From the Divisions of Medical and Biochemical
Microbiology, ¶ Molecular Infection Biology, and
Biophysics, Research Center Borstel, Center for Medicine and
Biosciences, Parkallee 22, D-23845 Borstel, Germany
Received for publication, July 11, 2002, and in revised form, October 28, 2002
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ABSTRACT |
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Burkholderia cepacia is a bacterium
with increasing importance as a pathogen in patients with cystic
fibrosis. The deep-rough mutant Ko2b was generated from B. cepacia type strain ATCC 25416 by insertion of a kanamycin
resistance cassette into the gene waaC encoding
heptosyltransferase I. Mass spectrometric analysis of the
de-O-acylated lipopolysaccharide (LPS) of the mutant showed that it consisted of a bisphosphorylated glucosamine backbone with two
3-hydroxyhexadecanoic acids in amide-linkage, 4-amino-4-deoxyarabinose (Ara4N) residues on both phosphates, and a core oligosaccharide of the
sequence Ara4N-(1 Taxonomically, Burkholderia cepacia is a complex of
eight species or genomovars (1), which are all characterized as
nutritionally versatile as well as notoriously drug-resistant (2) and
all display a complex genome (3). Strains have been described to live
on penicillin G as the sole carbon source (4), others are able to
degrade chlorinated aromatic substrates, such as the principal
ingredient of the herbicide Agent Orange (5). Furthermore, the organism
can antagonize and repress many soil-borne plant pathogens and is
considered as a plant growth-promoting rhizobacterium (6). However,
regarding the pathogenic potential of B. cepacia, there has
been a controversial discussion about the use and especially the
liberation of this organism for biological control and soil decontamination purposes (6-9).
First described as a phytopathogen associated with soft rot on onion
bulbs (10), B. cepacia has recently emerged as an
opportunistic pathogen in humans (3). Most important is its role as a
pulmonary pathogen in patients with cystic fibrosis (CF) (2, 11, 12) or
chronic granulomatous disease (CGD) (13, 14) and in immunocompromised patients. The so-called cepacia syndrome occurs in ~20% of CF patients infected with B. cepacia, and is characterized by
fever, acute necrotizing pneumonia, and bacteremia with a high
mortality (12). In CGD patients B. cepacia pneumonia is the
second leading cause of death (13, 14).
The genome of B. cepacia consists of two to four chromosomes
with an overall genome size of 5-9 Mb and is characterized by a large
number of insertion elements (15, 16). These mobile elements could
easily facilitate the horizontal transfer of virulence genes from other
pathogenic bacteria and thus enhance the pathogenicity of B. cepacia (11). Previous studies have demonstrated that B. cepacia strains are able to invade macrophages and respiratory epithelial cells (17, 18) and can persist in macrophages; however,
without significant bacterial replication (19). This property could
contribute to the pathogenicity of the bacteria in lung damage and
their systemic spread. A number of virulence factors are known for
B. cepacia. In addition to extracellular products such as
hemolysins, proteases, and lipases, iron-binding siderophores play a
major role for virulence (20) as well as mucin binding cable pili,
which adhere to mucin in lungs of CF patients (21). Another important
virulence determinant is the lipopolysaccharide
(LPS),1 which is likely to
contribute to the inflammatory nature of B. cepacia
infections in cystic fibrosis patients. Clinical isolates as well as
environmental strains possess LPS of high inflammatory potential (22),
which induces much higher levels of tumor necrosis factor The common architecture of LPS and most steps of its biosynthesis are
well known (27). One sugar constituent of the inner core region, found
in all LPS structures, is
3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), which
is directly linked to the lipid A. Most LPS contain an
Although most clinical isolates belong to genomovar III, so far the
type strain ATCC 25416 (genomovar I) is the only strain of the B. cepacia complex with a known LPS core structure (29). Whether Ko
is a constituent of the LPS in other genomovars, as well, remains to be
investigated. We were interested in whether it was possible to generate
a deep-rough, heptose-less mutant of the type strain in order to find
out whether the mutant also contained a Ko residue in its truncated
LPS. To incorporate this unusual sugar the bacterium has to use an
additional biosynthetic pathway or at least an additional modifying
enzyme. To learn something about the reasons why bacteria replace the
conserved Kdo residues partially with Ko, the biological activity of
the mutant was compared with that of the wild type. The answers to some
of these questions are reported here.
Bacterial Strains, Plasmids, and Growth Conditions--
All
bacterial strains and plasmids used are listed in Table
I. B. cepacia was grown at
30 °C in nutrient broth, which was supplemented with 150 µg/ml
kanamycin for growth of the mutant B. cepacia Ko2b.
Recombinant C. glutamicum strains were cultivated at
30 °C in 2× YT medium supplemented with kanamycin (30 µg/ml). E. coli was grown at 37 °C in Luria-Bertani medium
supplemented with the appropriate antibiotics.
General Cloning Techniques, Gene Library Construction, and DNA
Sequence Analysis--
Manipulation of recombinant DNA was according
to standard protocols (37). DNA was transformed by electroporation
using a BioRad Gene Pulser. For gene library construction, chromosomal DNA of B. cepacia ATCC 25416 was partially digested with
Sau3AI. Fragments ranging from 1.5 to 6 kb were ligated into
the BamHI site of cloning vector pZErO2.1
(Invitrogen) and transformed into E. coli TOP10F', yielding
~6 × 105 colonies from which recombinant plasmids
were prepared. The digoxygenin-11-dUTP system (Roche Diagnostics) was
used for DNA labeling and detection in Southern experiments according
to the instructions of the manufacturer. Determination of the sequence
was carried out by cycle sequencing with custom-made primers
(MWG-Biotech) and fluorescent dye terminators on an ABI 377 sequencing
instrument (PerkinElmer Life Sciences). The computer programs Geneworks
(Intelligenetics) and Basic Local Alignment Search Tool (TBLASTN;
National Center for Biotechnology Information) were used to analyze DNA
and deduced amino acid sequences.
Cloning of waaC and Plasmid Constructions--
Approximately 2 µg of the gene library were used for in vivo
complementation of Salmonella enterica serovar
Typhimurium LT2 deep-rough strain SA1377 (waaC630) (38) as
described previously (39), and recombinant clones were tested for the
expression of smooth type LPS (S-LPS). From this library, plasmid
pBC1/1377 was isolated, and waaC was identified within its
insert. To inactivate waaC by inserting a
kanamycin-resistance cassette, the kanamycin resistance of the vector
part of pBC1/1377 had to be exchanged for a chloramphenicol resistance.
Thus, the chloramphenicol acetyltransferase gene was amplified with
primers CAT1Spe
(5'-CGCAGAATACTAGTATCCTGGTGTCC-3', SpeI site in
boldface type) and CAT2BspHI
(5'-TTTGCGTTCATGAGCTTGGCAC-3', BspHI site in
boldface type) using plasmid pHSG397 as a template and ligated with two
fragments of pBC1/1377: one HindIII-SpeI fragment, containing the waaC gene, and one
HindIII-BspHI fragment containing the origin of
replication. After ligation of the three fragments, the resulting
plasmid was opened with EcoRI cut inside of waaC,
and a non-polar kanamycin resistance cassette, obtained by
EcoRI digestion of plasmid pUC4K, was inserted. The final
plasmid was termed pBCwaaC::kan. The
inactivated waaC gene was excised as a
EcoRV-SphI fragment from plasmid
pBCwaaC::kan and inserted into the
suicide plasmid pCVD442 digested with SmaI and
SphI. The resulting plasmid
pCVDwaaC::kan was amplified in E. coli SM10
For subcloning, the waaC gene was amplified by PCR with
primers BcewaaC1 (5'-GGCGGATCCTCAGCGTGCAAAAGAT-3',
BamHI site in boldface type) and BcewaaC2
(5'-GTATCTGCAGGGCGTTACAGGAGACCG-3', PstI site in
boldface type) using plasmid pBC1/1377 as template. After digestion
with BamHI and PstI the amplificate was ligated into shuttle vector pCB20, digested with the same enzymes. The resulting plasmid pBCC was used for transformation of S. enterica mutants SA1377 (waaC630) and SL3789
(waaF511) and C. glutamicum R163 for
complementation and preparation of cell-free extracts, respectively.
For complementation of mutant Ko2b the waaC gene was excised
as a BamHI-PstI fragment from plasmid pBCC and
ligated into pBBR1Tp, a vector with a broad host range carrying a
trimethoprim resistance. Deep-rough mutant Ko2b was transformed with
the resulting plasmid pBBR1Tp/waaC, and recombinant clones
were analyzed for expression of the S-form LPS in silver-stained
SDS-polyacrylamide gels.
A PCR cloning strategy based on data from the ongoing genome sequencing
project of B. cepacia genomovar III strain J2315 at the
Sanger Center (www.sanger.ac.uk) was employed for cloning of the Kdo
transferase (waaA). Based on sequence homologies to other
waaA genes, primers BcwaaA1 (5'-TTGAGCGCGCGCCTGCTTCG-3') and
BcwaaA2 (5'-ACGCGAGTCCGGGCGGCATCG-3') were designed to amplify by PCR a
waaA-encoding fragment from chromosomal DNA of B. cepacia ATCC 25416. The amplificate was directly cloned into
vector pT-Adv using the AdvanTAge PCR cloning kit
(Clontech) and expressed in E. coli
XL1-Blue employing blue/white screening. Sequence analysis was carried
out on both strands of five different clones as well as on a direct
amplificate of waaA from chromosomal DNA. For in vitro analysis waaA was amplified by PCR using primers
BcwaaAATG (5'-GCATGCTGCGGGTGATCTATCGCG-3'),
BcwaaAStop (5'-CGTCTATGCGTCGTCGGCAGCGTC-3') and Pfu polymerase. After purification the amplificate was
phosphorylated with polynucleotide kinase (Stratagene) and ligated as a
blunt-end fragment into vector pJKB20 (40), digested with
EcoRV, and transformed into E. coli XL1-Blue.
Resulting transformants were analyzed for correct orientation by
restriction with BamHI. One construct was termed pQB2,
transformed into C. glutamicum R163 and sequenced.
Construction and Genetic Characterization of the Chromosomal
Knockout Mutation--
B. cepacia was transformed with
plasmid pCVDwaaC::kan by
electroporation, and recombinant clones were selected on agar plates supplemented with 200 mg/liter kanamycin sulfate. The resulting chromosomal knockout mutant was termed B. cepacia Ko2b and
was characterized by Southern hybridization and analytical PCR using chromosomal DNA as template. For this purpose the primer pair BCCKO1
(5'-GTGCAAAAGATCCTGATCG-3') and BCCKO2 (5'-TTACAGGAGACCGAAGCC-3') was
used to amplify the complete waaC gene, whereas primers
SacB1 (5'-TAACCTTTACTACCGGACTG-3') and SacB2
(5'-TTGTCCTTGTTCAAGGATGC-3') were used to identify remaining vector
sequences of pCVDwaaC::kan by
amplification of the sacB gene. Three different probes were used for Southern experiments. The first probe was an EcoRI
fragment of pUC4K containing the kanamycin resistance cassette
introduced into the waaC gene, the second harbored the
sacB gene from pCVD442 on a PstI fragment and the
third contained nucleotides 73-724 of the waaC gene
including the insertion site of the kanamycin resistance cassette as
EcoRV-EcoRI fragment. The chromosomal DNA isolated from B. cepacia Ko2b was digested with
EcoRI, PstI, or EcoRV-SphI, respectively.
Nucleotide Sequence Accession Numbers--
The sequences
encoding the Kdo transferase (WaaA) and the heptosyltransferase I
(WaaC) of B. cepacia ATCC 25416 were
deposited at GenBankTM/EBI and are available under
accession numbers AJ420777 and AJ302064, respectively.
In Vitro Analysis of Cloned Glycosyltransferases--
Cell-free
extracts containing Kdo transferase or heptosyltransferase I from
B. cepacia were prepared from C. glutamicum
strains R163/pQB2 and R163/pBCC, respectively, as described previously (41). The in vitro tests for Kdo transfer were carried out
in a total volume of 20 µl containing HEPES (50 mM, pH
7.5), MgCl2 (10 mM), and Triton X-100 (3.2 mM). CMP-Kdo was generated in situ from Kdo (2 mM) and CTP (5 mM) using purified CMP-Kdo
synthetase as described (40). The synthetic tetraacylated lipid A
precursor of E. coli, compound 406 (42), was used as lipid
acceptor for Kdo transfer at a concentration of 100 µM.
As Kdo transferases for control reactions cell-free extracts of
R163/pJKB16 (E. coli) or R163/pCB23
(Haemophilus influenzae) were used (41). A crude cell-free extract obtained from B. cepacia ATCC 25416 served
as additional control. For heptose transfer, reactions were carried out
in two steps; first, Kdo2-406 was generated by incubation with WaaA from E. coli for 1 h at 37 °C and stopped
by boiling for 10 min. Thereafter, ADP-heptose and cell-free extract
containing a heptosyltransferase were added to the mixture, another
incubation of 1 h at 37 °C followed, and reactions were stopped
by freezing at SDS-PAGE--
Proteinase K-digested cell-free lysates were
prepared from recombinant strains (44), separated by SDS-PAGE, and
detected with alkaline silver nitrate (48).
Preparation and Chemical Analysis of LPS--
B.
cepacia wild-type LPS was extracted according to the phenol/water
method (49). B. cepacia Ko2b was cultivated for 24 h at
30 °C in nutrient broth supplemented with 150 mg/liter kanamycin sulfate. The cells were dried as described (50), and LPS was extracted
using a phenol/water/sarkosyl procedure followed by treatment of the
aqueous phase material with phenol/chloroform/light petrol ether (51).
Analyses of glucosamine, Kdo, and phosphate were performed as described
(52). Analysis of complete fatty acids was carried out according to
Ref. 53. LPS was de-O-acylated by mild hydrazinolysis (54),
phosphate groups were removed with 48% hydrofluoric acid (4 °C;
48 h), and the resulting product was dialyzed, reduced with
NaBH4, and subjected to strong alkaline hydrolysis to
achieve de-N-acylation (54). After neutralization and
desalting single oligosaccharides were separated by analytical high
performance anion exchange chromatography on a Dionex DX300 system as
described previously (50). De-O-acylated LPS
(LPSde-O-Ac) and isolated oligosaccharides were subjected
to mass spectrometry (MS). To determine the position of Ara4N in the
core oligosaccharide of mutant Ko2b, LPSde-O-Ac was
subjected to mild methanolysis followed by N-acetylation and
permethylation for analysis by gas chromatography-MS (GC-MS) as
described by Isshiki et al. (29).
Mass Spectrometry--
Mass spectrometric analysis was performed
in the negative ion mode using an electrospray Fourier-transform ion
cyclotron resonance (ESI-FT-ICR) mass spectrometer (APEX II, Bruker
Daltonics, Billerica, MA) equipped with a 7 Tesla actively
shielded magnet and an Apollo ion source. Mass spectra were acquired
using standard experimental sequences as provided by the manufacturer.
Samples were dissolved at a concentration of ~20 ng/µl in a
50:50:0.001 (v/v/v) mixture of 2-propyl alcohol, water, and
triethylamine and sprayed at a flow rate of 2 µl/min.
Capillary entrance voltage was set to 3.8 kV, and dry gas temperature
to 150 °C. Capillary skimmer dissociation (CSD) was induced by
increasing the capillary exit voltage from Isolation and Cultivation of Human Monocyte-derived
Macrophages--
Mononuclear cells were isolated from the peripheral
blood of healthy volunteers by density gradient centrifugation (55). Lymphocytes and monocytes were separated by counterflow elutriation as
described previously (56). Highly purified (consistently >95%)
monocytes were cultivated for 7 days in teflon culture bags (CellGenix,
Freiburg, Germany) in RPMI 1640 (Biochrom, Berlin, Germany) in the
presence of 2% (v/v) heat-inactivated human AB-serum, 2 ng/ml human
M-CSF (R&D Systems, Wiesbaden, Germany), 2 mM
L-glutamine (Biochrom), 100 units/ml penicillin G, and 100 µg/ml streptomycin (both Biochrom). Viable cells were determined by
trypan blue staining. Macrophages were phenotyped by cell surface
marker expression profile (CD14, macrophage mannose receptor,
carboxypeptidase M, HLA-DR) as described previously (57). Macrophages
were cultivated in 24-well flat-bottom microtiter plates (Nunc,
Roskilde, Denmark) in RPMI 1640 containing 10% (v/v) heat-inactivated
fetal calf serum (Biochrom) and 2 mM
L-glutamine. Cultures were incubated at 37 °C in a
humidified atmosphere containing 5% CO2.
Macrophage Invasion Assay--
Invasion assays were carried out
as described earlier (18). Briefly, B. cepacia was grown at
30 °C to late-exponential phase, cells were washed and diluted in
phosphate-buffered saline, and then added to 4 × 105
human macrophages (at a concentration of 8 × 105 per
ml) at a multiplicity of infection (m.o.i.) of 1-7 bacteria per
macrophage. After 1 h of incubation at 37 °C macrophages were washed vigorously with warm Hank's balanced salt solution (HBSS) to
remove extracellular bacteria. Fresh medium containing ceftazidime (1 mg/ml) and amikacin (1 mg/ml) was added to kill extracellular bacteria,
and cultures were incubated for an additional 2 h at 37 °C. To
determine bacterial uptake macrophages were lysed after 1 and 3 h
of incubation by addition of 0.1% saponin. Lysates were serially
diluted and plated on agar. As controls, bacteria were cultivated
without macrophages and antibiotics to monitor growth rates at
37 °C.
Stimulation of Human Macrophages and Detection of TNF- Cloning and Sequence Analysis of waaA from B. cepacia--
DNA
sequences from the ongoing genome sequencing project of B. cepacia genomovar III strain J2315 were searched for homologies with known Kdo transferase genes. Two contigs were identified including
the start and the end of a putative Kdo transferase gene. The putative
waaA gene was amplified by PCR from chromosomal DNA of
B. cepacia ATCC 25416 (type strain; genomovar
I). The resulting amplificate of ~1500 bp was directly cloned into
vector pT-Adv and sequenced. Sequence analysis of both strands revealed
an open reading frame encoding a protein of 452 amino acids and a
predicted molecular mass of 48 kDa. Its amino acid sequence showed
highest homology to the Kdo transferases from Bordetella
pertussis and Bordetella bronchiseptica (43%
identity, 54% similarity). The protein contained a glycosyltransferase
I motif at position 309-413 according to the Pfam data base, which is
present in all Kdo transferases investigated so far (39, 59). After
subcloning into the shuttle vector pJKB20 both strands of five
different clones were sequenced. For comparison, a direct amplificate
of waaA from chromosomal DNA was sequenced. One clone showed
a point mutation and was discarded; all other sequences were identical.
Cloning and Sequence Analysis of waaC from B. cepacia--
The
waaC gene from B. cepacia was cloned from a gene
library by functional complementation of S. enterica mutant
SA1377 (waaC630) (38). Selection of transformants with
novobiocin and phage Ffm (39) resulted in the discovery of plasmid
pBC1/1377. As shown on silver-stained SDS-polyacrylamide gels of
proteinase K-treated whole cell lysates, the plasmid was able to
restore the biosynthesis of smooth LPS in S. enterica SA1377
(waaC630) (data not shown). Sequence analysis of pBC1/1377
revealed an open reading frame encoding a protein of 331 amino acids
and a predicted molecular mass of 36 kDa. The deduced amino acid
sequence contained a heptosyltransferase motif (Pfam data base
accession no. PF01075, amino acids 76-330) and possessed significant
homology to other known heptosyltransferases I; highest homology was
shown to WaaC of P. aeruginosa (42% identity, 57%
similarity). Downstream of the waaC gene more putative open reading frames were identified; however, no functions could be assigned. After subcloning of the waaC gene in a shuttle
vector the resulting plasmid pBCC was transformed into S. enterica mutants SA1377 (waaC630) and SL3789
(waaF511) for complementation studies. As shown in Fig.
1 the enzyme was able to restore
expression of S-form LPS in strain SA1377 (lane 4) but not
in SL3789 (lane 5), indicating that WaaC of B. cepacia is functionally equivalent to other heptosyltransferases I
(39, 40, 43, 60).
In Vitro Characterization of the Cloned Kdo Transferase and
Heptosyltransferase I of B. cepacia--
The putative open reading
frame of the waaA gene was amplified by PCR and cloned into
the shuttle vector pJKB20 to give plasmid pQB2. Plasmids pQB2 and pBCC
were used to transform C. glutamicum R163. Cell-free
extracts were prepared from both strains and subjected to in
vitro assays. The activity of WaaA was tested as described (39),
using the synthetic lipid acceptor 406, Kdo, CTP, and CMP-Kdo
synthetase from E. coli. As controls the Kdo transferases from E. coli and H. influenzae were included as
well as the cell-free extract from B. cepacia ATCC 25416. All transferases converted acceptor 406 into one or more polar
products, which reacted with mAb A20, indicating the transfer of at
least one Kdo residue (Fig. 2A). The cloned Kdo
transferase of B. cepacia is clearly bifunctional comparable
to that of E. coli and also the crude extract from B. cepacia generated a product that reacted with mAb A17,
demonstrating the presence of a 2
The activity of WaaC from B. cepacia was investigated using
synthetic lipid acceptor 406 in a coupled enzyme assay with Kdo transferase from E. coli and a low molecular mass filtrate
from E. coli WBB06 (44) as the source of ADP-heptose (40).
The cloned WaaC of E. coli was used as a reference (40).
Both transferases led to the formation of a more polar product than the
acceptor structure Kdo2-406 after separation of reaction
products on TLC plates (Fig. 3,
lanes 2 and 3). Bands were either
visualized with mAb S36-20, specific for Rd2-type LPS (47),
to identify reaction products (Fig. 3, panel B), or with mAb
A20 (45), recognizing a terminal Kdo residue, to identify the reaction
intermediate Kdo2-406 (Fig. 3, panel A). When
either heptosyltransferase I or ADP-heptose were omitted from the
reaction mixture no Rd2-type LPS was generated (Fig. 3,
lanes 4 and 5).
Construction and Genetic Characterization of the Deep-rough B. cepacia Mutant Ko2b--
The deep-rough LPS mutant Ko2b was
constructed by allelic replacement of the waaC gene in the
genome of B. cepacia. For this purpose, the cloned
waaC gene on plasmid pBC1/1377 was inactivated by insertion
of a non-polar kanamycin resistance cassette derived from vector pUC4K.
The resulting construct was subcloned into the suicide vector pCVD442,
which possesses a pir-dependent origin of
replication and needs the presence of the Chemical Characterization of LPS from B. cepacia Wild Type and
Strain Ko2b--
B. cepacia wild type LPS was extracted
according to the phenol/water method (49), whereas preliminary
experiments had shown that extraction of mutant Ko2b gave highest
yields when 2% N-lauroyl sarkosyl sodium was added to the
phenol/water mixture (51). For further purification, the material was
extracted with phenol/chloroform/light petrol ether, and the LPS
obtained was dissolved in water and precipitated with ethanol/acetone.
Separation of LPS on SDS-polyacrylamide gels (Fig.
4) clearly showed that B. cepacia wild type expressed S-form LPS with a typical ladder
pattern (lane 2), whereas the mutant Ko2b appeared as
deep-rough Re-type LPS (lane 3) comparable to that of
S. enterica strain SA1377 (lane 1). When mutant
Ko2b was transformed with plasmid pBBR1Tp/waaC, the ability to express complete S-form LPS was restored (data not shown).
Chemical analyses of Ko2b LPS identified GlcN, Kdo, and phosphorous, as
well as the fatty acids 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid, tetradecanoic acid, and hexadecanoic acid.
To elucidate the chemical structure of the core region of the mutant
LPS in more detail LPS was de-O-acylated by mild
hydrazinolysis (LPSde-O-Ac). Negative ion ESI-FT-ICR mass
spectra of LPSde-O-Ac showed several single-, double-, and
triple-charge molecular ion peaks. For easier interpretation the charge
deconvoluted spectrum is given in Fig.
5A comprising two prominent
molecular ion peaks [M-H]
That the Ara4N linked to the core was exclusively linked to the
terminal Ko is shown in Fig. 7. When
analyzed by GC-MS after mild methanolysis followed by
N-acetylation and permethylation of
LPSde-O-Ac, fragment ions at m/z
291
Mass spectrometric analyses of the core oligosaccharides obtained after
reduction, dephosphorylation and de-N-acylation of LPSde-O-Ac, and separation by high performance anion
exchange chromatography revealed three fractions with molecular masses of [M-H] Infection of Human Macrophages with B. cepacia Wild Type and Mutant
Ko2b--
The capability of B. cepacia mutant Ko2b to
invade human monocyte-derived macrophages was investigated in
comparison to that of the wild type strain. Since both strains differ
only in the architecture of their LPS we wanted to find out whether a
deep-rough mutant was still able to invade macrophages and to survive
at comparable rates. Human macrophages were infected and plated after 1 and 3 h of incubation post-infection (see Table
II). The rate of infection, presented as
the percentage of the bacterial inoculum that was detected in
macrophage lysates after 1 h of infection, varied between 0.7 and
1.3% for the wild type and between 1.0 and 4.9% for the mutant.
Furthermore, the intracellular survival of B. cepacia,
depicted as percentage of bacteria that survived 2 h of
extracellular antibiotic treatment, was between 7.5 and 46% and
between 6.3 and 33% for the wild type and the mutant, respectively.
The relatively large scatter regarding the survival rates was most
likely due to the fact that no macrophage cell line was used but
primary cells that were derived from isolated monocytes of individual
blood donors. It is known that human individuals usually show a broad
range of reaction profiles when tested in biological assays; however,
data obtained from the same donor depict a high degree of consistency.
From a total of four experiments our results clearly showed that the
truncation of the LPS did not impair the mutant in its capacity to
invade macrophages or to survive inside the macrophages when compared
with the wild type strain. Thus, no major difference could be pointed
out between the behavior of the wild type and the mutant B. cepacia strain.
TNF- The main chain of the inner core region in the LPS of B. cepacia consists of a trisaccharide with two heptose and one Kdo residues. However, the substitution at position 4 of the Kdo is a Ko
residue and not a second Kdo residue as in
Enterobacteriaceae, nor a phosphate group as in
Vibrio, Haemophilus, or Bordetella. This sugar has been found in other species as well (30, 34, 63), but
only in B. cepacia ATCC 25416 Ko was found to replace Kdo in
stoichiometric amounts. So far it is not known whether Ko is the
product of a separate biosynthetic pathway and is transferred by a
specific Ko transferase or whether it is generated by oxidation of free
Kdo, CMP-Kdo, or after incorporation into the LPS. To answer these
questions, it was necessary to determine first whether the Kdo
transferase of B. cepacia is a mono- or bifunctional enzyme, since a monofunctional Kdo transferase would strongly support the
incorporation of Ko by a specific Ko transferase. Cell-free extracts,
prepared from B. cepacia ATCC 25416, were used as a source
for Kdo transferase and tested in in vitro experiments. These extracts exhibited a bifunctional action, generating a
disaccharide linked to the lipid A acceptor. However, it was not
possible to exclude the presence of a putative Ko transferase in these
assays and because it is not known whether the antibodies used were
able to distinguish between Ko and Kdo in the reaction product. To investigate the Kdo transferase in detail the enzyme was cloned and
expressed in the Gram-positive host C. glutamicum. The
cloned enzyme displayed a bifunctional activity when Kdo was included in the assay. Interestingly, it was not possible to clone
waaA from a gene library of B. cepacia in analogy
to other Kdo transferases (39, 64, 65) in E. coli strain
CJB26, harboring an intact copy of the waaA gene on a
temperature-sensitive plasmid (64). For the monofunctional Kdo
transferase from H. influenzae it was shown that
the enzyme supported growth of E. coli CJB26 (66). However,
in another system a viable mutant could only be generated when the Kdo
kinase was also present (50). One explanation why the Kdo transferase
from B. cepacia was not able to support growth could be the
large phylogenetic difference between both species; E. coli
belongs to the To study the biosynthesis of the inner core region more closely it was
necessary to generate a genetically defined deep-rough mutant of
B. cepacia. We decided to do so by inactivation of
heptosyltransferase I. The waaC gene was cloned from a gene
library by in vivo complementation and tested. It displayed
a monofunctional activity in vivo and in vitro,
comparable to all other WaaC enzymes investigated so far (39, 40, 43,
67-69). Thus, the heptosyltransferase of B. cepacia acted
as well on acceptors containing Ko or Kdo, indicating that the enzyme
is not able to distinguish between Kdo and Ko in the acceptor molecule.
By inserting a kanamycin resistance cassette into waaC the
gene was inactivated in vitro and introduced into B. cepacia where the plasmid was integrated into the chromosome by
homologous recombination. The instable, merodiploid intermediate had to
undergo a second recombination event, and thus the active copy of
waaC and additional vector sequences were excised, leaving the inactivated copy of heptosyltransferase I on the chromosome. The
resulting mutant Ko2b depicted a deep-rough LPS phenotype in
silver-stained gels (Fig. 4) and could be complemented to express complete S-form LPS by transformation with a plasmid harboring waaC from B. cepacia (data not shown).
We constructed a deep-rough mutant in order to obtain an LPS with the
lowest complexity to facilitate the structural analysis, which
indicated that the mutant also contains Ko as a substituent for Kdo.
Therefore, it is concluded that the incorporation of Ko is achieved
before the first heptose residue is added to the LPS. From earlier
investigations on B. cepacia LPS (70) it was known that only
minor amounts of Kdo were detectable and that at least one phosphate
group of the lipid A was substituted with Ara4N. Our data showed that
both phosphate groups are substituted with Ara4N, confirming
assumptions by Helander et al. (71) that in B. cepacia also the glycosidically linked phosphate is substituted with Ara4N. Further structural investigations revealed the presence of
the trisaccharide Ara4N (1 Attachment of Ara4N residues to the phosphate groups of lipid A leads
to polymyxin resistance in a number of bacteria (72-75). Recently, the
enzyme responsible for the transfer of Ara4N to both phosphate groups
of lipid A has been identified in S. enterica and E. coli and was termed ArnT (76). A homolog of the arnT gene could also be identified in the genome of B. cepacia; however, it remains to be elucidated whether the same
transferase incorporates Ara4N also into the inner core region. Our
results showed, that in B. cepacia the substitution with
Ara4N always occurs at the outer sugar, mostly the Ko. In contrast,
structural investigations of LPS on various serotypes of the genus
Proteus showed a substitution of the inner Kdo residue with
Ara4N (77). It will be interesting to investigate and compare all those
Ara4N transferases for the substrate specificity they need to
accomplish their different tasks. It also remains to be elucidated how
Ko is incorporated into the LPS. This becomes even more challenging
since Ko was found to be a component of the LPS more often in a variety
of bacteria (30, 34, 63). Whether these organisms share a specific Ko
transferase and a synthesis pathway to generate Ko or they possess
machinery to transform Kdo into Ko will be the subject of future studies.
The fact that LPS is a pathogenicity factor and might play an important
role during an infection with B. cepacia led us to compare
the wild type strain ATCC 25416 and the mutant Ko2b in biological test
systems. First, the capacity to infect human monocyte-derived macrophages was analyzed. It turned out that the mutant showed infection rates comparable to the wild type (Table II). Thus, the
truncation of the LPS does not seem to impair the organism to interact
with macrophages and infect them. However, growth rates were slightly
lower at 37 °C compared with the wild type. In a second set of
experiments LPS preparations from both strains were tested for their
ability to induce TNF- 8)
D-glycero-D-talo-oct-2-ulosonic acid
(Ko)-(2
4)3-deoxy-D-manno-oct-2-ulosonic
acid (Kdo). The mutant allowed investigations on the biosynthesis of
the LPS as well as on its role in human infection. Mutant Ko2b showed
no difference in its ability to invade human macrophages as compared with the wild type. Furthermore, isolated LPS of both strains induced the production of tumor necrosis factor
from macrophages to
the same extent. Thus, the truncation of the LPS did not decrease the
biological activity of the mutant or its LPS in these aspects.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF-
)
in monocytes than the LPS from Pseudomonas aeruginosa (23,
24) and stimulates the up-regulation of other proinflammatory cytokines
(25, 26).
(2
4)-linked Kdo disaccharide in their inner core region;
however, several bacteria like Haemophilus,
Vibrio, and Bordetella depict one phosphorylated
Kdo in this position (28). In case of LPS from B. cepacia
ATCC 25416 it was revealed that the inner core region consists of a
D-glycero-D-talo-oct-2-ulosonic acid (Ko)
(2
4) Kdo disaccharide where the Ko residue is
substituted non-stoichiometrically with 4-aminoarabinose (Ara4N) in
position 8 (29). The unusual sugar Ko has been found earlier as a
substitute for Kdo in the LPS of Acinetobacter (30-32) and
Tatlockia (33) and was recently identified in the LPS of
Yersinia pestis (34), but so far nothing is known about the
significance of this substitution. Interestingly, the core region of
Burkholderia caryophylli does not contain Ko (35). A lot of
data exist about the biosynthesis and transfer of Kdo into the LPS
(36); however, no data are available about the incorporation of Ko. One
could assume that a specific enzyme introduces a hydroxyl function at
position 3 of the side branch Kdo. On the other hand, Ko could be
synthesized by an independent pathway and transferred by a specific
transferase. To answer these questions we investigated the early
glycosyltransferases involved in the biosynthesis of the LPS in
B. cepacia ATCC 25416. First, the Kdo transferase (WaaA) of
this strain was cloned and characterized to determine whether it is a
mono- or bifunctional enzyme. We show here that B. cepacia
harbors a bifunctional Kdo transferase comparable to WaaA from
Escherichia coli. Second, the gene encoding
heptosyltransferase I (waaC) was cloned from B. cepacia, expressed in Corynebacterium glutamicum and
characterized as a monofunctional heptosyltransferase in
vitro and in vivo.
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DISCUSSION
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Strains and plasmids used in this study
pir.
20 °C. The heptose donor ADP-heptose was provided
by a partially purified (43) cell extract of the
heptosyltransferase-deficient E. coli strain WBB06 (44). The
control heptosyltransferase was prepared from R163/pJKB10 (E. coli) (40). Aliquots were spotted on silica-60 TLC plates (Merck)
and developed in a solvent mixture of chloroform/pyridine/88% formic
acid/water (30:70:16:10, v/v). Blotting and immunological
identification of the reaction products was performed as described (40)
using monoclonal antibodies A20 (45), A17 (46), and S36-20 (47),
recognizing a terminal Kdo residue, a Kdo disaccharide (Re-type LPS),
and Rd2-type LPS, respectively.
100 V to
350 V. Infrared-multiphoton dissociation of isolated parent ions was performed
with a 35 watt, 10.6 µm CO2 laser (Synrad, Mukilteo, WA).
The unfocused laser beam was directed through the center of the ICR
cell for 40 ms, and the fragment ions were detected after a delay of
0.5 ms.
Release--
4 × 105 human macrophages (at a
concentration of 8 × 105 per ml) were stimulated with
LPS isolated from B. cepacia (wild type and mutant Ko2b) and
S. enterica serovar Friedenau as control. Culture
supernatants of macrophages were harvested 4 h post-stimulation and stored at
20 °C until further analysis. For detecting TNF-
in culture supernatants a sandwich-ELISA was performed as recommended by the manufacturer (H. Gallati, c/o Intex, Muttenz, Switzerland) (58).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
LPS phenotype of S. enterica deep-rough mutants complemented with plasmid
pBCC encoding WaaC from B. cepacia. Protein-free
whole cell lysates of the strains were separated by SDS-PAGE and
detected with alkaline silver nitrate. Lane 1, SA1377
(Re-type LPS); lane 2, SL3789 (Rd2-type LPS);
lane 3, SL3770 (smooth LPS); lane 4, SA1377/pBCC;
lane 5, SL3789/pBCC.
4-linked Kdo disaccharide (Fig.
2B). Only the transferase from H. influenzae
displayed mostly monofunctional activity.
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Fig. 2.
In vitro activity of the Kdo
transferase (WaaA) from B. cepacia. Reaction
products were separated by TLC and detected with monoclonal antibodies
after blotting on polyvinylidene difluoride membrane. Reaction products
were visualized with mAb A20, recognizing a terminal Kdo residue
(A) and mAb A17, recognizing a 2 4-linked Kdo
disaccharide (B), respectively. Lane 1, enzyme
reaction with WaaA from H. influenzae; lane 2,
enzyme reaction with WaaA from E. coli; lane 3,
enzyme reaction with WaaA from B. cepacia; lane
4, enzyme reaction with a cell-free extract from B. cepacia. The asterisk indicates reaction products that
are based on an underacylated contaminant of the acceptor 406.
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Fig. 3.
In vitro activity of
heptosyltransferase I (WaaC) from B. cepacia.
Reaction products were separated by TLC and detected with monoclonal
antibodies after blotting on polyvinylidene difluoride membrane. Lipid
acceptor Kdo2-406 and reaction products were visualized
using mAb A20, recognizing a terminal Kdo residue (A) and
mAb S36-20, recognizing Rd2-type LPS (B),
respectively. Lane 1, Kdo2-406; lane
2, enzyme reaction with WaaC from E. coli; lane
3, enzyme reaction with WaaC from B. cepacia;
lane 4, as lane 3 but without WaaC; lane
5, as lane 3 but without ADP-heptose. The
asterisk indicates reaction products that are based on an
underacylated contaminant of the acceptor 406.
-protein of phage lambda
to replicate (61). When introduced into B. cepacia, only those cells survived selection on kanamycin, which had integrated the
plasmid into the chromosome by homologous recombination. To enforce
excision of the wild type allele and remaining vector sequences from
the chromosome, a counter selection on sucrose was carried out. The
sacB gene from Bacillus subtilis, also present on
plasmid pCVD442, encodes a levansucrase that synthesizes a polymer from
sucrose, which is lethal in many bacteria (62). When cultivated on agar
plates containing sucrose and kanamycin only those mutants survived in
which the wild type allele had been replaced by the inactivated copy of
waaC, and vector sequences were no longer present on the
chromosome. One of the isolated mutants was termed B. cepacia Ko2b and further analyzed. To confirm the construction of
mutant Ko2b analytical PCRs were carried out using primers BCCKO1 and
BCCKO2 as well as SacB1 and SacB2. Primers BCCKO1 and BCCKO2 bound to
the 5'- and 3'- region of the waaC gene, respectively, and
led to amplification of the complete gene. Using chromosomal DNA from
wild type B. cepacia as a template, an amplificate of ~1
kb was generated, consistent with the length of waaC (996 bp). However, when chromosomal DNA from mutant Ko2b was used as
template, the sole amplificate showed a length of ~2.3 kb, indicating
the presence of the kanamycin resistance cassette (1282 bp) within the
sequence of waaC (996 bp) (data not shown). Primer pair
SacB1 and SacB2 were designed for amplification of the sacB
gene. Using DNA from plasmid pCVD442 as template, a PCR product of
~1.3 kb was obtained, consistent with the length of sacB
(1347 bp). When either chromosomal DNA from B. cepacia wild type or mutant Ko2b was used as template, no amplificate could be
obtained, indicating that the vector sequence had been excised from the
chromosome (data not shown). To further verify the results, Southern
hybridization was carried out using three different probes. Hybridization of chromosomal DNA from either wild type or mutant Ko2b
with probes against (i) the kanamycin resistance cassette, (ii) the
sacB gene, and (iii) the waaC gene gave signals
(a) only with Ko2b DNA, (b) only with control
plasmid, and (c) with all DNA. However, with Ko2b DNA the
hybridizing fragment was ~1.3 kb larger than that of wild type DNA
consistent with the insertion of the kanamycin resistance cassette
(1282 bp) (data not shown). Taken together, the results indicate that
the waaC gene on the B. cepacia chromosome was
successfully replaced by an inactivated copy.
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Fig. 4.
Characterization of the LPS from mutant
B. cepacia Ko2b. Purified LPS (10 µg per lane)
was separated by SDS-PAGE and detected with alkaline silver nitrate.
Lane 1, S. enterica strain SA1377 (Re-type LPS);
lane 2, B. cepacia wild-type; lane 3,
B. cepacia mutant Ko2b.
1 at m/z
1725.7 and 1856.8, which were in agreement with the calculated molecular weights (1726.68 and 1856.74) of the expected molecular structure depicted in Fig. 6 consisting
of one Ko, one Kdo, two glucosamines, two phosphate groups, two
hydroxyhexadecanoic acids, and two or three Ara4N residues,
respectively. Both peaks were accompanied by peaks of low intensity
originating from the exchange of Ko by Kdo
(
m/z
16) and from sodium adduct ions
(
m/z + 22). Tandem mass spectrometric anaylsis
using infrared multiphoton dissociation (IRMPD) of the two main
components exhibited several fragment ions of diagnostic importance due
to the cleavage of the linkage between lipid A and the core
oligosaccharide, the loss of Ara4N units
(
m/z
131), and the loss of phosphate residues (
m/z
98 and
80). The MS/MS spectrum (Fig.
5B) obtained from the LPS carrying three Ara4N residues
(m/z 1856.8) as parent ion comprises four diacyl
species (m/z 1856.8, 1725.7, 1594.7, and 1643.7),
three diacyl lipid A fragment ions (m/z 1269.6, 1138.6, and 1007.5) and two core fragments (m/z
455.1 and 587.2), which differ from one another by
m/z
131. Furthermore, cleavage of phosphate
was observed from diacyl lipid A carrying no Ara4N substitution or one
Ara4N residue but not from lipid A carrying two Ara4N residues indicating that both phosphates must have been substituted by Ara4N.
MS/MS from LPS carrying only two Ara4N (spectrum not shown) exhibits
the same lipid A fragment ions but only one core fragment ion at
m/z 455.1 indicating that LPS carries three Ara4N
residues, lipid A two, and the core, in non-stoichiometric amounts, one Ara4N residue. These results were further confirmed by capillary skimmer dissociation, which generated the same fragment ions (data not
shown).
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Fig. 5.
Negative ion ESI-FT-ICR mass spectra of
de-O-acylated LPS from B. cepacia
mutant Ko2b. A, charge deconvoluted mass
spectrum; B, IRMPD-MS/MS of the molecular ion at
m/z 1856.8.
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Fig. 6.
Structure of the LPS from B. cepacia mutant Ko2b as derived from mass spectrometry and
GC-MS analysis. The substitution of Ko with Ara4N is
non-stoichiometric. In a minor fraction, Ko is replaced by Kdo.
FA, 3-hydroxyhexadecanoic acid.
259 (
MeOH) and 216
156 (- HOAc) indicated the
presence of Kdo at the reducing end and Ara4NAc at the nonreducing end
of the oligosaccharide. The two fragment ions at
m/z 522 and 597 were in agreement with Ara4NAc
Ko and Ko
Kdo moieties of the trisaccharide, respectively. The
same fragmentation pattern was previously described by Isshiki et
al. (29). Chemical ionization MS revealed a pseudomolecular ion
(M+NH4)+ at m/z 847, which was in accordance with the molecular mass of the structure
depicted in Fig. 7 (data not shown).
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Fig. 7.
Electron impact mass spectrum of an
oligosaccharide obtained from B. cepacia mutant
Ko2b. De-O-acylated LPS was subjected to mild
methanolysis, N-acetylation, and permethylation as described
elsewhere (29).
1 at m/z 781.27, 797.27, and 928.33, representing a glucosamine-glucosaminol backbone
substituted with a Kdo disaccharide, a Ko-Kdo disaccharide and an
Ara4N-Ko-Kdo trisaccharide, respectively (data not shown).
Infection of human macrophages of four different donors with B. cepacia
ATCC 25416 and mutant Ko2b
Release of Human Macrophages in Response to LPS of B. cepacia--
To examine the potential of isolated wild type and mutant
LPS to activate macrophages, the induction of TNF-
was determined. Isolated LPS from S. enterica serovar Friedenau served as
control (Fig. 8). The results
demonstrated that LPS of B. cepacia is a potent inductor of
TNF-
, which is in accordance with data obtained from other groups
(19, 23, 26). In comparison to LPS from S. enterica the
preparation of B. cepacia mutant Ko2b is somewhat less
active; however, saturation of TNF-
induction is reached at a
concentration of 10 ng of LPS/ml. The fact that the preparation of
B. cepacia wild type LPS is about 10-fold less active than Ko2b LPS can be explained by the large difference in the molecular weight of both molecules. When fatty acid contents of similar amounts
of LPS were determined, B. cepacia wild type LPS contained only one-tenth of the amount obtained from mutant Ko2b LPS (data not
shown). This significant difference in molecular weight should make up
the difference found in TNF-
induction as seen in Fig. 8. Our
results indicate that the truncated LPS of the deep-rough mutant Ko2b
can induce TNF-
release in human macrophages to a similar extent as
the wild type LPS from B. cepacia.
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Fig. 8.
TNF- released by
human macrophages stimulated with LPS preparations from B. cepacia. Macrophages were stimulated with LPS
preparations from B. cepacia wildtype (open
circle), B. cepacia mutant Ko2b (filled
circle), or S. enterica serovar Friedenau as control
(filled triangle) at concentrations indicated. Culture
supernatants obtained after 4 h of stimulation were assayed for
TNF-
. Means of duplicates plus S.D. of one representative experiment
of four is shown.
DISCUSSION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-group of proteobacteria with a G + C content of
52% and B. cepacia belongs to the
-group with a G + C
content of 64%. The waaA gene has a G + C content of
73%.
8) Ko (2
4) Kdo in the core region of strain ATCC 25416; however, the substitution of Ko with Ara4N
was estimated to about 25% (29). We show here that the trisaccharide
Ara4N-Ko-Kdo is also present in the deep-rough mutant Ko2b. In
addition, an unsubstituted Ko-Kdo disaccharide was found as well as
minor amounts of a Kdo disaccharide. For unknown reasons the latter
appears in most but not all preparations investigated and may depend
upon the growth conditions. The major fraction contains two Ara4N
residues in the lipid A moiety, whereas about 40% carry a third Ara4N
residue located at the Ko (Fig. 5). The higher degree of substitution
in the deep-rough mutant in comparison to the wild type might enhance
the stability of the LPS in the absence of an outer core region and
O-antigen.
release in human macrophages (Fig. 8). It is
obvious that LPS from B. cepacia is a potent inducer of
TNF-
, although the control LPS of S. enterica is
approximately two orders of magnitude more active. If corrected for the
mass difference between wild type and mutant Ko2b LPS, there is no
significant variation in the biological activity of the LPS of both
strains. Considering these results, mutant Ko2b appears to be suited
for further biological testing. However, whether the introduced
mutation impairs its capacity to survive in an in vivo model
has to be investigated.
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ACKNOWLEDGEMENTS |
---|
We thank S. Kusomoto, Japan, for providing synthetic lipid A precursor 406, P. Kosma, Austria, for Kdo, H. Moll and R. Engel for help with GC-MS, W. Brabetz for helpful discussions, and I. J. von Cube, H. Lüthje, and V. Susott for expert technical assistance.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 470/A1 (to H. B. and S. G.) and LI 448/1-1 (to B. L.).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/EBI Data Bank with accession number(s) AJ302064 and AJ420777.
§ To whom correspondence should be addressed: Division of Medical and Biochemical Microbiology Research Center Borstel, Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany. Tel.: 49-4537-188469; Fax: 49-4537-188419; E-mail: sgronow@fz-borstel.de.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M206942200
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ABBREVIATIONS |
---|
The abbreviations used are:
LPS, lipopolysaccharide;
Ara4N, 4-amino-4-deoxy-L-arabinose;
Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid;
Ko, D-glycero-D-talo-oct-2-ulosonic
acid;
mAb, monoclonal antibody;
m.o.i., multiplicity of infection;
MS, mass spectrometry;
TNF-, tumor necrosis factor
;
ESI-FT-ICR, electrospray Fourier-transform ion cyclotron resonance.
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