From the
Lipopolysaccharide (LPS) is a major component of the bacterial
outer membrane, and for Rhizobium spp. has been shown to play
a critical role in the establishment of an effective nitrogen-fixing
symbiosis with a legume host. Many genes required for O-chain
polysaccharide synthesis are in the lps
Lipopolysaccharides (LPSs)
Structural regions of the LPS from R. leguminosarum and R. etli have been defined according to fragments
released from purified LPSs by mild acid hydrolysis. The structures of
the core oligosaccharides from R. etli strain CE3, and from
R. leguminosarum biovar trifolii and bv.
viciae
(4, 6, 7, 8, 9, 10) are:
On-line formulae not verified for accuracy
STRUCTURE Iand
On-line formulae not verified for accuracy
STRUCTURE II
The lipid A portion of these LPSs has a trisaccharide glycosyl
backbone consisting of one each of galacturonosyl (GalA),
glucosaminosyl (GlcN), and 2-aminogluconosyl (GlcN-onate) residues; the
latter 2 residues being O- and N-acylated with
Synthesis of the core oligosaccharide
and O-chain portion of the LPS requires lps genes
from at least five regions of the R. etli CE3 genome, as
defined by cosmid genetic cloning
(5, 14, 15) .
Most of the genes that have been identified are located in a stretch of
the chromosome, termed the lps
As in the CE3 parent LPS, the
GalA/tetramer/trimer ratio for all the mutant LPSs, except that from
CE358 in which OS6 replaces OS3 and OS4, is 1:1:1. Thus, of the strains
examined in this report, only strain CE358 (from complementation group
I) appears to be altered in the core oligosaccharides that are released
from the LPS by mild acid hydrolysis.
Negative ion FAB-MS analysis
(spectrum not shown) of the mild acid hydrolysate of CE358 LPS gave two
major ions; one of [M-H]
Small amounts of the LPSs were
methylated and carboxymethyl reduced. This was followed by the
preparation and GLC-MS analysis of the PMAAs. Fig. 6shows the
results for the LPSs from strains CE350, 357, and 358 which represent
the three types of LPSs observed. The LPSs from the other mutants had
the same glycosyl linkages as shown for strain CE357. Peak 1, which was
present only in the LPS from strain CE350, had a retention time and
fragmentation pattern consistent with the PMAA of terminal Gal (m/z = 205, 162, 118, 161). Peak 2 was present only in the LPS
from strain CE358 and was due to the PMAA of 6-linked Man (m/z = 233, 189, 162, 118). Peak 3, in the case of CE350 LPS,
was due to the PMAA of terminal GalA (m/z = 235, 191,
162, 118) in which the carboxyl group had been reduced with NaBD
The
linkages of the Kdo residues for several of the LPSs, e.g. from strains CE357 and CE358, were determined by methylation,
carboxymethyl reduction (lithium triethylborodeuteride), mild acid
hydrolysis (0.1 M trifluoroacetic acid at 80 °C for 30
min), reduction (sodium borodeuteride), normal acid hydrolysis (2
M trifluoroacetic acid at 121° for 2 h), reduction (sodium
borodeuteride), and preparation of the PMAAs. Three types of Kdo
residues were found in these LPSs; terminally linked Kdo (primary
fragments of m/z 89, 205, 206, 250, and 366), 5-linked Kdo
(m/z 89, 206, and 394), and 4,5-linked Kdo (m/z 89,
and 422). The 4,5- and 5-linked Kdo residues are consistent with the
tri- and tetrasaccharide core oligosaccharide structures, and the
terminal Kdo is presumably due, in part, to the Kdo residue that is
attached to O-6 of the core tetrasaccharide Gal residue. Thus,
it is likely that the core structure for the LPSs from CE3 and its
mutants, except for CE350, contains terminal Kdo attached to
O-6 of the Gal residue.
In summary, these methylation
results from the intact LPSs showed (a) that the intact core
region of these LPSs contain a Kdo residue attached to O-6 of
the Gal residue, (b) that this Kdo residue is the likely site
of O-chain attachment, and (c) that the LPS from
mutant CE350 lacks this Kdo residue.
Alditol acetate analysis of the
intact LPSs from CE350, 356, 357, 359, 360, and 121 show that they all
contain small amounts of glycosyl residues previously reported
(12) to be in the O-chain polysaccharide, e.g. GlcA, methylated Rha, Fuc, methylated Fuc, and quinovosamine
(Qvn). Since PAGE and immunoblot analyses
(30) showed that the
monoclonals only bind to LPS I, III, IV, or V, and not to LPS II, these
results indicate that the minor amounts of LPS III, IV, and/or V that
are present in these mutants are forms of the LPS that contain various
truncated O-chains. This was confirmed by purifying a small
amount of LPS IV from strain CE121 using gel filtration chromatography
in the presence of DOC. Analysis of this LPS IV showed the presence of
GlcA/2-O-MeRha/Fuc/2,3-di-O-MeFuc/Man/Qvn =
1:1:1:1:1:1. The LPS from the parent strain, CE3, has these sugars in
roughly a 4:4:4:1:1:1 ratio with the exception that a 2,3,4-tri-MeFuc
residue replaces the 2,3-di-O-MeFuc residue. The repeating
unit of this O-chain is comprised of
GlcA/2-O-MeRha/Fuc in a 1:1:1 ratio.
The precise arrangement of the core components in the
intact LPS is not known. It is possible that the tri- and
tetrasaccharides are present on two different types of LPS molecules.
However, their presence in approximately a 1:1 ratio in both the parent
and in all of the mutant LPSs indicates that there may be a single LPS
species which contains all of the core elements. Further work is in
progress to structurally characterize the intact core region of these
LPSs.
The two mutants which vary in their core structures are CE350
and CE358. The core region from strain CE350 lacks the Kdo residue that
is normally attached to O-6 of Gal, and the core region from
strain CE358 lacks the GalA residue that is normally attached to
O-4 of the Man residue. The missing Kdo residue in CE350 LPS
suggests that the defective gene in this mutant may encode a specific
CMP-Kdo transferase. It should be noted that glycosyl linkage analysis
of the CE350 LPS suggests that a small portion of the Gal residues has
Kdo at O-6. Thus, this mutation does not lead to a complete
lack of this Kdo residue. That some of the CE350 LPS molecules have
this Kdo residue, and that a truncated version of the O-chain
is attached to that residue, is supported by the fact that CE350 LPS
preparations have LPS III, contain small amounts of O-chain
sugars, and bind the JIM26 monoclonal antibody. On the other hand the
LPS from strain CE358 seems to consist only of LPS II, and completely
lacks O-chain sugars, suggesting that (a) the GalA
residue that is missing from the O-4 position of the Man
residue may be required for transfer of O-chain to the core
region, and (b) that the defective gene in CE358 may encode a
UDP-GalA transferase.
Other than those of CE350 and CE358, LPSs
analyzed in this study have all of the core components and contain
various forms of truncated O-chain as evidenced by the
presence of typical O-chain glycosyl residues. Since all of
these mutants are symbiotically
defective
(14, 16, 30) , it is apparent that a
complete core region alone is not sufficient for R. etli to
effectively nodulate its legume host.
We thank John Kim for performing the PAGE analyses.
region of the
CE3 genome; this region may also carry lps genes required for
core oligosaccharide synthesis. The LPSs from several strains mutated
in the
region were isolated, and their mild acid released
oligosaccharides, purified by high performance anion-exchange
chromatography, were characterized by electrospray- and fast atom
bombardment-mass spectrometry, NMR, and methylation analysis. The LPSs
from several mutants contained truncated O-chains, and the
core region consisted of a
(3-deoxy-D-manno-2-octulosomic acid)
(Kdo)-(2
6)-
-Galp-(1
6)-[
-GalpA-(1
4)]-
-Manp-(1
5)-Kdop (3-deoxy-D-manno-2-octulosomic acid)
(Kdo)pentasaccharide and a
-GalpA-(1
4)-[
-GalpA-(1
5)]-Kdop trisaccharide. The pentasaccharide was altered in two mutants in
that it was missing either the terminal Kdo or the GalA residue. These
results indicate that the lps
region, in addition to
having the genes for O-chain synthesis, contains genes
required for the transfer of these 2 residues to the core region. Also,
the results show that an LPS with a complete core but lacking an
O-chain polysaccharide is not sufficient for an effective
symbiosis.
(
)
are important
determinants of the surface characteristics and ecology of
Gram-negative bacteria. Current understanding of LPS and its biological
roles comes mainly from studies of enteric bacteria. To gain a greater
appreciation of the role of LPS in nature, it is important to
supplement the enteric bacterial paradigm with studies of bacteria in
different phylogenetic groups and with distinct physiology.
Rhizobium etli(1) and Rhizobium leguminosarum are closely related species in the
group of the
Proteobacteria
(2, 3) . The biology of these bacteria has
been extensively studied, particularly their nitrogen fixing symbioses
with certain legumes, and LPS structure is critical in the development
of these symbioses. The LPS structure
(4) and genetics
(5) of several strains of these species are known in some
detail.
-hydroxymyristate, -palmitate, -pentadecanoate, -stearate, and
27-hydroxyoctacosanoate
(11) . Both the lipid A and the core
regions are structurally very different from those of the enteric
bacteria, in which the lipid A is comprised of an acylated
bis-1,4`-phosphorylated
-1,6-glucosamine disaccharide, while the
core oligosaccharide usually contains heptose and lacks uronosyl
residues. The remaining LPS structural region is the distal
O-chain polysaccharide that, when present, is the dominant
antigen of the LPS and the bacterial cell. R. leguminosarum and R. etliO-chains, as released by mild
hydrolysis, are polysaccharides that contain Kdo at their reducing
ends
(12, 13) . This Kdo may be the outermost core
glycosyl residue to which the O-chain is transferred during
the biosynthesis of the LPS.
region
(5) , in
which nine complementation groups have been identified within 18
kilobases of DNA
(16) (Fig. 1). It appears that this
region carries genes for synthesis of at least the strain-specific
O-chain glycosyl residues and linkages
(17) and at
least one gene necessary for core oligosaccharide synthesis as
well
(9, 18) .
Figure 1:
The location of the various mutations
in the region of the R. etli CE3 genome. All of the
mutants designated by the filled circle either lack or have
much less than normal LPS I. The letters refer to
complementation groups, presumably representing operons, defined by Tn5
insertion mutagenesis. The distance shown is between the Tn5 insertions
at either end of the region.
R. leguminosarum, R.
etli, and Bradyrhizobium japonicum mutants that are
deficient in the LPS O-chain polysaccharide elicit incomplete
infections and root nodule development on their legume
hosts
(5, 14, 19, 20) . Although all LPS
mutants that are defective in symbiosis have deficient or altered
O-chain-containing LPS, it had not been ruled out in previous
studies that these mutants might also be defective in the core
oligosaccharides. In fact, the LPSs from two R. etli mutants
have truncated core structures
(9, 18) . Therefore, one
motive for this study was to determine whether or not an intact core
portion of the LPS is sufficient for symbiotic proficiency. Another
motive was to infer specific biosynthetic functions for particular
genetic loci within lps region by correlating mapped
mutations with LPS structural defects. In addition, the results of this
study suggest a definition for the biosynthetic core and the linkage
between a particular core sugar and the Kdo residue which is at the
reducing end of the O-chain polysaccharide.
Bacterial Strains
The bacterial strains used in
this study are given in . Bacteria were grown in TY medium
with added calcium as described previously
(12) .
LPS Isolation
Bacteria were harvested by
centrifugation and the pellets extracted using the hot phenol-water
extraction procedure as described previously
(12, 21) .
The LPSs were further purified from the aqueous layer, as described
previously
(12, 21) , by digestion with RNase and DNase,
followed by extensive dialysis against deionized water using
12,000-14,000 MWC dialysis tubing, and freeze-dried. The LPS from
the parent strain, CE3, had also been further purified by
gel-filtration chromatography using Sepharose 4B in and
EDTA/triethylamine buffer at pH 7
(12, 22) . Separation
of higher from lower molecular weight forms of LPS was accomplished by
gel filtration chromatography on Sephadex G-150 in the presence of
deoxycholate (DOC) as described
previously
(23, 24, 25) .
Polyacrylamide Gel Electrophoretic Analysis
(PAGE)
PAGE analysis was performed using 18% acrylamide gels
with DOC as the detergent
(26) . The gels were silver stained as
described
(27) .
Isolation of Core Oligosaccharides from the
LPSs
Each LPS was dissolved in deionized water (10 mg/ml),
acetic acid was added to 1%, and the solution heated at 100 °C for
1 h. This procedure hydrolyzes the ketosidic bond between the
polysaccharide Kdo residue and the lipid A, which
precipitates
(28) . The lipid A was removed by centrifugation,
and the carbohydrate was further purified by HPAEC on a CarboPac PA1 column (DIONEX) using a gradient comprised of 1 M
NaOAc (A) and 100 mm NaOH (B); 10% A and 90% B for 10 min and then to
50% A and 50% B by 40 min. The various carbohydrate peaks were
collected, the acetate was removed by passage through DIONEX OnGuard H
cartridges, and the eluants freeze-dried.
Analysis of the Glycosyl Residues
Glycosyl
compositions were determined by the preparation and gas liquid
chromatographic (GLC)-mass spectrometric (MS) analysis of alditol
acetates, or trimethylsilyl methyl glycosides
(29) , and glycosyl
linkages were determined by methylation analysis using the Hakomori
procedure as described by York et al.(29) . For the
isolated core oligosaccharides, it was necessary to reduce the samples
with NaBD prior to methylation. Also, for certain samples,
after methylation, it was necessary to reduce the carboxymethyl groups
of the acidic sugars with lithium triethylborodeuteride (Superdeuteride
from Aldrich)
(29) . Alditol acetates of the methylated samples
were prepared by hydrolysis, reduction with NaBD
, and
acetylation with acetic anhydride in pyridine as described by York
et al.(29) . Combined GLC-MS was performed using an
HP5890-5970 GLC-MSD system equipped with a 30-m SP2330 fused
silica column from Supelco for the alditol and partially methylated
alditol acetates (PMAAs), or with a 30-m DB-1 column from J&
Scientific for trimethylsilyl methyl glycosides.
Mass Spectrometry Analysis
Fast atom
bombardment-mass spectrometry (FAB-MS) was performed using a VG ZAB-SE
instrument at an accelerating voltage of 8 kV. Approximately 2-10
µg of sample was placed on the probe. Thioglycerol was used as the
matrix. Electrospray mass spectrometry (ES-MS) was performed using a
SCIEX API-III mass analyzer operated in the positive mode with an
orifice of 50 V. Samples were dissolved in 20% aqueous acetonitrile and
pumped into the mass spectrometer at a rate of 3 µl/min.
NMR
Samples were exchanged several times with
DO, dissolved in D
O and analyzed at 295 °K
using a Bruker AM500 spectrometer. Chemical shifts were measured
relative to the HOD resonance, which, in turn, was measured relative to
sodium 3-trimethylsilylpropionate-2,2,3,3-d
.
RESULTS
PAGE Analysis of R. etli LPSs
The LPSs from
R. etli CE3 and various mutants were analyzed by DOC-PAGE
(Fig. 2). LPS I and LPS II were the major components of the
parent (CE3) LPS. The LPS I band was detected in greatly reduced amount
in the LPS from CE359 and not detected in any LPSs from the other
mutants examined. All of the mutant LPS preparations contained LPS II
and, in addition, other low molecular weight forms of LPS designated
LPS III (CE350), LPS IV (CE121, CE356, CE357), and LPS V (CE359,
CE360). The only exception was the LPS preparation from CE358 which
contained only LPS II. These different forms of LPS were
distinguishable by their differing abilities to bind four monoclonal
antibodies (JIM26, JIM27, JIM28, and JIM29); i.e. it was
previously reported that LPS IV and V bind all four monoclonals while
LPS III binds only JIM26, and LPS II does not bind to any of the
monoclonals
(30) .
Figure 2:
The DOC-PAGE profile (silver stain) of
LPSs from the various mutants of R. etli CE3. LPS I
is the high molecular weight form of the LPS which contains the
O-chain. LPS II lacks the O-chain. LPSs III, IV, and
V are various forms of LPS that are distinguished from one another
slightly in their electrophoretic mobility, and in their ability to
interact with the monoclonal antibodies, JIM26, 27, 28, and
29.
Analysis of the R. etli CE3 LPS Oligosaccharides by High
Performance Anion-exchange Liquid Chromatography
(HPAEC)
Analysis of the mild acid hydrolysate from CE3 LPS by
HPAEC showed (Fig. 3A) the presence of five components,
OS1-OS5. Fractions OS1 and OS2 were identified as monomeric Kdo and
GalA, respectively, by comparing retention times to those of authentic
standards. The monomeric Kdo, OS1, eluted as several peaks due to the
formation of various anhydro forms during the mild acid hydrolysis
procedure
(31, 32) . Subjection of standard Kdo to the
mild acid hydrolysis conditions resulted in the same peaks as those
observed for OS1 from the LPS samples. Preparation and GLC-MS analysis
of the trimethylsilyl methylglycosides of OS2 showed that it was
composed of only GalA. Proton NMR analysis of OS3 and 5 showed that
their spectra (not shown) matched those published
(18) for the
major tetra- and trisaccharide components, respectively, from this LPS;
therefore, these previously reported structures (Structures I and II
shown above) can be assigned to OS3 and OS5. Oligosaccharide OS4 had
the same glycosyl composition as the tetrasaccharide, OS3; namely,
GalA, Man, Gal, and Kdo. The relative molar ratio of OS2 (GalA)/OS3
+ OS4 (tetramers)/OS5(trimer) was 1:1:1 and was determined using
molar response factors of standard monomeric GalA for OS2 and of a
pectic trisaccharide for OS3, OS4, and OS5.
Figure 3:
Analysis
by HPAEC of the LPS core oligosaccharides obtained from Rhizobium
etli CE3 LPS (A), and CE358 LPS (B).
Oligosaccharide OS4 Is a Tetrasaccharide with an Anhydro
Kdo Residue at Its Reducing End
As stated above, the glycosyl
composition of OS4 was the same as that for OS3. Methylation analysis
also showed that OS4 had the same glycosyl linkages as OS3; namely,
terminal-GalA/terminal-Gal/4,6-linked Man in a 1:1:1 ratio. The
methylation procedure used for this analysis destroyed the Kdo residue
and prevented its analysis; however, the various types of Kdo linkages
in these LPSs are described below. Analysis by negative FAB-MS of the
oligosaccharides prior to purification by HPAEC showed
[M-H] ions of m/z 589, 719, and
737. The ions of m/z 589 and 737 are due to the tri- and
tetrasaccharides (Structures II and I, respectively). The ion at
m/z 719 is consistent with a tetrasaccharide that lacks a
water molecule, i.e. minus 18 atomic mass units, and could be
due a lactone or anhydro version of this molecule. Fractions OS3 and
OS4, purified by HPAEC, were reduced with NaBD
,
permethylated, and analyzed by ES-MS, Fig. 4, A and
B. Fraction OS3 gave the ions expected for a molecule derived
from a tetrasaccharide that contained the expected reducing Kdo
pyranose residue; namely, [M+NH
]
and
[M+NH
+Na
]
of m/z 983 and 1005, respectively. Analysis of OS4,
Fig. 4B, resulted in ions of m/z 937 and 959,
[M+NH
]
and
[M+NH
+Na
]
,
respectively. These latter ions were also present in OS3 indicating
that this fraction was contaminated with some anhydro or lactone form
of the tetrasaccharide. The 46 atomic mass units difference between OS3
and OS4 is not consistent with a lactone which would have been reduced
with NaBD
; however, it is consistent with an anhydro-Kdo
derivative.
Figure 4:
Analysis by
ES-MS of the purified OS3 (A), and OS4 (B), after
reduction with NaBD and
permethylation.
NMR analysis of OS4 gave a complex spectrum (not shown).
The complexity of the spectrum may indicate that OS4 contained more
than one type of anhydro-Kdo residue. The typical resonances for the
methylene protons from the reducing Kdo pyranose residue of OS3 (
1.8 and 2.1) were absent. This apparent lack of the Kdo methylene
proton resonances can occur for oligosaccharides containing certain
anhydro forms of Kdo
(33) . In a 4,7- or 4,8-anhydro-Kdo residue,
the methylene geminal protons are adjacent to a C-2 carbonyl rather
than to the hemi-ketal C-2 of a normal reducing Kdo pyranose and are,
therefore, quite acidic and easily exchanged with deuterium during
preparation of the sample for NMR analysis. Additionally, if not fully
exchanged with deuterium, the chemical shifts of such geminal methylene
protons are shifted far downfield (e.g. to
2.90 and 3.3
(33)) compared to their resonances in the tetramer with a normal Kdo
pyranose residue (e.g.
1.8 and 2.1). This combination of
deuterium exchange and downfield chemical shift into the region near
the glycosyl ring protons, make these methylene protons difficult to
observe under the best conditions. Thus, glycosyl composition, glycosyl
linkage, mass spectrometry, and NMR analyses support the conclusion
that OS4 is a version of the tetrasaccharide that contains either a
4,7- or a 4,8-anhydro-Kdo residue at its reducing end; i.e.
-Gal-(1
6)-[
-GalA-(1
4)]-
-Man-(1
5)-[4,7-
or 4,8-anhydro]-Kdo.
Characterization of the Core Oligosaccharides Purified
from the LPSs of R. etli Mutants
Fig. 3B shows
the HPAEC profile of the mild acid hydrolysates from mutant CE358. The
HPAEC profiles for strains CE350, CE357, CE356, CE121, CE359, and CE360
were identical to that of CE3 (Fig. 3A). For strain
CE358, OS3 and 4 were replaced by OS6. The relatively short retention
time of OS6 indicates that it is not as acidic as the other
oligosaccharide components. This was confirmed by chemical analysis
which is described further below.
m/z = 589 which was due to the GalA
Kdo
trisaccharide (OS5, ), and one of
[M-H]
m/z = 561 which was
consistent with a trisaccharide consisting of 2 hexosyl (Hex) and 1 Kdo
residues, Hex
Kdo. These results were confirmed by ES-MS
analysis of the reduced permethylated oligosaccharides, Fig. 5.
Figure 5:
Analysis by ES-MS of the core
oligosaccharides released from CE358 LPS by mild acid hydrolysis. The
sample was reduced with NaBD, and permethylated prior to
analysis.
The HexKdo trisaccharide (OS6) was isolated by HPAEC and
its structure deduced by glycosyl composition, linkage, and NMR
analyses. Composition analysis showed that it consisted of Gal, Man,
and Kdo. Glycosyl linkage analysis of the neutral sugars showed the
presence of terminal Gal and 6-linked Man. The proton NMR spectrum of
OS6 (spectrum not shown) matched that previously reported for a
Hex
Kdo trisaccharide from another
region mutant,
CE109 (18). This result was consistent with the fact that HPAEC
analysis of the mild acid hydrolysate of CE109 LPS also showed the
presence of OS6 (data not shown). Thus NMR, FAB-MS, and HPAEC analyses
strongly suggest that OS6 from CE358 has the same structure as that
previously reported (9, 18) CE109 trisaccharide; namely,
-Gal-(1
6)-
-Man-(1
5)-Kdo.
Analysis of the Intact LPSs from the R. etli
Mutants
The above results showed that only one mutant, CE358,
was altered in the core oligosaccharides released by mild acid
hydrolysis of its LPS. However, it was necessary to examine the mutant
LPSs prior to mild acid hydrolysis in order to determine if there were
other differences that may not have been detectable due to the mild
acid hydrolysis conditions.
prior to hydrolysis and acetylation. Minor ions of m/z = 233 and 189 were present in peak 3 from CE350 LPS and
indicated the presence of small amounts of 6-linked Gal. In the case of
the LPSs from CE358 and CE357, as well as the other remaining mutant
LPSs, terminal galactose (peak 1) was not detected, and peak 3
consisted of a mixture of the PMAAs derived from 6-linked Gal and
terminal GalA; m/z = 235 (233), 191
(189) , 162,
118. The ratio of the 233:235 (or 189:191) ion intensities is somewhat
reflective of the 6-linked Gal/terminal GalA ratio and was 0.077, 0.15,
and 0.23 for the LPSs from CE350, CE357, and CE358, respectively. The
larger ratio for CE358 compared with that for CE357 LPS was consistent
with the fact that the former LPS lacks one of the GalA residues. Peak
4 was the PMAA of 4,6-linked Man (m/z = 261,118) and
was found in the LPSs from all the mutants except CE358 which contained
only 6-linked Man (peak 2).
Figure 6:
The
GC profiles of the partially methylated alditol acetates derived from
the LPSs from CE350 (A), CE357 (B), and CE358
(C). Comparison of the retention times to authentic standards
and the mass spectral data show that peak 1 is the PMAA of
terminal Gal, peak 2 of 6-linked Man, peak 3 of a
mixture of 6-linked Gal and terminal GalA, and peak 4 of
4,6-linked Man. Peak * was identified as phthalate, a common
contaminant. The remaining small peaks observed are most likely other
non-carbohydrate contaminants.
With one exception, these data are
consistent with structures of the oligosaccharides released by mild
acid hydrolysis. The exception is that the Gal residue in the mild acid
released oligosaccharides is terminally linked while it is 6-linked in
the intact LPSs, except for that from CE350 in which it is largely
terminally linked. Thus, in these LPSs, except for the LPS from CE350,
the Gal of the core tetrasaccharide has a mild acid labile residue,
presumably Kdo, attached at O-6. Since it is known that the
O-chain polysaccharide purified by mild acid hydrolysis has a
Kdo residue at its reducing end, it is likely that it is this residue
which is attached to O-6 of Gal in these intact LPSs.
(
)
Thus, the reduced level of these sugars in LPS IV and its
faster PAGE mobility compared with that for LPS I are consistent with
the concept that LPS IV has a truncated O-chain.
DISCUSSION
The results described above suggest that the
``rough'' LPSs (i.e. LPS II) from the parent and
mutant strains have the structures shown in Fig. 7. The site of
attachment of the core region to the lipid A, and the lipid A
structure, have been described in a previous report
(11) . The
LPS II core region is comprised of the previously reported tetra- and
trisaccharide molecules (9, 18) (Structures I and II), with a Kdo
residue linked to O-6 of the tetrasaccharide Gal residue. This
Kdo residue may be the site of O-chain attachment.
Figure 7:
A schematic representation showing the
possible structure of the LPS from R. etli CE3 and its various
mutants. Mutant CE358 lacks the GalA residue -linked to
O-4 of Man, as well as the O-chain polysaccharide.
Mutant CE350 lacks the Kdo residue attached to O-6 of Gal.
However, this mutant is somewhat leaky in that a small percentage of
its LPS contains this Kdo residue as well as a truncated portion of the
O-chain polysaccharide. Mutants CE356, CE357, CE359, CE360,
and CE121 all contain a complete core region as well as various
truncated versions of the O-chain polysaccharide. The
shaded circle indicates that it is not yet known how these
various core oligosaccharides are linked together in the complete
molecule. The X represents a moiety or chemical environment
which renders the GalA glycoside bond labile to mild acid. The core
region is attached via a mild acid labile substituent (presumably Kdo)
to O-6 of the lipid A GlcN residue
(11).
The
results described above also show that mild acid hydrolysis releases
monomeric GalA from all of the LPSs. Thus, the core region must contain
a ``GalA-1 X'' in which X is an
unidentified substituent or chemical environment which renders the GalA
glycoside bond labile to mild acid. The mechanism by which monomeric
GalA is released from the LPS by mild acid hydrolysis is not yet
understood.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.