). Restriction enzymes were purchased from either Life Technologies, Inc. (Burlington, Ontario), New England Biolabs (Mississauga, Ontario), or Boehringer Mannheim (Laval, Quebec). Plasmids were introduced into E. coli strains by using CaCl2-competent cells (11) or by electroporation using conditions described elsewhere (12) and a Gene Pulser from Bio-Rad (Mississauga, Ontario). Chromosomal DNA isolation was performed using the Qiagen genomic DNA isolation kit, and plasmid DNA was prepared using QIAprep plasmid spin columns (Qiagen Inc., Santa Clarita, CA). Where necessary, DNA fragments were isolated from agarose gels using the Geneclean kit from Bio/Can Scientific (Mississauga, Ontario).
PCR and Sequencing Techniques-- Oligonucleotides were synthesized using a Perkin-Elmer 394 DNA synthesizer, and sequencing was performed using an ABI 377 DNA sequencing apparatus (Perkin-Elmer) at the Guelph Molecular Supercentre (University of Guelph). PCR was performed using a GeneAmp PCR System 2400 from Perkin-Elmer. The "expand high-fidelity enzyme mix" (Boehringer Mannheim) was used as the polymerase enzyme in PCR reactions where products were greater than 5 kb. For product sizes of less than 5 kb, PwoI DNA polymerase (Boehringer Mannheim) was used. PCR amplification of the 14-kb fragment flanked by the waaC and waaA genes was performed as follows: one initial cycle at 94 °C for 1 min; 20 cycles at 94 °C for 15 s and 68 °C for 12 min; 16 auto cycles at 94 °C for 15 s and 68 °C for 12 min, with an auto-extension at 68 °C for 15 s per cycle; a final cycle at 72 °C for 10 min. The oligonucleotide primers were based upon similar regions of sequence between E. coli K-12 and S. enterica in the waaC and waaA genes and are as follows: (i) forward primer, 5'-ACGTTGCCCGCACTCACTGA-3' and (ii) complementary reverse primer, 5'-TTCGGTGGCAGGTAAGGTTC-3'. PCR products were purified using the QIAquick PCR purification kit from Qiagen. To ensure error-free sequencing, the sequence of each of the DNA strands was determined from the product of separate PCR runs. In the rare instances where a mismatch in sequence between strands occurred, a small region surrounding the mismatch was reamplified and resequenced.
In Vitro Mutagenesis and Gene Replacement--
The E. coli F632 waaK gene was mutated in vitro by
insertion of a gentamicin-resistance cassette (the aacC1
gene from Tn1696). The cassette was isolated on a 835-bp
SacI fragment from plasmid pUCGM, blunt-ended with T4 DNA
polymerase, and inserted into the unique EcoRV site in the
waaK coding region of plasmid pWQ900 (Fig.
2). The
waaK::aacC1 gene was then recovered on
a 1.8-kb SacI fragment which was inserted into the
SmaI site of the suicide delivery vector pCVD442 (13).
Plasmid pCVD442 carrying the
waaK::aacC1 gene was maintained in the
mobilizing strain SM10pir and transferred to E. coli F632
by conjugation. E. coli F632
waaK::aacC1 was obtained by sucrose
selection in the absence of NaCl at 37 °C. Resulting colonies were
tested for gentamicin resistance and ampicillin sensitivity. The
presence of the waaK::aacC1 mutation was confirmed by Southern hybridization and PCR, followed by sequencing the junction
sites of waaK-aacC1 on the amplified fragment.
The E. coli F632 waaL gene was mutated in
vitro by replacement of an internal 1.2-kb
HpaI-MfeI fragment from the waaL
coding region of pWQ900 (Fig. 2) with the gentamicin resistance
cassette present on a SmaI fragment from plasmid pUCGM. This
essentially removes the complete waaL coding region. The
waaL::aacC1 gene was recovered on a
2.5-kb BstEII-BstXI fragment which was
blunt-ended with Klenow enzyme and T4 DNA polymerase and inserted into
the unique EcoRV site of the suicide delivery vector pMAK705
(14). E. coli F632 was transformed with pMAK705 carrying the
waaL::aacC1 gene, and chromosomal gene
replacement was carried out by a procedure described elsewhere (15).
The presence of the waaL::aacC1
mutation in E. coli CWG302 was confirmed by sequencing the
junctions of waaL-aacC1 in an amplified PCR
fragment.
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Computer Analysis-- Sequence data were edited and analyzed using AssemblyLIGN and MacVector software (International Biotechniques Inc., New Haven, CT). Hydrophilicity plots of predicted amino acid sequences were performed using the MacVector software package and the method of Kyte-Doolittle, with a hydrophilicity window of 7 and an amphiphilicity window of 11. Homology searches of nucleotide and amino acid sequences in the National Center for Biotechnology Information data bases were done with the BLAST (basic local alignment search tool) server analysis program (16). Pairwise nucleotide sequence alignments and percentage identity scores were obtained using the NALIGN program of the PC/GENE software package (IntelliGenetics Inc, Mountain View, CA) with an open gap cost of 25 and a unit gap cost of 5. Pairwise protein alignments and percentage identity and similarity scores were obtained using the PALIGN program of PC/GENE with an open gap cost of 5 and a unit gap cost of 5. Multiple sequence alignments were performed using CLUSTALX (version 1.62b). Protein secondary structure was predicted using the GARNIER and GGBSM programs present in the PC/GENE software package and by hydrophobic cluster analysis (HCA) using the HCA plot program (Doriane Informatique, Le Chesnay, France).
Lipopolysaccharide Analysis by SDS-PAGE-- Small scale LPS preparations were made from SDS-proteinase K whole cell lysates by the method of Hitchcock and Brown (17). Large scale preparations used the hot phenol/water extraction as described elsewhere (18). LPS was separated on 10-20% gradient SDS-Tricine polyacrylamide gels that were obtained from Novex (San Diego, CA). Polyacrylamide gel electrophoresis (PAGE) conditions were those recommended by the manufacturer. Silver staining (19) and Western immunoblotting procedures have been described (15), as has production of polyclonal rabbit anti-D-galactan I serum (20). Throughout this study, LPS from an equivalent number of cells was loaded in each gel lane.
Generation of Core Oligosaccharides-- Water-insoluble LPSs were obtained by hot water/phenol extraction of E. coli F632 and CWG300 cells (18) and treated with 1% acetic acid at 100 °C to cleave the acid-labile ketosidic linkage between the core and lipid A. The water-insoluble lipid A was isolated from the hydrolysate as a pellet by centrifugation (5000 × g, 5 °C). The supernatant containing core OS was purified through passage on a column of Bio-Gel P-2 (1 m × 1 cm) with water as eluent. The lipid A-free core OS eluted after the void volume and was detected by the phenol/sulfuric acid assay (21).
Sugar Composition and Methylation Linkage Analyses-- Sugar composition analysis was performed by the alditol acetate method (22). Hydrolysis of glycosidic bonds was achieved by using 4 M trifluoroacetic acid at 100 °C for 4 h. The samples were then reduced in H2O with NaBD4 and acetylated with acetic anhydride using residual sodium acetate as the catalyst. Characterization of the alditol acetate derivatives was performed by gas-liquid chromatography-mass spectrometry using a Hewlett-Packard chromatograph equipped with a 30-m DB-17 capillary column (210 °C (30 min) to 240 °C at 2 °C/min). Mass spectrometry in the electron impact mode was recorded using a Varian Saturn II mass spectrometer. Enantiomeric configurations of the individual sugars were determined by the formation of the respective 2-(S)- and 2-(R)-butyl glycosides (23). Methylation linkage analysis was carried out by the Ciucanu and Kerek (NaOH/Me2SO-methyl iodide) procedure (24). The permethylated alditol acetate derivatives were fully characterized by gas-liquid chromatography-mass spectrometry in the electron impact mode using a column of DB-17 operated isothermally at 190 °C for 60 min.
Fast Atom Bombardment-Mass Spectrometry-- A fraction (25%) of the methylated sample was used for positive ion fast atom bombardment-mass spectrometry. This was performed by using a Jeol JMS-AX505H mass spectrometer with glycerol/thioglycerol as the matrix and a tip voltage of 3 kV.
Nuclear Magnetic Resonance (NMR)
Spectroscopy--
1H and 13C NMR spectra of
the core OSs were recorded on a Bruker AMX 500 spectrometer at 300 K
using standard Bruker software. Prior to performing the NMR
experiments, the samples were lyophilized three times with
D2O (99.9%). The internal references for 1H
and 13C NMR were the HOD peak (H 4.786) and
acetone (
C 31.4), respectively.
Glycosyltransferase Assays-- Incorporation of radiolabel from UDP-[14C]GlcNAc into LPS was used as a measurement of glycosyltransferase activity. Membranes were prepared as described previously (25) from 500 ml of log phase E. coli CWG300 (waaK::aacC1) cells, to provide the acceptor LPS. Membrane-free soluble enzyme extracts of each strain were prepared by collecting ultracentrifugation supernatants from cell-free lysates. Each reaction contained an equal amount of acceptor (20 µg of membrane protein) and an aliquot of soluble enzyme extract (15 µg protein) in a final volume of 0.1 ml. The buffer comprised 50 mM Tris-HCl, 10 mM MgCl2, and 1 mM dithiothreitol. The reaction was started by addition of 0.025 µCi of UDP-[14C]GlcNAc (specific activity 10.2 mCi/mmol; ICN), and the glycosyltransferase assays were performed at 37 °C. To measure incorporation into LPS, aliquots of the reaction mixture were separated by descending paper chromatography. High molecular weight radiolabeled LPS was retained at the origin after descending paper chromatography using ethanol (95%) and 1 M ammonium acetate (7:3) (26). Unincorporated substrate migrates in this system and the origins from the chromatogram were excised and counted in a scintillation counter.
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RESULTS |
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Genetic Determinants for Outer Core OS Biosynthesis in E. coli K-12, R2, and S. enterica-- As a starting point for this study, the sequence of the core OS biosynthesis region of the chromosome in E. coli F632 (R2 core prototype) was determined. In E. coli K-12 and S. enterica, the waaC and waaA genes encode the heptosyltransferase I and the bifunctional Kdo transferase, respectively, for inner core biosynthesis (3). Similarities in the waaC and waaA genes (and gene products) between E. coli K-12 and S. enterica and preliminary Southern hybridization experiments (data not shown) suggested that these genes would likely be conserved in other E. coli core types. This predicted conservation was used to design PCR primers to amplify the region containing the outer core OS biosynthesis genes from E. coli F632. The complete nucleotide sequence of the resulting 14-kb PCR amplification fragment was determined, revealing a general organization typical of those seen in E. coli K-12 and S. enterica (Fig. 1B). The organization and function of the core OS biosynthesis regions in S. enterica and E. coli K-12 have been reviewed previously (2). However, the sequence information for S. enterica was incomplete and, in some regions contained errors, effectively limiting comparisons with the region from E. coli K-12. These problems were resolved by sequencing PCR amplification products spanning gaps in S. enterica sequences and resequencing regions where some ambiguities remained. The structures of the completed regions from the three bacteria are shown in Fig. 1B as are the sequence relationships between the genes and their predicted gene products.
Although the functions of some outer core OS biosynthesis enzymes have been established in biochemical analyses, others are inferred from structures of mutant core OSs resulting from defects in various genes (for reviews see Refs. 2 and 3). The relationships among predicted polypeptides representing the core OS backbone glycosyltransferases in S. enterica, E. coli K-12, and E. coli R2 are consistent with the structures of their respective core OSs. All three core types have a Glcp-
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A New Family of -Glycosyltransferases: the WaaIJ
Family--
WaaI, and to a lesser extent WaaJ, of S. enterica have been established as the HexII and HexIII
glycosyltransferases, respectively, involved in assembly of the outer
core OS portion of the LPS molecule. The HexII and HexIII
glycosyltransferases of E. coli K-12, WaaO, and WaaR have
been identified, although direct biochemical evidence conclusively
identifying their functions is currently limited. These proteins share
four highly conserved regions of primary sequence (labeled I, II, III,
and IV) which are highlighted in Fig. 3. BLASTP searches of the data
bases using the WaaI, -J, -O, or -R proteins identify a number of other
known or putative
-glycosyltransferases in a variety of other
prokaryotes and one protein from a eukaryote (Table III). Secondary
structure predictions using a number of programs within the PC/GENE
software package as well as HCA analysis (35) predict the following:
(i) sequence I is present in an
-helix; (ii) sequence II lies within
a
-strand; (iii) sequence III is present in undetermined secondary
structure; and (iv) sequence IV is part of random coil structure.
Characterization of the E. coli R2 waaK Gene--
The E. coli R2 and S. enterica core OSs both have a side
branch 1,2-linked GlcpNAc substitution on the terminal
Glcp residue (Fig. 1A). A Hepp residue
occupies the same position in E. coli K-12 which would
explain the absence of a waaK homolog in its core OS
biosynthesis gene cluster. S. enterica mutations mapping to
waaK lack GlcNAc in their outer core OS (36), and the
waaK gene has been identified in this organism (7). The
predicted products of the WaaK homologs from E. coli R2 and
S. enterica share 75.3% identity (83.2% total similarity)
(Fig. 1B). The predicted molecular mass of the E. coli R2 WaaK protein is 42.8 kDa based on sequence analysis, and
the cloned R2 waaK coding region (pWQ901) directs synthesis
of a protein with a calculated molecular mass of 43 kDa in
Coomassie-stained SDS-polyacrylamide gels (data not shown). The WaaK
proteins from E. coli F632 and S. enterica have the E(X7)E
-glycosyltransferase motif also
found in WaaG and WaaB. The motif identified in E. coli R2
WaaK is
E288AFCMVAVE296,
identical to that of S. enterica WaaK. Sequence data and
structural similarities in the core OSs are consistent with WaaK
homologs encoding
1,2-linked GlcpNAc transferases.
Unambiguous assignment was achieved by structural and biochemical
analyses of a precisely defined waaK mutant in the R2 core
OS prototype strain, E. coli F632 (see below).
The E. coli R2 waaK Gene Product Encodes a
UDP-N-Acetylglucosamine:(Glucosyl) LPS
1,2-N-Acetylglucosaminyltransferase for Outer Core OS
Assembly--
Insertional inactivation of the R2 waaK
coding region in E. coli F632 gave strain CWG300
(waaK::aacC1). In SDS/Tricine-PAGE, the
LPS lipid A-core band of CWG300 migrates slightly faster than that of
the wild-type parent (Fig. 4), reflecting
a core truncation. Introduction of plasmid pWQ901 (which carries the R2
waaK gene) into CWG300 yields LPS with the same migration as
the wild-type R2 strain (F632), confirming that the defect results only
from the expected single mutation.
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The Role of WaaK in Ligation of O-PS-- An S. enterica mutant with the waaK953 allele (SL733) does not produce any detectable S-LPS in silver-stained SDS-PAGE (7). The same result is shown in Fig. 6A, lane 2. Introduction of a plasmid containing the complete waaK coding region from S. enterica was able to complement the waaK953 phenotype by restoring synthesis of O-PS in this strain (7). As might be expected given the similarities in core OS structures and WaaK homolog sequences, the R2 waaK gene carried on pWQ901 could functionally replace the waaK gene of S. enterica SL733, leading to restoration of S-LPS formation (Fig. 6A, lane 3).
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Characterization of the E. coli F632 WaaL Gene Product-- The waaL homolog in E. coli F632 was initially identified by its occupation of a similar position within the core OS biosynthesis cluster as those in E. coli K-12 and S. enterica. The predicted R2 WaaL protein is, however, much more similar to the S. enterica WaaL protein (81.1% total similarity) than the E. coli K-12 WaaL protein (33.6% total similarity). Hydrophilicity plots of the three WaaL homologs show significant similarity in their predicted structures, and those for S. enterica and E. coli R2 are virtually identical (Fig. 7). Computer analysis predicts that all three WaaL proteins contain at least eight membrane spanning domains. The distribution and the sizes of the transmembrane segments and surface-exposed loops are similar. The predicted E. coli K-12 WaaL protein is slightly larger in size (419 amino acids, 46,874 Da) than WaaL homologs of either E. coli R2 (405 amino acids, 46,048 Da) or S. enterica (404 amino acids, 46,031 Da).
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DISCUSSION |
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Variations in outer core OS structures currently determine five different core types in the LPSs of E. coli and one in S. enterica. The data presented here establish that the E. coli R2 core OS biosynthesis gene cluster is a hybrid of those of E. coli K-12 and S. enterica. The predicted glycosyltransferases (WaaG, -O, and -R) for assembly of the outer core OS backbone are highly similar in E. coli K-12 and R2. In contrast, the products of the waaK and waaL genes of R2, which are involved in the completion of the core OS and ligation of O-PS, are highly conserved with homologs in S. enterica.
Relatively little is known about the mechanism of action of
glycosyltransferases, and models lean heavily on the more extensive literature for glycosylhydrolases. As more sequences are available it
is apparent that there are several families of - and
-glycosyltransferases. WaaI and -J provide the prototype for a new
family of
-glycosyltransferases. Members of the family identified in
prokaryotes contain four conserved regions of primary sequence (Fig. 3)
located in regions of common secondary structure. Interestingly, one
eukaryotic member of the family (protein T10M13.14 of Arabidopsis
thaliana) lacks the sequence III motif. It is striking that where
substrates for these proteins are known, the WaaIJ family proteins use
UDP-hexose (Galp or Glcp), and many are involved
with the core region of an LPS or lipo-oligosaccharide molecule (Table
III). One, WbbM, is involved in O-PS
synthesis, and only one (GspA) has been identified from Gram-positive
bacteria. It is not possible to assign catalytic and/or binding
residues without more extensive biochemical analysis, but the
identification of conserved residues in this family provides the
foundation on which such strategies will be based.
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There are some potential open reading frames in E. coli K-12
whose functions remain obscure (reviewed in Ref. 2). Based primarily on
SDS-PAGE data, the waaS gene has been proposed to play a
role in an alternate form of LPS which is separate from those molecules
which will become an acceptor for O-PS (2). The "waaS"
regions in E. coli R2 and K-12 are relatively poorly conserved, and only remnants remain in S. enterica. Analysis
of the structure of the predicted R2 WaaS protein indicates that it is
an additional member of the WaaIJ family of -glycosyltransferases (Fig. 3). These motifs are absent in the K-12 WaaS protein. Examination of the available structural data for the R2 and K-12 core OSs indicates
they have non-stoichiometric substitutions of the KdoII residue with
either Galp or Rhap residues, respectively (38). Structural features of the R2 WaaS protein are consistent with a
transferase that uses UDP-Galp as a donor. The absence of a similar modification in S. enterica core OS is consistent
with the absence of a complete open reading frame equivalent to
waaS. Ultimately, the waaS genes may require
unique designations, but this should await experimental determination
of their precise roles in core OS assembly. Two additional open reading
frames, waaZ and waaY, are conserved in all three
core types. Their role in core assembly is presently unknown although
the waaZ gene may also play a role in the production of an
alternate form of LPS (2). The role of WaaQ is also unknown although it
may function as a HeppIII transferase for inner core
biosynthesis, based on limited resemblance to the sequences of other
heptosyltransferases (3). Mutations in waaP influence inner
core phosphorylation, but its precise role is not known (3). The
waaQ and waaP genes (and gene products) are
highly conserved in all three core types. The identification of
conserved sequences for these largely uncharacterized genes in E. coli R2 does not shed light on the function of their gene
products, and since they are not considered to influence outer core OS
carbohydrate structure, their roles are not addressed here. However,
structural and genetic information is now available to systematically
address these additional questions in core OS assembly.
In the core OS of S. enterica, addition of the terminal
1,2-linked GlcpNAc side branch requires WaaK (7, 36, 39). Membranes of S. enterica are known to incorporate
GlcpNAc from UDP-GlcpNAc (40), but the data
directly linking WaaK to the glycosyltransferase activity has been
circumstantial. Here, we show that the E. coli R2 WaaK
homolog is the UDP-N-acetylglucosamine:(glucosyl) LPS
1,2-N-acetylglucosamine transferase for outer core OS
assembly. Structural analysis of the S. enterica core OS
(36) indicate this substitution is stoichiometric. However, in E. coli R2 (this study and Ref. 8), this terminal GlcNAc substitution
is non-stoichiometric. Possible reasons for this minor difference
remain unclear. The literature for the corresponding HexIII
substitution in E. coli K-12 indicates the presence of
terminal
-1,6-heptose, referred to as HepIV (38). As predicted from
the structures, the K-12 core OS biosynthesis gene cluster does not
contain a homolog of waaK. In E. coli K-12, the
gene which we have renamed waaU (originally this gene was
also referred to as rfaK) occupies the identical location to
waaK. It has previously been suggested that WaaU might still
be a GlcpNAc transferase but one involved in transfer of GlcpNAc to an undefined location in the inner core OS (2,
37). Interestingly, WaaU contains two
-glycosyltransferase motifs, E228QIKVIYQE235
and
E261IETLPFDE269,
resembling the the two closely occurring E(X7)E
motifs found in the C-terminal third of WaaC and WaaF proteins of
E. coli K-12, R2, and S. enterica. This is in
contrast to the hexosyltransferases such as WaaG, -B, and -K, which
contain only a single copy of the motif. Furthermore, BLASTP searches
identify regions of local similarity shared by WaaU and a variety of
known and predicted heptosyltransferases, including WaaC and WaaF
proteins. Thus, whereas the identity of the glycosyltransferase for the
terminal
1,6-linked Hepp (HepIV) side branch in the
E. coli K-12 core is equivocal, waaU is the most
likely candidate.
Currently, little is known of the mechanism by which O-PSs are ligated to the lipid A-core molecule. Insights into the ligation reaction could lead to novel therapeutic agents that prevent the attachment of O-PS and lead to a higher degree of complement-mediated killing by the host. The ligase enzyme is envisioned as a glycosyltransferase with a complex (lipid-linked oligosaccharide) substrate requirement. Motifs found in currently known glycosyltransferases are absent in the ligase protein, as might be expected since the ligase substrate is not a nucleotide diphospho sugar. There is little conservation in the primary sequences of the ligases of E. coli R2/S. enterica and E. coli K-12, although their secondary structures appear to be a conserved feature. Ligases from E. coli K-12, R2 (see above,) and S. enterica (data not shown) all interact with and efficiently ligate the reporter O-PS, D-galactan I, to their respective core OS molecules. Furthermore, the ability of the K-12 ligase protein to interact with a variety of different polysaccharide structures is interesting and suggests a relaxed specificity for the ligated structure. To the extent that biosynthetic data are available, all of the polysaccharides currently known to be ligated to lipid A-core by WaaL are assembled on an undecaprenyl pyrophosphoryl lipid intermediate (reviewed in Refs. 1 and 41). The precise details of the trans-cytoplasmic assembly pathways can vary considerably, and the E. coli K-12 ligase efficiently ligates O-PS products from the three currently known pathways (1). The undecaprenyl pyrophosphoryl carrier may provide the conserved feature in the ligated substrate for ligase function.
We are interested in the structural requirements for the acceptor in the O-PS ligation reaction. Core OS structure has a profound effect on ligase specificity. Prior to this work, the only known ligases were those from S. enterica and E. coli K-12. Comparative analysis of these ligases is complicated by core OS structures that differ in both backbone sequence and terminal side branch substituents. Also, a full collection of precise mutations in core OS assembly as well as individually cloned and expressed genes have not been available to address directly the structural requirements. These limitations prompted the analysis reported here.
Previous cross-complementation data indicate that the waaL
gene from E. coli K-12 cannot complement a ligase-defective
mutant S. enterica, suggesting structural specificity in
terms of the core OS acceptor (37). Studies involving a prototype
S. enterica waaK mutant (SL733) indicate that absence of the
terminal 1,2-linked GlcNAc abrogates attachment of most O-PS; only
trace amounts remained (36). As reported here, and in previous work by
others (7), there is no evidence of residual S-LPS in the currently
available isolate of S. enterica SL733. The nature of the
waaK953 allele is unknown, leading to questions concerning
whether WaaK activity is essential for ligation or only important for
ligation efficiency. Complementation of the ligation defect by
waaK genes from the same organism (7) and from E. coli R2 (this work) indicates that the defects are confined to
waaK in S. enterica SL733. In a defined
waaK null mutant of E. coli F632 (strain CWG300),
the ligation of a reporter O-PS to the R2 core OS is eliminated, and a
terminal side group substitution is therefore essential for ligation
activity in E. coli R2 and probably in S. enterica. Consistent with this conclusion, there is one report of
the structure of an O-PS (serotype O104) attached to an R2 core OS, and
the WaaK-directed GlcNAc side branch was present in stoichiometric
amounts in the linkage region of the resulting S-LPS (42). The ability
of the E. coli R2 waaK gene product to
efficiently complement the S. enterica WaaK-mediated
ligation defect rules out any role for core OS backbone structure in
determination of acceptor specificity in the group of ligase enzymes
examined here, since these core OSs differ at the HexII position
(i.e. Gal in S. enterica and Glc in E. coli R2).
Interestingly, in previous work from others, some ligation activity was restored when a plasmid containing waaL and waaU from E. coli K-12 was introduced into an S. enterica waaK mutant (37). Inactivation of the waaL coding region of this plasmid eliminated its inability to complement the waaK phenotype of S. enterica. This suggests that the "complementation" of the waaK defect in fact represented replacement of enough S. enterica core OS terminus with that of E. coli K-12, to allow the K-12 ligase to functionally replace the S. enterica chromosomally encoded WaaL. Unfortunately, the structure of the resulting core was not determined. In light of the published lack of complementation of the S. enterica waaL mutant with the cloned E. coli K-12 waaL gene (37), the requirement for a precise side group appeared to be absolute. However, using a plasmid carrying only the R2 waaL structural gene, we were able to restore low levels of ligation activity in the E. coli K-12 waaL mutant (CS2334). The differences in the two studies could reflect differences in sensitivity of detection methods or levels of waaL expression; previous studies did not use a defined inducible promoter in complementation plasmids. Based on the data presented here, a terminal side group is critical for this group of ligases, although the precise nature of the residue clearly affects efficiency of ligation. The different efficiencies of the R2 WaaL protein in ligation to the K-12 and R2/S. enterica core OSs presumably reflect steric hindrance resulting from the replacement of the GlcpNAc side branch (present in the native R2 core OS) with a Hepp residue (in K-12 core OS).
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ACKNOWLEDGEMENTS |
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We thank K. E. Sanderson and C. Schnaitman for strains; P. Reeves for discussions regarding nomenclature; and W. Wakarchuk for sharing sequence data prior to publication. We also thank A. Clarke for discussions regarding protein analysis and for critically reviewing the manuscript. The excellent technical assistance provided by Karen Amor is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported in part by funding awarded (to C. W.) by the Canadian Bacterial Diseases Network and by the Natural Sciences and Engineering Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF026386 and AF019375.
§ Recipient of a Natural Sciences and Engineering Research Council postdoctoral fellowship.
To whom correspondence should be addressed. Tel.: 519-824-4120 (ext. 3478); Fax: 519-837-1802; E-mail: cwhitfie{at}uoguelph.ca.
1 The abbreviations used are: LPS, lipopolysaccharide; PAGE, polyacrylamide gel electrophoresis; S-LPS, smooth LPS; R-LPS, rough LPS; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; kb, kilobase pairs; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; O-PS, O-polysaccharide; core OS, core oligosaccharide; PCR, polymerase chain reaction; HCA, hydrophobic cluster analysis.
2 Since most data supports the presence of a single core structure in the genus Salmonella and the majority of genetic and biochemical data in this area is confined to Salmonella enterica serovar Typhimurium, this bacterium will be simply be referred to as S. enterica in this communication.
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REFERENCES |
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