Departments of Medicine, and Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA1
Department of Veterans Affairs Medical Center, Atlanta, GA 30033, USA2
The Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA3
Author for correspondence: David S. Stephens. Tel: +1 404 728 7688. Fax: +1 404 329 2210. e-mail: dstep01{at}emory.edu
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ABSTRACT |
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Keywords: meningococcus, lipopolysaccharides, heptose biosynthesis
Abbreviations: ABC, ATP-binding cassette; ES-MS, electrospray mass spectrometry; Hep, L-glycero-D-manno-heptose; Kdo, 3-deoxy-D-manno-2-octulosonic acid; LOS, lipooligosaccharide; NHS, normal human serum
The GenBank accession number for the sequence of the ice-2 operon in Neisseria meningitidis NMB is AF036242.
a Present address: BioResource International, Inc., 840 Main Campus Drive, Suite 3560, Raleigh, NC 27606, USA.
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INTRODUCTION |
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Meningococcal LOS is structurally distinct from the lipopolysaccharide (LPS) of other Gram-negative organisms in that, among other features, it lacks the repeating O-antigen side chains. Meningococcal LOS is composed of a conserved inner-core structure: a membrane-associated hexa-acylated lipid A moiety attached to two molecules of 3-deoxy-D-manno-2-octulosonic acid (Kdo) and two molecules of L-glycero-D-manno-heptose (Hep). Attached to the Hep2-Kdo2-lipid A inner-core structure in LOS are oligosaccharide chains of varying length and composition. In N. meningitidis and Neisseria gonorrhoeae LOS, the oligosaccharides are composed of glucose, galactose, N-acetylglucosamine and N-acetylneuraminic acid.
The biochemical and genetic aspects of LPS synthesis have been extensively studied in Escherichia coli and Salmonella. The isolation and characterization of heptose-deficient inner-core mutants in E. coli and Salmonella have helped identify genes necessary for heptose transfer (rfaC, rfaF) (Chen & Coleman, 1993 ; Sirisena et al., 1992
) and biosynthesis (gmhA, rfaE, rfaD) (Brooke & Valvano, 1996a
, b
; Drazek et al., 1995
; Eidels & Osborn, 1974
; Nichols et al., 1997
; Sirisena et al., 1992
, 1994
). rfaC encodes heptosyltransferase I, which transfers the first heptose from the ADP-L-glycero-D-manno-heptose donor molecule to the Kdo2-lipid A substrate to create Hep-Kdo2-lipid A, and rfaF encodes heptosyltransferase II, which transfers the second heptose from ADP-L-glycero-D-manno-heptose to the Hep-Kdo2-lipid A substrate to create the inner-core structure Hep2-Kdo2-lipid A (Chen & Coleman, 1993
; Kadrmas & Raetz, 1998
; Sirisena et al., 1992
).
Over the last 5 years a number of studies, aided by data from meningococcal and gonococcal genome sequencing projects, have identified meningococcal and gonococcal homologues of E. coli and Salmonella LPS inner-core assembly and biosynthesis genes (Gotschlich, 1994 ; Jennings et al., 1995
; Kahler et al., 1996a
, b
; Lee, F. K. N. et al., 1995
; Zhou et al., 1994
; for a review, see Kahler & Stephens, 1998
). These studies have also helped to define unique features of meningococcal inner-core LOS, such as differences in lipid A phosphorylation and differences in the composition and attachment of acyl chains on lipid A (Kahler & Stephens, 1998
). Nevertheless, issues regarding compartmentalization and modification of LOS and LPS inner-core structures remain unresolved and questions still remain about the genetics and biochemistry of heptose biosynthesis and incorporation in E. coli, Salmonella (Valvano et al., 2000
) and N. meningitidis.
In this study, we have identified and characterized a novel gene, gmhX, which, based on genetic and biochemical evidence, is required for the incorporation of heptose into meningococcal LOS and appears to be involved in heptose biosynthesis. We demonstrate that gmhX is encoded in a polycistronic operon (ice-2; LOS inner-core extension, locus 2), which also contains a gene involved in meningococcal phospholipid biosynthesis. While gmhX is required for heptose incorporation, it is distinct from the kinase and ADP-heptose synthetase domains encoded by rfaE, which in N. meningitidis are found as two separate genes (rfaE and aut), and from the ADP-heptose isomerase (rfaD). Homologues of gmhX are found associated with heptose biosynthesis operons in other species.
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METHODS |
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LOS isolation and SDS-PAGE.
LOS was examined by proteinase K digestion and Tricine/SDS-PAGE on a mini Protean apparatus (Bio-Rad) (Lesse et al., 1990 ; Schagger & von Jagow, 1987
).
Composition and linkage analysis of LOS.
Purified LOS was prepared from N. meningitidis strains by a modified version of the procedure described by Galanos (Galanos et al., 1969 ; Kahler et al., 1996a
, b
), and analysed by electrospray mass spectrometry (ES-MS) as previously described (Kahler et al., 1996a
, b
).
DNA preparation and transformation procedures.
Chromosomal DNA was prepared from NMB by the method of Nath (1990) . Plasmid DNA was extracted from E. coli as described by Engebrecht & Brent (1996)
. NMB and E. coli were transformed with DNA as described by Chung et al. (1989)
and Janik et al. (1976)
. Restriction digests and ligation reactions were performed under conditions specified by the manufacturer (New England Biolabs).
Single specific primer-PCR cycle sequencing.
This was used to amplify chromosomal DNA flanking the transposon insertion in 469 as described by Shyamala & Ames (1989) . The amplified products were purified with the QIAquick PCR Purification Kit (Qiagen) and sequenced with the AmpliCycle Sequencing Kit (Perkin-Elmer) or by automated sequencing at the Emory Core DNA Sequencing Facility. Nucleotide and amino acid sequence analysis was performed using either the GCG Wisconsin Package version 8.1 (Genetics Computer Group) or the DNASTAR sequence analysis package.
Southern blotting techniques.
Chromosomal DNA was prepared from NMB as described by Nath (1990) and digested with SspI, ClaI or EcoRI under conditions specified by the manufacturer (New England Biolabs). The DNA fragments were separated on a 0·7% agarose gel, run at 30 V for 6 h, transferred to a nylon membrane (Micron Separation), hybridized with a gmhX-specific probe, and developed using the Genius chemiluminescence system (Boehringer Mannheim).
Construction of polar and non-polar gmhX mutants.
An internal fragment of gmhX containing a unique SspI restriction site was amplified from NMB and cloned in pCR2 (Invitrogen). To create a polar mutation in gmhX, an SpR cassette excised from pHP45
(Prentki & Krisch, 1984
) was inserted into SspI-linearized pGS1620 to create pGS114. The gmhX::
SpR construct was excised from pGS114 and inserted into vector pHGS298 to create pGS123, which was used to transform NMB, resulting in GS152.7. To create a non-polar mutation in gmhX, aphA-3 was amplified from pUC18K, cut with SmaI, and inserted into SspI-linearized pGS128 (pGS1620
StyI to delete KnR marker). The resulting gmhX::aphA-3 construct (pGS137) was used to transform NMB to create GS158.
Construction of rfaC::lacZermC reporter in NMB and integration into the genome of N. meningitidis.
A unique BamHI restriction site was created in rfaC based on the method described by Hughes & Andrews (1996) . Primers RN9 (5'-CGGTTTTGCAGTTACTTAAAGGC-3') and RN12 (5'-TTACAAACGGATCCAACCGTG-3') were used to PCR-amplify an internal 5' section of rfaC from NMB, and RN11 (5'-ATTGCACGGTTGGATCCGTTTG-3') and RN14 (5'-GTCTGCAATCTGTTCCGCACG-3') were used to PCR-amplify an internal 3' section of rfaC from NMB. Complementary primers RN11 and RN12 are mutagenic primers that introduce a unique BamHI site in the rfaC amplification product. Equimolar amounts of the amplification products were combined and used as a template in a second round of PCR amplification with nested primers RN10 (5'-GCAGGTTATCGTGAATGAAGCG-3') and RN13 (5'-CCCCAAGGCAGATAAACATTGC-3'). The resulting 900 bp amplification product containing rfaC with a unique BamHI site was then cloned in pGEM-T (Promega) to create pRN1013. A BamHI fragment from pAEermC (encoding promoterless ß-galactosidase and erythromycin resistance) was inserted into the unique internal BamHI site in rfaC in pRN1013 to create pRN1013#1. The rfaC::lacZermC cassette was amplified from pRN1013#1 and used to transform NMB and GS158 to create the GS159 and GS160 reporter strains, respectively.
Quantification of ß-galactosidase activity in rfaC reporter strains.
ß-Galactosidase activity of the GS159 and GS160 reporter strains was measured by a whole cell ELISA protocol adapted from Takahashi et al. (1992) . Briefly, Maxisorp microtitre plates (Nunc) were incubated overnight at 4 °C with mouse anti-ß-galactosidase mAb (Promega) in 0·1 M carbonate buffer and blocked with 10% calf serum in TBST (0·01 M Tris base, 0·015 M NaCl, with 0·1% Tween 20, pH 8·0). A standardized bacterial cell suspension (2·5x108 c.f.u. ml-1) was lysed by repeated freeze-thawing; 50 µl of each lysate was then added to the wells and incubated at 37 °C for 1 h. Rabbit anti-ß-galactosidase mAb (ICN) in TBST was added to the wells and incubated at 37 °C for 1 h, followed by incubation with goat anti-rabbit IgG conjugated to peroxidase (ICN) in TBST at room temperature for 1 h. The O-phenylenediamine dihydrochloride substrate (Sigma) in citrate/phosphate buffer (0·05 M citric acid, 0·1 M Na2HPO4, with 0·72% H2O2) was added, incubated for 30 min at room temperature and stopped with 2·5 M H2SO4. Absorbance at 490 nm was measured on a microtitre plate reader (Bio-Tek).
Complementation of htrB mutant.
To test whether the gmhX gene product exhibits MsbA activity and suppresses the temperature-sensitive phenotype of an htrB mutant, E. coli htrB::Tn10 mutant MLK53 (Polissi & Georgopoulos, 1996 ) was transformed with pGEM-TgmhX and grown at 30 °C overnight on LB plates with the appropriate selection. Twenty-four colonies of each transformant were patched onto duplicate plates containing the appropriate selective antibiotic and incubated at 30 or 42 °C, respectively.
4'-Phosphatase assay.
Whole-membrane extracts of NMB and GS158 were analysed for 4'-phosphatase activity with the kind assistance of Shib Basu and C. R. H. Raetz. Membrane extracts were incubated with [4'-32P](Kdo)2-IVA as described by Price et al. (1995) ; the products were then separated by TLC and analysed using a phosphoimager.
Polymyxin sensitivity assays.
104 and 105 c.f.u. of each meningococcal strain or mutant were resuspended in GC broth and spotted in duplicate on GC agar plates containing serial twofold dilutions of the agent to be tested. The concentration ranges for polymyxin B were 15·5500 µg ml-1. The plates were incubated overnight at 37 °C and the MIC was determined as described by Shafer et al. (1984) .
Serum bactericidal assay.
Serum bactericidal assays with 10, 25 and 50% normal human serum (NHS) were performed on meningococcal strains as previously described (Kahler et al., 1998 ). In this study, bactericidal assays were performed at 5, 15 and 30 min at 37 °C.
RT-PCR.
RT-PCR experiments were performed as previously described (Kahler et al., 1996b ). Histidine auxotrophy was examined as previously described (Erwin & Stephens, 1995
).
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RESULTS |
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The third gene in this cluster, nlaB (242 aa), shared homology (27% identity, 46% similarity over 242 aa) with E. coli lysophosphatidic-acid acyltransferases gene, plsC (Coleman, 1992 ). We have shown that nlaB encodes a meningococcal enzyme with in vivo and in vitro lysophosphatidic-acid acyltransferase activity (Shih et al., 1999
).
Since the Tn916 mutant in mutant 469 was demonstrated to have a polar effect on nlaB expression, we presumed that gmhX and nlaB were transcriptionally linked and that the promoter for nlaB must lie upstream of gmhX. RT-PCR of the orfCnlaB region demonstrated that orfC, gmhX and nlaB were co-transcribed in an operon (Fig. 4). Because these genes constitute the second operon involved in LOS inner-core biosynthesis identified in our laboratory, we named the operon ice-2 (LOS inner-core extension, locus 2).
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Construction and analysis of the meningococcal gmhX mutant
To determine which gene(s) were responsible for the LOS phenotype of mutant 469, specific mutations were constructed in orfC, gmhX or nlaB and the mutations were introduced into the parent strain NMB by homologous recombination. LOS prepared from NMBgmhX::SpR polar mutant GS152.7 and NMBgmhX::aphA-3 non-polar mutant GS158 demonstrated the truncated LOS phenotype of mutant 469 (Fig. 2a
), whereas GS156 (NMB nlaB::
SpR) exhibited the parental LOS phenotype (data not shown). Composition and linkage analysis of the LOS from gmhX mutants GS152.7 and GS158 confirmed that both mutants exhibited a Kdo2-lipid A LOS structure with variable phosphorylation at the 1' and 4' positions of lipid A, similar to mutant 469. To confirm that the mutant LOS phenotype in the GS158 mutant was caused by insertion of a non-polar cassette into gmhX and not due to a second site orfC mutation in the GS158 background, the nucleotide sequences of orfC in NMB and GS158 were examined and found to be identical. Although the predicted gmhX gene product shared homology with the N-terminal histidinol-phosphate phosphatase domain of E. coli HisB (Chiariotti et al., 1986
) and its homologues in H. influenzae (YaeD, accession number P46452) and Salmonella typhimurium (HisB, P10368), neither mutant 469 nor meningococcal gmhX mutants GS152.7 and GS158 demonstrated histidine auxotrophy.
Southern hybridization of the NMB chromosome with a gmhX-specific probe confirmed that gmhX was present as a single copy in the NMB chromosome. To demonstrate that the gmhX mutation in GS158 was responsible for the LOS inner-core defect, we restored the gmhX LOS mutant to the parental LOS phenotype by homologous recombination with a wild-type copy of gmhX. Non-polar gmhX mutant GS158 was transformed with a 4 kb PCR product amplified from GS156 (Shih et al., 1999 ), encompassing wild-type copies of orfC and gmhX, in addition to nlaB::
SpR. Spectinomycin-resistant, kanamycin-sensitive transformants were isolated and tested for mAb 3F11 reactivity. All transformants tested had a restored mAb LOS phenotype (3F11+). We concluded that the gmhX mutation was responsible for the truncated LOS mutant phenotype in mutant 469.
Phenotype of gmhX mutants
Similar to other deep rough mutants (Wilkinson et al., 1972 ), gmhX mutants demonstrated decreased growth rates at 30 °C and 37 °C based on optical density measured at 550 nm (data not shown). Outer-membrane profiles in mutant 469 appeared to be identical to parent strain NMB. The gmhX mutants demonstrated increased sensitivity to the antimicrobial agent polymyxin B. The MICs to polymyxin B were 250 µg ml-1 for parent strain NMB and 62·5 µg ml-1 for gmhX mutants.
We have previously shown that, compared to the serum-resistant encapsulated parent strain NMB, encapsulated mutant 469 was more sensitive to killing by 25 and 50% NHS (P<0·002) (Kahler et al., 1998 ). However, mutant 469 was less sensitive to killing in 25% NHS at 30 min (data not shown for the 5 and 15 min time points) when compared to encapsulated serum-sensitive LOS mutants containing heptose (Table 2
) (CMK2, P<0·001; CMK1, P<0·001). The serum sensitivity of mutant 469 was not due to a polar effect on nlaB or the deletion of orfC, since specific nlaB mutants were resistant to killing by NHS, and polar and non-polar gmhX mutants were not significantly different from 469. Mutations in other genes (e.g. gmhX, rfaD, aut) which resulted in the expression of Kdo2-lipid A LOS structures demonstrated similar survival in NHS.
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To study the possibility that inactivation of gmhX affected heptose transfer to the Kdo2-lipid A by affecting transcription of the heptosyltransferase I gene rfaC, an rfaC::lacZermC reporter construct was inserted into the wild-type background (GS159) and the non-polar gmhX mutant (GS160). No differences in ß-galactosidase activity were observed in GS159 and GS160 (data not shown), suggesting that the gmhX gene product did not affect rfaC transcription, and by inference, RfaC activity.
In Rhizobium leguminsarum, the 4'-phosphate substitution on lipid A is removed by a phosphatase before further additions (e.g. heptose) to the structure can be made. Therefore, we investigated whether gmhX could encode a phosphatase involved in Kdo2-lipid A assembly. As noted, analysis of the lipid A portion of 469 LOS by ES-MS demonstrated variable phosphorylation of the 4' position of lipid A. We tested whether whole-membrane extracts of NMB and GS158 contained lipid A 4'-phosphatase activity (Price et al., 1995 ). No release of 4'-phosphate from the [4'-32P](Kdo)2-IVA substrate was observed in either parent strain NMB or the gmhX mutant GS158.
Recently, msbA, first identified as a multicopy suppressor of htrB mutants defective in LPS synthesis, was proposed to transport nascent corelipid A molecules across the inner membrane (Polissi & Georgopoulos, 1996 ; Zhou et al., 1998
). Because the msbA gene product contains Walker box motifs and is predicted to be an ABC transporter involved in LPS biosynthesis, we postulated that gmhX could play a similar role in LOS biosynthesis and tested the ability of gmhX to complement an E. coli htrB mutant. None of the htrB mutants transformed with a multicopy plasmid expressing the gmhX gene product restored the temperature-sensitive phenotype of the htrB mutant. In addition, searches of the meningococcal and gonococcal sequence databases revealed a homologue of msbA that was distinct from gmhX.
The gmhX homologues in Helicobacter pylori [Rv0114 (CAA17308)] and Streptomyces coelicolor genomes were found with clusters of heptose biosynthesis genes (e.g. gmhA, rfaE, rfaD). In Synechocystis and Mycobacterium tuberculosis, gmhX was adjacent to gmhA homologues. Because the heptose biosynthetic pathway has not been fully characterized, we considered whether the gmhX gene product was a functional homologue of rfaD, rfaE or aut. The meningococcal homologues of rfaD and aut were inactivated by insertional mutagenesis (Table 1) and the LOS phenotypes of these mutants determined by Tricine/SDS-PAGE analysis. In the parental background (gmhX intact), meningococcal rfaD and aut isogenic mutants both exhibited heptose-deficient truncated LOS structures similar to that observed for the meningococcal gmhX mutants, demonstrating that rfaD and aut were required for heptose incorporation into LOS and were distinct in function from gmhX.
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DISCUSSION |
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Only two genes involved in bacterial heptose biosynthesis have been fully characterized (Fig. 5). gmhA (formerly called lpcA) encodes the phosphoheptose isomerase, which converts sedoheptulose 7-phosphate into D-glycero-D-manno-heptose 7-phosphate (Brooke & Valvano, 1996b
). The second characterized gene, rfaD (recently designated gmhD), encodes the ADP-L-glycero-D-manno-heptose epimerase, which converts ADP-D-glycero-D-manno-heptose to ADP-L-glycero-D-manno-heptose (Coleman, 1983
; Pegues et al., 1990
), the substrate for the heptosyltransferase reaction (Chen & Coleman, 1993
; Kadrmas & Raetz, 1998
; Sirisena et al., 1992
). Based on evidence for the initial substrate (sedoheptulose 7-phosphate) and end product (ADP-L-glycero-D-manno-heptose), a putative pathway for heptose biosynthesis was proposed to include a mutase step for conversion of the D-glycero-D-manno-heptose 7-phosphate to a D-glycero-D-manno-heptose 1-phosphate and an ADP-heptose synthetase step for formation of the activated heptose sugar ADP-D-glycero-D-manno-heptose (Eidels & Osborn, 1974
) (Fig. 5a
). ADP-heptose synthetase, rather than ADP-heptose synthase, more accurately describes the enzymatic reaction at this step and we propose the adoption of this nomenclature.
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Based on the predicted enzyme activities of RfaE and Aut, two additional models for heptose biosynthesis have recently been proposed (Valvano, 1999 ; Valvano et al., 2000
). One model predicts that the intermediate steps involve dephosphorylation of D-glycero-D-manno-heptose 7-phosphate to create D-glycero-D-manno-heptose, which is subsequently phosphorylated to produce D-glycero-D-manno-heptose 1-phosphate (Fig. 5b
). An alternative model proposes that an additional phosphate on D-glycero-D-manno-heptose 1-phosphate is added to create D-glycero-D-manno-heptose 1,7-diphosphate, which is then converted to ADP-D-glycero-D-manno-heptose (Fig. 5c
).
In this study, we describe the identification of a novel gene, gmhX, which plays a role in heptose assembly, yet is distinct from heptose biosynthesis genes characterized to date. Meningococcal mutants defective in gmhX, rfaE, aut or rfaD each produced a truncated, heptose-deficient Kdo2-lipid A LOS structure. Although gmhX homologues are present in several other species, the effect of this gene on LPS or LOS structure has not been documented previously. Based on the predicted ATP-binding and phosphatase properties of GmhX, the predicted protein is a candidate for the enzyme that removes the 7-phosphate from D-glycero-D-manno-heptose 7-phosphate (Fig. 5b) or from ADP-D-glycero-D-manno-heptose 7-phosphate (Fig. 5c
). Further investigation at the biochemical level will be necessary to determine the precise roles for GmhX, RfaE and Aut in heptose assembly. Progress in elucidating this pathway has been hampered by the lack of available substrates necessary for the development of specific enzymic assays. However, it is clear that the meningococcal rfaE, rfaD, aut and gmhX homologues encode distinct enzymes that are functionally conserved for heptose assembly.
Mutations in gmhX and other genes (aut, rfaD) yielding Kdo2-lipid A LOS structures resulted in meningococci that are more sensitive to killing by normal human sera. However, Kdo2-lipid A mutants are not as sensitive as capsule-deficient or inner-core LOS mutants containing heptose. Loss of LOS epitopes recognized by bactericidal antibodies or different alterations in outer-membrane topology created by the different truncated LOS structure are possible explanations for these results.
The juxtaposition and co-transcription of LOS and phospholipid genes in the ice-2 operon in N. meningitidis may also be important. The previously described ice-1 operon encodes two enzymes involved in the addition of glucose and N-acetylglucosamine, respectively, to Hep2-Kdo2-lipid A LOS inner core (Kahler et al., 1996b ). Upstream of these LOS glycosyltransferases in gonococcal strain FA1090 are homologues of fabF and fabG, genes that encode fatty acid biosynthetic enzymes in E. coli (C. M. Kahler and others, unpublished data). Other meningococcal operons also contain genes associated with LOS assembly adjacent to genes associated with phospholipid assembly. For example, a fabG homologue is located downstream of the lgtAE genes necessary for LOS biosynthesis (Kahler & Stephens, 1998
) and the lpxDfabZlpxA gene cluster contains the genes for lipid A (lpxD, lpxA) and fatty acid (fabZ) biosynthesis (Steeghs et al., 1997
). Since LOS constitutes a large proportion of the outer membrane in meningococci, the coordinated regulation of membrane phospholipid genes with genes involved in LOS assembly would facilitate synchronized synthesis of these critical outer-membrane components and efficient adaptation to various environmental changes.
In summary, we have identified a novel gene required for the incorporation of L-glycero-D-manno-heptose into the LOS of N. meningitidis. This gene is distinct from other heptose biosynthesis genes. It is a candidate for the phosphatase removing 7-phosphate from ADP-D-glycero-manno-heptose or D-glycero-manno-heptose of the heptose biosynthesis pathway. Our finding supports the hypothesis that the heptose biosynthesis pathway in N. meningitidis and other species is more complex than originally proposed. Furthermore, mutations in gmhX and other genes that result in Kdo2-lipid A structures demonstrate increased sensitivity to NHS. Finally, genes encoding LOS and phospholipid assembly in N. meningitidis appear to be coordinately regulated, which may facilitate adaptation to environmental changes.
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ACKNOWLEDGEMENTS |
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Received 10 January 2001;
revised 20 March 2001;
accepted 4 April 2001.