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J Biol Chem, Vol. 273, Issue 41, 26310-26316, October 9, 1998
§,¶,
,, and**
From the Department of Microbiology, University of
Guelph, Guelph, Ontario N1G 2W1, Canada and the Institute for
Biological Sciences, National Research Council, Ottawa,
Ontario K1A OR6, Canada
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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The waaY, waaQ, and waaP genes are located in the central operon of the waa (formerly rfa) locus on the chromosome of Escherichia coli. This locus contains genes whose products are involved in the assembly of the core region of the lipopolysaccharide molecule. In the R1 core prototype strain, E. coli F470, there are nine genes in this operon, and all but waaY, waaQ, and waaP have been assigned function. In this study, the waaY, waaQ, and waaP genes were independently mutated by insertion of a non-polar antibiotic resistance cassette, and the structures of the resulting mutant core oligosaccharides were determined by chemical analyses and phosphorus-nuclear magnetic resonance spectroscopy. All three of these mutations were shown to affect the modification of the heptose region of the core, a region whose structure is critical to outer membrane stability. Mutation of waaY resulted in a core oligosaccharide devoid of phosphate on HepII. Mutation of waaQ resulted in loss of the branch HepIII residue on HepII and impeded the activity of WaaY. Mutation of waaP resulted in loss of phosphoryl substituents on HepI and obviated WaaQ and WaaY activity. Only mutation of waaP resulted in hypersensitivity to novobiocin and sodium dodecyl sulfate, a characteristic of deep-rough mutations.
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INTRODUCTION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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The outer membrane of Gram-negative bacteria is a barrier to many antibiotics and host defense factors (1). The outer leaflet of this membrane is composed almost exclusively of lipopolysaccharide (LPS),1 a unique glycolipid with structural features essential to outer membrane stability. In Escherichia coli and Salmonella enterica, the LPS molecule is conceptually divided into three distinct regions: 1) a hydrophobic membrane anchor designated lipid A; 2) a short chain of sugar residues with multiple phosphoryl substituents, referred to as the core oligosaccharide (core OS); and 3) a structurally diverse polymer composed of oligosaccharide repeats, termed the O antigen. Lipid A and the core OS are synthesized together as a single unit (lipid A-core, Fig. 1), which serves as an acceptor for preformed O antigen to yield the completed LPS molecule (2, 3).
Five distinct core OS structures have been identified in E. coli (core types K-12, R1, R2, R3, and R4), and two more are known for S. enterica (4, 5). The genes responsible for biosynthesis of the core OS in these bacteria are clustered on the chromosome in the waa (formerly rfa) locus near 81 min on the E. coli K-12 and S. enterica linkage maps. Mutations in many of the glycosyl transferases encoded by this locus result in the production of LPS lacking O antigen (termed rough or R-LPS) since O antigen cannot be ligated to an incomplete lipid A-core acceptor molecule. Strains that produce only R-LPS are more susceptible to complement-mediated serum killing than their wild-type counterparts (reviewed in Ref. 6). Mutations in the waa locus that specifically affect the phosphoryl substitution of the core OS heptose region (Fig. 1) can significantly alter outer membrane permeability, giving rise to a pleiotropic phenotype called deep-rough (7, 8). Characteristics of the deep-rough phenotype include the following: 1) hypersensitivity to detergents and hydrophobic antibiotics, 2) sloughing of LPS from the outer membrane, 3) leakage of periplasmic proteins into the culture medium, and 4) a marked decrease in the protein content of the outer membrane (reviewed in Ref. 9). The phosphoryl substituents in the heptose region are postulated to be so critical to outer membrane stability because their negative charge allows neighboring LPS molecules to be cross-linked by divalent cations (1, 10). Given its crucial role in outer membrane stability, the assembly of the heptose portion of the LPS molecule provides potential targets for novel therapeutic compounds. Development of such therapeutic strategies is limited, however, because the enzymology involved in the heptose region assembly is poorly understood.
Previous studies with S. enterica serovars Minnesota (11, 12) and Typhimurium (7) and with E. coli K-12 (13) have implicated waaP in the phosphorylation of both HepI and HepII (Fig. 1). Mutation of waaP in these organisms was also reported to cause characteristics of the deep-rough phenotype. Furthermore, in E. coli K-12, waaP has been implicated in the addition of HepIII (Fig. 1). Interpretation of these data, however, has been complicated by their reliance on strains with poorly defined or polar mutations. The goal of this study was to identify unequivocally the genes involved in modification of the inner core HepI and HepII residues and to determine the precise contribution of the corresponding gene products to the biosynthesis of the inner core heptose region.
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The R1 core is the most prevalent among clinical isolates of E. coli (14, 15), and since its structure is known (16) and the genetics of its outer portion have been resolved (see Fig. 2A)2 studies were performed using the prototype E. coli R1 strain, F470. A precise, non-polar insertion mutation was made in the waaP gene of E. coli F470 to unequivocally establish the role of its gene product in modification of the heptose region. Furthermore, additional functions originally assigned to the waaP gene product were shown to be encoded by two genes (waaY and waaQ) whose functions were previously unknown.
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EXPERIMENTAL PROCEDURES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Bacterial Strains and Growth Conditions-- E. coli F470 is an R-LPS derivative of an O8:K27 strain (18) and serves as the prototype for R1 core OS studies. Derivatives of F470 used in this study include CWG296 (F470 with a waaP::aacC1 insertion), CWG297 (F470 with a waaQ::aacC1 insertion), and CWG312 (F470 with a waaY::aacC1 insertion). Bacteria were grown in Luria-Bertani (LB) broth at 37 °C. Growth media were supplemented with ampicillin (100 µg/ml), gentamicin (15 µg/ml), or chloramphenicol (30 µg/ml) when necessary.
In Vitro Mutagenesis and Gene Replacement-- The waaY, waaQ, and waaP genes of the R1 core OS biosynthesis region were independently mutated by insertion of a non-polar gentamicin resistance cassette (the aacC1 gene from Tn1696). Briefly, each of the genes (with some flanking DNA) was individually PCR amplified, cloned into the pBluescript II SK(+) phagemid (Stratagene), and sequenced to ensure error-free amplification. The aacC1 gene from plasmid pUCGM (19) was then inserted into an appropriate site (near the middle) of the coding region of each gene (Fig. 2B). The DNA fragment containing the insertionally inactivated gene was subsequently cloned into the suicide delivery vector pMAK705 (20), and chromosomal gene replacement was performed as described previously (21). Mutations were verified by PCR amplification of the mutated gene from the chromosome and confirmed by sequencing.
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DNA Methods-- Restriction endonuclease digestion and ligation were performed as described by Sambrook et al. (22). Restriction enzymes were purchased from Life Technologies, Inc., New England Biolabs, or Boehringer Mannheim. Plasmids were introduced into E. coli strains by electroporation using a Gene Pulser from Bio-Rad. Chromosomal DNA isolation was performed using the Qiagen genomic DNA isolation kit, and plasmid DNA was prepared using QIAprep Miniprep spin columns (Qiagen Inc., Santa Clarita, CA). When 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) in the Guelph Molecular Supercentre at the University of Guelph. PCR amplification was performed using a GeneAmp PCR System 2400 from Perkin-Elmer. The PwoI DNA polymerase enzyme (Boehringer Mannheim) was used as recommended by the manufacturer.
Cloning and Expression of waaP for Complementation-- Oligonucleotide primers for the amplification of waaP from the F470 chromosome were designed to introduce appropriate restriction sites for cloning. The forward primer (5'-TGTGGATccAAATAGTGGGCACTCA-3') introduced a BamHI site (underlined) 32 base pairs downstream of the waaP stop codon, and the complementary reverse primer (5'-GGGTGGTCcatATGGTTGAACTTAA-3') introduced an NdeI site (underlined) overlapping the waaP start codon (bases shown in lowercase indicate mismatches between the primer and chromosomal sequences). The coding region for waaP alone was subsequently isolated as a BamHI-NdeI fragment and cloned behind the arabinose-inducible PBAD promoter of the expression vector pBAD18 (23) to generate plasmid pWQ909. L-Arabinose was used at a final concentration of 0.002% to induce expression of WaaP from plasmid pWQ909.
Computer Analyses-- Homology searches of nucleotide and amino acid sequences in the National Center for Biotechnology Information data bases were performed using the BLAST (basic local alignment search tool) or PSI (position-specific iterated)-BLAST server analysis programs (24, 25). Pairwise nucleotide sequence alignments and percent 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. Multiple protein alignments and percentage identity and similarity scores were obtained using the PC/Gene program CLUSTALV, with an open gap cost of 10 and a unit gap cost of 10. Routine DNA and protein sequence manipulations were performed using the MacVector and AssemblyLIGN software packages (International Biotechnologies, Inc., New Haven, CT).
SDS and Novobiocin Sensitivity Testing--
2-Fold serial
dilutions of SDS (200 
0.1 mg/ml) and novobiocin (200 1.6 µg/ml) were made in 5-ml volumes of LB. Each series of tubes was
point inoculated from an overnight culture of the strain to be tested
and then incubated with shaking at 37 °C. Growth was scored as
positive if after 8 h the culture was visibly turbid
(i.e. OD600 > 0.2).
Preparation of Core OS-- Water-soluble LPS were obtained by the hot phenol/water extraction of cells (26) and then treated with 2% acetic acid at 100 °C to cleave the acid-labile ketosidic linkage between the core OS and lipid A. The water-insoluble lipid A was removed by centrifugation (5000 × g, 30 min), and the supernatant was passed through a column of BioGel P-2 (1 m × 1 cm) with water as eluent. The core-containing fractions were collected and lyophilized.
Sugar Composition and Methylation Linkage Analyses-- Sugar composition analysis was performed by the alditol acetate method (27). Hydrolysis of glycosidic bonds was achieved with 4 M trifluoroacetic acid at 100 °C for 4 h. The samples were then reduced in water with sodium borodeuteride and acetylated with acetic anhydride using residual sodium acetate as the catalyst. Characterization of the alditol acetate derivatives was performed by gas-liquid chromatography (GLC)-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. Linkage data were interpreted based upon the previously published structure of the R1 core (4), as described for analysis of the R1 outer core assembly.2 Methylation linkage analyses were carried out by the procedure of Ciucanu and Kerek (28). The permethylated alditol acetate derivatives were fully characterized by GLC-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 (FAB-MS)-- A fraction of the methylated sample from the linkage analysis was taken for positive ion FAB-MS, which was performed using a Jeol JMS-AX505H mass spectrometer with glycerol/thioglycerol as the matrix and a tip voltage of 3 kV.
31P NMR Spectroscopy-- 31P NMR spectra were recorded with a Bruker DRX 400 MHz instrument at 161.98 MHz with ortho-phosphoric acid as the external reference (0.0 ppm) and with pI = 30 in the proton-decoupling mode. Prior to performing the NMR experiments, the samples were lyophilized three times in 2H2O (99.9%). The p2H was adjusted with triethylamine when necessary.
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RESULTS |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Structural Analysis of the Core OS from the R1 Prototype Strain F470-- The established structure of the F470 core OS (Fig. 2A) served as a framework for the interpretation of our results. Sugar composition analysis detected the presence of all the expected core OS sugars (glucose, galactose, and heptose; data not shown). Methylation linkage analysis of the core OS from F470 showed all of the permethylated alditol acetate derivatives from the outer core residues in the ratios expected (Fig. 3A and Table I). No peaks were observed in the GLC chromatogram (Fig. 3A) for the derivatives from either HepI or HepII because the phosphoryl substituents attached to these residues (Fig. 2A) made their derivatives too polar to elute from the column. This proved useful in subsequent analyses because it allowed the loss of phosphoryl substituents from HepI and HepII to be monitored as the appearance of either a 3-substituted or 3,7-disubstituted heptose derivative peak in the GLC chromatogram.
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10 ppm are characteristic of a
diphosphodiester (PPEtN on HepI) (29). At lower solvent p2H
values, the peak at 5 ppm was shifted upfield (data not shown), confirming its assignment as a phosphomonoester (29). The ratio of P to
PPEtN in the F470 core OS was estimated by the relative peak heights in
Fig. 3E. As expected, there was more P than PPEtN, as HepII
is only substituted with P while HepI is substituted with either P or
PPEtN.
Sequence Analysis and Mutagenesis of waaY-- Pairwise nucleotide alignments of the waaY coding region from E. coli K-12, R1, R2, and R4 and S. enterica serovar Typhimurium (all of the core types for which DNA sequence of the waa loci was available) showed identities ranging from 59.4 to 99.0%. Multiple alignment of the predicted WaaY proteins showed high total similarity (85.3%) but much lower identity (43.1%). In BLASTP searches of the available data bases, WaaY showed no significant homologies to other characterized proteins. However, using the PSI-BLAST server analysis program, queries with the various WaaY protein sequences suggested biologically relevant similarities to a large number of kinases (data not shown). This circumstantial data suggested a potential role for WaaY in phosphorylation reactions.
To test this possibility, strain CWG312 was derived by insertion of the aacC1 gene into the unique SnaBI site in the waaY coding region of F470 (Fig. 2B). The LPS from the resulting mutant migrated similarly to that of the F470 parent in SDS-polyacrylamide gel electrophoresis (data not shown), precluding any significant effect of the mutation on core OS extension. However, the minimum inhibitory concentrations of SDS and of novobiocin for CWG312 were slightly less than for F470 (Table II), suggesting subtle changes in outer membrane properties.
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Structural Analysis of the CWG312 Core OS and Assignment of WaaY Function-- The structure of the deduced CWG312 core OS is shown in Table I. Sugar composition analysis detected the presence of all the core OS sugars found in F470 (data not shown). Linkage analysis of CWG312 resulted in a GLC chromatogram that was identical to that from the F470 parent (compare panels A and B in Fig. 3) except for the appearance of a peak (peak 9, Fig. 3B) corresponding to a 3,7-disubstituted heptose derivative (Table I). Based on the F470 structure, this derivative must result from unphosphorylated HepII. 31P NMR spectroscopy further corroborated the loss of P from the CWG312 core OS; in the F470 parent, there was more P than PPEtN, but in CWG312 there was slightly more PPEtN than P (compare panels E and F in Fig. 3). The observed shift in the ratio of P to PPEtN is explained by the loss of P from HepII, leaving only P and PPEtN on HepI. Therefore, given the disappearance of P from HepII in the CWG312 core OS and the biologically significant sequence similarity of WaaY to some kinases, WaaY was concluded to be involved in the phosphorylation of HepII.
Sequence Analysis and Mutagenesis of waaQ-- In E. coli K-12, R1, R2, and R4 and in S. enterica serovar Typhimurium, waaQ is the first gene of the central operon of the core OS biosynthesis region (Fig. 2B). Pairwise nucleotide alignments of the waaQ genes from these core types showed identities ranging from 68.5 to 98.6%. Multiple sequence alignment of the predicted WaaQ proteins reflected the homology observed at the nucleotide level with a total similarity of 88.1% (64.0% identity). The predicted WaaQ proteins showed limited homology to both WaaC (40.6% total similarity, 10.8% identity) and WaaF (50.1% total similarity, 16.5% identity), the HepI (30) and HepII (31, 32) transferases, respectively (Fig. 2A).
Strain CWG297 was derived by insertion of the aacC1 gene into the unique NruI site in the waaQ coding region of F470 (Fig. 2B). The LPS from the resulting mutant migrated similarly to that of the F470 parent in SDS-polyacrylamide gel electrophoresis (data not shown), but the minimum inhibitory concentrations of SDS and of novobiocin for CWG297 were slightly less than for F470 (Table II) and comparable with the values for the waaY mutant, CWG312.Structural Analysis of the CWG297 Core OS and Assignment of WaaQ Function-- Sugar composition analysis detected the presence of all the core OS sugars found in F470 (data not shown). The structure of the CWG297 core OS shown in Table I was deduced from methylation linkage analysis and 31P NMR spectroscopy as follows. First, methylation linkage analysis of the CWG297 core OS showed all of the outer core derivatives in the expected ratios (Table I), but the terminal heptose derivative (resulting from HepIII) was completely absent (note the absence of peak 6 in Fig. 3C). The disappearance of the HepIII side branch was also confirmed by FAB-MS (data not shown). Given the similarity of WaaQ to WaaC and WaaF (the HepI and HepII transferases, respectively; Fig. 2A) and the complete absence of HepIII in the CWG297 core OS, it was concluded that waaQ encodes the transferase for the branch HepIII residue. Interestingly, the appearance of a 3-linked heptose derivative in the linkage analysis of CWG297 (peak 8, Fig. 3C) also indicated the loss of a phosphoryl substituent from either HepI or HepII (both are 3-linked after the loss of HepIII) due to the waaQ mutation. To resolve which heptose residue was lacking, its phosphoryl substituent, the 31P NMR spectrum of the CWG297 core OS, was compared with the spectra from F470 and CWG312 (waaY). Given the similar P to PPEtN ratios for CWG297 and CWG312 (compare panels F and G in Fig. 3), it was concluded that the same phosphoryl substituent (P on HepII) was absent in both strains. If the waaQ mutation had affected phosphoryl substitution of HepI, the ratio of P relative to PPEtN would have increased rather than decreased. The observed loss of P from HepII in CWG297 did not contradict the assignment of WaaQ as the HepIII transferase since WaaY had already been assigned as the enzyme responsible for the phosphorylation of HepII (see above). Rather, the transfer of HepIII by WaaQ seems to be a prerequisite to the phosphorylation of HepII catalyzed by WaaY.
Sequence Analysis and Mutagenesis of waaP-- In E. coli K-12, R1, R2, and R4 and in S. enterica serovar Typhimurium, waaP occurs as the third gene of the central operon of the core OS biosynthesis region (Fig. 2B). Pairwise nucleotide alignments indicated that the waaP gene from these core types was highly conserved (71.8-98.1% identity). Similarity at the protein level was even higher, with multiple sequence alignment of the predicted WaaP proteins showing a total similarity of 93.3% and an identity of 75.9%. In BLASTP searches of the available data bases, WaaP showed no significant similarity to other characterized proteins, as previously reported (9, 13). However, the recently developed PSI-BLAST server analysis program again identified biologically relevant similarities shared between the various WaaP proteins and a large number of kinases (data not shown).
Strain CWG296 was derived by insertion of the aacC1 gene into the unique NruI site in the waaP coding region of F470 (Fig. 2B). The resulting mutant was hypersensitive to novobiocin and SDS (Table II), as expected based on results from previous studies (7, 33). Introduction of multicopy waaP (on plasmid pWQ909) into CWG296 restored wild-type levels of resistance to these hydrophobic agents (Table II).Structural Analysis of the CWG296 Core OS and Assignment of WaaP
Function--
Sugar composition analysis detected the presence of all
the core OS sugars found in F470 (data not shown). The structure of the
CWG296 core OS shown in Table I was deduced from methylation linkage
analysis and 31P NMR spectroscopy as follows. First,
31P NMR spectroscopy showed a complete lack of phosphorus
in the CWG296 core OS (Fig. 3H), indicating that mutation of
waaP resulted in the loss of all phosphoryl substituents
from HepI and HepII. The GLC chromatograms from the CWG296 and F470
methylation linkage analyses were then compared to determine any other
effects of the waaP mutation on the core OS structure. Of
note, the terminal heptose derivative from HepIII was almost completely
absent in the CWG296 core OS (note the absence of peak 6 in Fig.
3D). This lack of terminal heptose was also observed by
FAB-MS (data not shown) and implicated the waaP mutation in
the loss of HepIII in addition to the above-mentioned deficiency in
phosphoryl substituents. The absence of phosphoryl substituents and of
HepIII was further corroborated by the appearance of a large peak in
the CWG296 GLC chromatogram (peak 8, Fig. 3D) corresponding
to a 3-substituted heptose derivative from HepI and HepII. The
calculated molar ratio of this derivative (Table I) was somewhat lower
than expected, but the observed signal was clearly twice that from
CWG297 (compare peak 8 in Fig. 3, C and D; see
also Table I), as expected based on their predicted structures (the
prolonged retention time of the 3-substituted heptose derivative on the
GLC column may account for this slight discrepancy in molar ratios).
Finally, the amounts of terminal glucose (from the 
-Glc side branch)
and of 2,3-disubstituted glucose (from GlcII) were noticeably decreased
in CWG296 (compare peaks 1 and 7 in Fig. 3, A and
D; see also Table I). The decrease in 2,3-disubstituted
glucose was offset by the appearance of an approximately equal amount
of 2-substituted glucose (note the appearance of peak 3, Fig.
3D), reflecting GlcII lacking the -Glc substitution.
-Glc substituted GlcII. As WaaY and WaaQ had already been assigned in this
study as the enzymes responsible for the phosphorylation of HepII and
the transfer of HepIII, respectively (see above), and because WaaV had
been previously assigned as the -Glc transferase (see Fig.
2A),2 it was concluded that the enzyme encoded
by waaP was responsible for the phosphoryl substitution of
HepI. This conclusion is supported by the sequence similarity of WaaP
to known kinases, first reported in this study. The loss of other
substituents and sugar residues from the CWG296 core OS does not
contradict this assignment but suggests that the activity of WaaP is a
prerequisite to the efficient functioning of other enzymes.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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The data presented here identify WaaY as the enzyme that phosphorylates HepII, WaaQ as the transferase for the branch HepIII residue and WaaP as the enzyme responsible for the addition of either P or PPEtN to HepI. With these assignments, a function has now been ascribed to every gene in the waaQ operon of the core OS biosynthesis region in the E. coli R1 prototype strain, F470 (see Fig. 2A). Moreover, the deduced structures of the core OS from the waaY, waaQ, and waaP mutants are consistent with a sequence of events in the decoration of the heptose region of the core OS where 1) WaaP adds a phosphoryl substituent to HepI, 2) WaaQ adds HepIII to HepII, and 3) WaaY adds P to HepII.
The conclusion that the activity of WaaP is a prerequisite to the functioning of both WaaQ and WaaY is based on the fact that the core OS of CWG296 (waaP) does not have HepIII or a P substituent on HepII (Table I), even though functional copies of the waaQ and waaY genes are present on the chromosome. Likewise, the activity of WaaQ is concluded to be required prior to the functioning of WaaY because the core OS of CWG297 (waaQ) does not have the P substituent on HepII (Table I), even though the waaY gene in this mutant is fully functional. These results can be explained by a fastidious substrate requirement in each sequential reaction, such that WaaQ can only effect the transfer of HepIII to a lipid A-core acceptor with P or PPEtN on HepI, and WaaY can only phosphorylate HepII if the lipid A-core acceptor has both HepIII and a phosphoryl substituent on HepI. Alternatively, these data could reflect a requirement for specific protein-protein interactions to provide a functional multi-enzyme complex.
Re-evaluation of previous structural studies in E. coli K-12 (13), in light of the data presented here, further confirms the assignment of WaaY and WaaP function. Parker et al. (13) created a polar mutation in E. coli K-12 that eliminated function of all genes downstream of waaQ in the waaQ operon and then determined the core OS structures (by tandem mass spectrometry) of the resulting mutant and of the mutant complemented with both waaG and waaP. As expected, the strain without a functional copy of the waaP gene had no phosphoryl substituents or branch HepIII residue in the heptose region. Complementation of the mutant with waaG and waaP, however, resulted in a core OS with both HepIII and P or PPEtN on HepI, but no P on HepII, again as expected given the absence of a functional waaY gene on the chromosome (due to the polarity of the mutation). Correct and complete interpretation of these results was not possible prior to the unequivocal gene assignments reported here.
The data presented here also provide an explanation for the multiple
functions attributed to waaP in the literature. For example, the suggested involvement of WaaP in the transfer of HepIII in E. coli K-12 (13) has now been resolved. WaaQ is the HepIII transferase but requires the prior functioning of WaaP. It had previously been suggested, based solely on limited predicted protein sequence similarities, that the product of waaQ might encode
the HepIII transferase (9). This study provides the first genetic and
structural data to support this claim. Another reported effect of
waaP mutations in S. enterica serovars Minnesota
(12) and Typhimurium (7) is a truncation of the core OS distal to GlcI (Fig. 1); however, the precisely defined, non-polar waaP
mutations derived in this study clearly show that core extension is
complete, although there is a reduction in the efficiency of branch
-Glc substitution. The core truncation observed in the
waaP mutants of S. enterica could be explained by
polar or multiple mutations, which eliminate the function of downstream
genes.
The contribution of the heptose region phosphoryl substituents to outer membrane stability is clearly shown by novobiocin and SDS sensitivity testing, as illustrated in Table II. The lack of P on HepII in CWG312 (waaY) and in CWG297 (waaQ) resulted in a minor increase in the sensitivity of these strains to the hydrophobic agents. However, the complete lack of phosphoryl substituents in the core OS of CWG296 (waaP) resulted in a 32-fold increase in the susceptibility to novobiocin and more than a 1000-fold increase in the susceptibility to SDS. Therefore, only the complete loss of phosphoryl substituents from the heptose region of the core was sufficient to cause the characteristic hypersensitivity to hydrophobic agents associated with the deep-rough phenotype. It is noteworthy that the negative charges required for LPS cross-linking need not come from phosphoryl substituents; for example, the core OS of Klebsiella pneumoniae is devoid of phosphate but contains negatively charged galacturonic acid residues instead (34, 35).
Another important question that remains unanswered is how the heterogeneity of phosphoryl substituents on HepI arises. Does WaaP catalyze the transfer of P or PPEtN to HepI? If WaaP is a typical kinase enzyme and catalyzes only the transfer of P, then another enzyme must exist (outside of the waa locus), which adds 2-aminoethyl phosphate (PEtN) to P on HepI to give PPEtN. However, if WaaP catalyzes the transfer of PPEtN, then a subsequent processing reaction must be invoked to explain the loss of PEtN from PPEtN to give P on HepI. There is preliminary evidence which indicates that phosphatidylethanolamine is the direct donor of the PEtN residues found in the core OS (17). Together with the similarity of WaaP to known kinases, this evidence suggests that WaaP likely transfers P to HepI with PPEtN arising from subsequent addition of PEtN. With the assignment of function to the waaQ, waaP, and waaY genes, these issues can now be addressed.
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ACKNOWLEDGEMENT |
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We gratefully acknowledge the technical assistance provided by Frank St. Michael.
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FOOTNOTES |
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* This work was supported in part by the Canadian Bacterial Diseases Network (Network of Centers of Excellence) (to C. W. and M. B. P.).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.
§ Recipient of a Natural Sciences and Engineering Research Council graduate scholarship.
¶ Recipient of postdoctoral fellowships from the Natural Sciences and Engineering Research Council and the Medical Research Council.
** To whom correspondence should be addressed. Tel.: 519-824-4120 (ext. 3478); Fax: 519-837-1802; E-mail: cwhitfie{at}uoguelph.ca.
The abbreviations used are: LPS, lipopolysaccharide; OS, oligosaccharide; R-LPS, rough-LPS; P, phosphate; PPEtN, 2-aminoethyl diphosphate; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acidHep, L-glycero-D-manno-heptoseGLC, gas-liquid chromatographyFAB-MS, fast atom bombardment-mass spectrometryPCR, polymerase chain reactionHex, D-hexose.
2 Heinrichs, D. E., Yethon, J. A., Amor, P. A., and Whitfield, C. (1998) J. Biol. Chem. 273, in press.
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REFERENCES |
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Abstract
Introduction
Procedures
Results
Discussion
References
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 M. S. Trent, C. M. Stead, A. X. Tran, and J. V. Hankins Invited review: Diversity of endotoxin and its impact on pathogenesis Innate Immunity, August 1, 2006; 12(4): 205 - 223. [Abstract] [PDF]
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 O. Sherlock, U. Dobrindt, J. B. Jensen, R. Munk Vejborg, and P. Klemm Glycosylation of the Self-Recognizing Escherichia coli Ag43 Autotransporter Protein. J. Bacteriol., March 1, 2006; 188(5): 1798 - 1807. [Abstract] [Full Text] [PDF]
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C. M. Taylor, M. Goldrick, L. Lord, and I. S. Roberts Mutations in the waaR Gene of Escherichia coli Which Disrupt Lipopolysaccharide Outer Core Biosynthesis Affect Cell Surface Retention of Group 2 Capsular Polysaccharides J. Bacteriol., February 1, 2006; 188(3): 1165 - 1168. [Abstract] [Full Text] [PDF]
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S. Gronow, W. Brabetz, B. Lindner, and H. Brade OpsX from Haemophilus influenzae Represents a Novel Type of Heptosyltransferase I in Lipopolysaccharide Biosynthesis J. Bacteriol., September 1, 2005; 187(17): 6242 - 6247. [Abstract] [Full Text] [PDF]
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E. Frirdich, C. Bouwman, E. Vinogradov, and C. Whitfield The Role of Galacturonic Acid in Outer Membrane Stability in Klebsiella pneumoniae J. Biol. Chem., July 29, 2005; 280(30): 27604 - 27612. [Abstract] [Full Text] [PDF]
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 P. De Spiegeleer, J. Sermon, K. Vanoirbeek, A. Aertsen, and C. W. Michiels Role of Porins in Sensitivity of Escherichia coli to Antibacterial Activity of the Lactoperoxidase Enzyme System Appl. Envir. Microbiol., July 1, 2005; 71(7): 3512 - 3518. [Abstract] [Full Text] [PDF]
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E. Frirdich and C. Whitfield Characterization of GlaKP, a UDP-Galacturonic Acid C4-Epimerase from Klebsiella pneumoniae with Extended Substrate Specificity J. Bacteriol., June 15, 2005; 187(12): 4104 - 4115. [Abstract] [Full Text] [PDF]
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E. Frirdich and C. Whitfield Review: Lipopolysaccharide inner core oligosaccharide structure and outer membrane stability in human pathogens belonging to the Enterobacteriaceae Innate Immunity, June 1, 2005; 11(3): 133 - 144. [Abstract] [PDF]
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J. W. Yoon, J. Y. Lim, Y. H. Park, and C. J. Hovde Involvement of the Escherichia coli O157:H7(pO157) ecf Operon and Lipid A Myristoyl Transferase Activity in Bacterial Survival in the Bovine Gastrointestinal Tract and Bacterial Persistence in Farm Water Troughs Infect. Immun., April 1, 2005; 73(4): 2367 - 2378. [Abstract] [Full Text] [PDF]
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N. A. Kaniuk, E. Vinogradov, J. Li, M. A. Monteiro, and C. Whitfield Chromosomal and Plasmid-encoded Enzymes Are Required for Assembly of the R3-type Core Oligosaccharide in the Lipopolysaccharide of Escherichia coli O157:H7 J. Biol. Chem., July 23, 2004; 279(30): 31237 - 31250. [Abstract] [Full Text] [PDF]
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E. Frirdich, E. Vinogradov, and C. Whitfield Biosynthesis of a Novel 3-Deoxy-D-manno-oct-2-ulosonic Acid-containing Outer Core Oligosaccharide in the Lipopolysaccharide of Klebsiella pneumoniae J. Biol. Chem., July 2, 2004; 279(27): 27928 - 27940. [Abstract] [Full Text] [PDF]
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M. Harper, A. D. Cox, F. St. Michael, I. W. Wilkie, J. D. Boyce, and B. Adler A Heptosyltransferase Mutant of Pasteurella multocida Produces a Truncated Lipopolysaccharide Structure and Is Attenuated in Virulence Infect. Immun., June 1, 2004; 72(6): 3436 - 3443. [Abstract] [Full Text] [PDF]
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L. Izquierdo, N. Coderch, N. Pique, E. Bedini, M. M. Corsaro, S. Merino, S. Fresno, J. M. Tomas, and M. Regue The Klebsiella pneumoniae wabG Gene: Role in Biosynthesis of the Core Lipopolysaccharide and Virulence J. Bacteriol., December 15, 2003; 185(24): 7213 - 7221. [Abstract] [Full Text] [PDF]
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unless otherwise
indicated. The data that identify the genetic determinants for the
modification of the heptose region (enclosed in boxes) are
reported in this study. The assignment of function to other genes has
been reported previously (