From the Centre of Microbial and Plant Genetics,
Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, Heverlee
B-3001, Belgium and the ** Complex Carbohydrate Research Center,
University of Georgia, Athens, Georgia 30602
Received for publication, February 6, 2001
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ABSTRACT |
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For O-antigen lipopolysaccharide (LPS) synthesis
in bacteria, transmembrane migration of undecaprenyl
pyrophosphate-bound O-antigen oligosaccharide subunits or
polysaccharide occurs before ligation to the core region of the LPS
molecule. In this study, we identified by mutagenesis an ATP-binding
cassette transporter in Rhizobium etli CE3 that is likely
responsible for the translocation of the O-antigen across the inner
plasma membrane. Mutant FAJ1200 LPS lacks largely the O-antigen, as
shown by SDS-polyacrylamide gel electrophoresis and confirmed by
immunoblot analysis. Furthermore, LPS isolated from FAJ1200 is totally
devoid of any O-chain glycosyl residues and contains only those
glycosyl residues that can be expected for the inner core region. The
membrane component and the cytoplasmic ATP-binding component of the
ATP-binding cassette transporter are encoded by wzm and
wzt, respectively. The Tn5 transposon in mutant
FAJ1200 is inserted in the wzm gene. This mutation resulted
in an Inf Rhizobiaceae are Gram-negative bacteria that are able to induce
the formation of nitrogen-fixing nodules on roots of leguminous plants.
For infection and differentiation of nodules, bacterial determinants
including surface polysaccharides are required.
LPS1 is the major structural
component of a Gram-negative bacterial outer membrane, and evidence for
its importance in plant-microbe interactions is appealing.
Various Rhizobium mutants with alterations in LPS are
defective in the symbiotic association at different stages of infection
and nodule development (1).
LPS consists of lipid A, which anchors it to the outer membrane, and a
polysaccharide portion that extends into the environment. The
polysaccharide portion contains an inner core region, conserved among
related strains, and an O-antigen region, whose structure varies in a
strain-dependent manner. The O-antigen region consists of
the repeating unit and a non-repeating sequence, also referred to as
the O-chain attachment region or outer core region (2-5). LPS II (or
rough LPS) refers to lipid A and the inner core, whereas LPS I (or
smooth LPS) refers to the complete structure. The recent elucidation of
the glycosyl sequence of the Rhizobium etli CE3 LPS
O-antigen completed the glycosyl sequence of the R. etli CE3 LPS (2-5). The O-antigenic polysaccharide was found to be a unique, relatively low molecular weight glycan of a fairly discrete size, with
surprisingly little variation in the number of repeating units (degree
of polymerization = 5). Each trisaccharide repeating unit
consists of glucuronic acid, fucose, and
3-O-methyl-6-deoxytalose (2).
No specific information on the biosynthetic mechanism leading to the
assembly of the O-chain polysaccharide in R. etli CE3 is
available. Nevertheless, despite the diversity in structures of
O-antigens, the mechanisms involved in their synthesis seem to be
conserved in those bacteria that have been studied to date (6). In
general, the activated sugar precursors are not transferred directly to
a growing LPS molecule. Instead, O-antigens are synthesized separately
on a lipid carrier, termed bactoprenyl phosphate. This polymerization
step can occur either in the cytoplasm or in the periplasm depending on
the assembly pathway. In each case, translocation of the O-antigen
across the inner plasma membrane is required. So far, three assembly
pathways are known for the polymerization and export of O-antigens.
These processes are designated the "Wzy-dependent" pathway, the "ABC transporter-dependent" pathway, and
the "synthase-dependent" pathway based on the proteins
that are involved in the pathways and the components involved in export
across the plasma membrane (7). Once completed, the O-antigen is
covalently ligated to a preformed acceptor composed of lipid A and the
inner core at the periplasmic face of the plasma membrane. After
ligation, the completed LPS molecule is translocated to the cell
surface by an unknown mechanism.
The genes involved in saccharide processing, including export,
polymerization, and assembly of complex polysaccharides such as LPS,
have already been identified in several Enterobacteriaceae. They have all been given names of the form wz* (8). In
Rhizobium, however, similar genes have not yet been
identified. Nevertheless, at least five genomic regions that seem to
have a role in the biosynthesis of LPS in Rhizobium were
identified (9-16). Most of the information is located in a stretch of
the R. etli CE3 chromosome, termed the lps In this work, we report on the isolation of two genes that are likely
involved in the export of rhizobial O-antigenic polysaccharides across
the inner membrane. The genetic analysis of a Tn5 mutant (FAJ1200) with a rough colony morphology on agar plates led to the
identification of two genes, wzm and wzt, the
deduced amino acid sequences of which show similarities to known ABC-2
transporters or traffic ATPases. Both genes coding for this ABC
transporter are located in the previously identified Bacteria and Growth Media--
Escherichia coli
strains were maintained on LB agar at 37 °C and grown in LB broth
supplemented with the appropriate antibiotics (17). R. etli
CE3, CE168, mutant FAJ1200, and the trans-conjugant FAJ1206 (mutant FAJ1200 with plasmid pFAJ1248 containing the R. etli wzm and wzt genes) were maintained on
Tryptone/yeast medium with added calcium dichloride (18) and
supplemented with the appropriate antibiotics. Triparental conjugations
were done as previously described (19).
Nucleic Acid Manipulations and Analysis--
Nucleic acid
manipulations were done as previously described (20, 21). Plasmid DNA
was prepared, and DNA sequencing was determined with a
Tn5 primer, the universal pUC primer, and the reverse
pUC primer on an ALF automated sequencer (Amersham Pharmacia Biotech, Uppsala).
PCR Amplification--
The 2.4-kb DNA fragment that contains the
wzm and wzt genes was amplified by PCR with
platinum Pfx DNA polymerase (Life Technologies, Inc.) using
two primers: 5'-AGGCGCGCCGCCGGGACAATACGCCGGGGCG-3' (RHI-120) and
5'-TCCCCCGGGGCATGCCTGCAGATCGACGCCCG-3' (RHI-141). The 2.4-kb PCR
product was ligated to the pFAJ1708 vector (22).
Data Base Searching, Multiple Alignments, and Generation of
Phylogenetic Trees--
Data base searches were performed using the
programs BLASTX and BLASTP (23). The ClustalW algorithm (24) was
used for multiple protein alignments, and the alignment layout
was prepared by the Genedoc program (25). Phylogenetic trees were
generated using the neighbor-joining method (26). Statistical
significance values were evaluated with the bootstrapping method (27).
Trees were displayed graphically using the software package Treecon for
Windows (28).
Plant Assays--
Phaseolus vulgaris cv. Negro Jamapa
seeds were surface-sterilized and germinated as described previously
(29). Bean seedlings were planted in jars containing a Jenssen medium
agar slant (30). The seedlings were inoculated with 200 µl of an
overnight rhizobial culture. The plants were maintained in a growth
chamber at 28 °C day and 24 °C night temperatures over a 12-h
photoperiod. After 3 weeks, plants were harvested. Uninoculated control
plants did not show any nodules or nodule-like structures.
Microscopic Analysis of Nodules--
Three-week-old nodules were
fixed and embedded in Technovit 7100 matrix (Heraeus Kultzer,
Wehrheim, Germany) as described previously (29). Sections of 3 µm were cut on an HM 360 microtome (Microm, Walldorf, Germany) and
subsequently stained with toluidine blue. Photographs were taken with a
Optiphot-2 microscope using an FX-35DX camera (Nikon, Tokyo).
LPS Purification--
LPS was purified by hot phenol/water
extraction, followed by size-exclusion chromatography in the presence
of deoxycholate (31) using Sephadex G-150 (32-34), unless otherwise stated.
Polyacrylamide Gel Electrophoresis (PAGE) and Immunoblot
Analyses--
The lipopolysaccharides that were extracted into both
the aqueous and phenol phases during hot phenol/water extraction were analyzed as described (35-37) and by immunoblotting using monoclonal antibodies (mAbs) JIM26, JIM27, JIM28, and JIM32 as previously described (38).
Analysis of the Core Oligosaccharides by High Performance Anion
Exchange Chromatography (HPAEC)--
The purified lipopolysaccharides
from CE3 and FAJ1200 were hydrolyzed in 1% acetic acid at 100 °C
for 1 h. The oligosaccharides released by mild acid hydrolysis
were analyzed by HPAEC as previously described (4).
Glycosyl Composition Analysis--
The compositions of the
purified lipopolysaccharides were determined by the preparation and gas
chromatographic-mass spectrometric analysis of trimethylsilyl
methylglycosides as previously described (39).
Isolation of the LPS Mutant FAJ1200--
From a large mutant
collection, we isolated a Tn5-induced mutant of R. etli CE3 (FAJ1200) that had a dry or rough appearance on
Tryptone/yeast agar and that also showed a tendency to agglutinate in
liquid medium. These properties are usually associated with lipopolysaccharide defects in Rhizobium.
Phenotypic Analysis of FAJ1200--
P. vulgaris cv.
Negro Jamapa seedlings were inoculated with wild-type R. etli CE3 and the FAJ1200 mutant to determine the nodulation
phenotype of the plant. The nodules induced by the mutant were small
and white and did not fix nitrogen. Microscopic analysis of 3-week-old
nodules was performed. After examination of toluidine blue-stained
3-µm sections, no infected cells could be observed in the nodules
induced by the mutant. Vascular bundles were located centrally (Fig.
1B) rather than peripherally
as in a normal nodule (Fig. 1A). This symbiotic behavior is
identical to that of the previously reported lps mutant
CE168 (Fig. 1C) (14).
Identification of wzt, Which Encodes the Cytoplasmic ATP-binding
Component--
To identify the gene disrupted by the Tn5
insertion, Southern hybridization was performed on
PstI-digested genomic DNA of the mutant with a probe
corresponding to the kanamycin resistance gene of Tn5. PstI
has a restriction site in the transposon but not the kanamycin
resistance gene. A 3.5-kb PstI hybridization signal was
detected. The corresponding fragment was cloned into pUC19, and the
resulting recombinant plasmid was designated pFAJ1220. The nucleotide
sequence of the DNA region flanking the Tn5 transposon was
determined with pFAJ1220 as the template DNA and a primer derived from
the Tn5 inverted repeat sequence. Sequence analysis was
further completed with subclones of the pFAJ1220 insert DNA in pUC19
vector DNA using the universal and reverse pUC primers. The determined
sequences revealed similarity to genes encoding the ATP-binding protein
of ABC-2 transporters or traffic ATPases. ABC-2 transporters are
constituents of the export system for polysaccharides in diverse
bacteria. They are composed of two proteins: an inner membrane protein
(Wzm) and a cytoplasmic ATP-binding protein (Wzt). Two subunits of each
protein probably compose a functional ABC-2 transport system
(40).
The identified ORF (ORF2 in Table I)
showed similarity to two distinct classes of proteins: those
responsible for LPS export and those responsible for capsular export.
The first class represents proteins essential for O-antigen transport
in Myxococcus xanthus, Pseudomonas aeruginosa,
Burkholderia pseudomallei, E. coli O9, Actinobacillus actinomycetemcomitans, and Aeromonas
salmonicida. The second class comprises proteins important for the
ATP-driven capsular polysaccharide export in E. coli K54,
Sinorhizobium meliloti, Haemophilus influenzae,
and Neisseria meningitidis. Based on these sequence
similarities, the gene corresponding to ORF2 in R. etli CE3
has been named wzt according to the newly implemented
bacterial polysaccharide gene nomenclature scheme (8) (Fig.
2). A phylogenetic tree based on the
sequence alignment (Fig. 3)
is represented in Fig.
4. Although not all the functions of the
listed gene products have been determined, two branches corresponding
to the two described classes of Wzt-like proteins can be observed.
Of all the ATP-binding proteins reported, the R. etli CE3
Wzt protein most closely resembles a KpsT-like protein of
Synechocystis sp. (29% amino acid identity) and an ABC
transporter of M. xanthus (26% amino acid identity). It
also shows good similarity to AbcA of A. salmonicida, which
forms an atypical ABC transporter for O-antigen transport (41). The
conserved sequence determinants, the Walker A and Walker B motifs (42)
common to all ATP-requiring proteins, are also present in Wzt of
R. etli (Fig. 3).
Identification of wzm, Which Encodes the Membrane
Component--
In other bacteria, the wzm gene is located
upstream of wzt. We knew from previous hybridization
experiments (data not shown) that a 7.8-kb EcoRI fragment is
located upstream of wzt. Therefore, we constructed in pUC19
a size-fractionated library with clones containing EcoRI
inserts of ~7.8 kb. Positive clones were selected by Southern
hybridization with a DNA probe containing the first 100 base pairs of
the wzt gene of R. etli CE3, which overlaps with
the 7.8-kb EcoRI fragment. Subcloned fragments from one
positive clone were subjected to automated sequence analysis and
revealed an ORF (ORF1 in Table I) that showed similarity to Wzm
proteins in other bacteria (Fig. 5). The
gene corresponding to ORF1 of R. etli CE3 has been named
wzm. The predicted gene product is similar to the KpsM-like
protein of Synechocystis sp. (27% amino acid identity), Wzm
of P. aeruginosa (22% identity), and Wzm of B. pseudomallei (22% identity) (43). As for Wzt, we postulate the
existence of two classes of Wzm-like proteins based on the results of a
phylogenetic analysis (Fig. 6) of the
amino acid sequences.
Although the sequence similarities of Wzm proteins are rather low, the
structure of Wzm is highly conserved. The hydrophobicity profile (data
not shown) indicates that R. etli CE3 Wzm contains six
putative membrane-spanning domains, with its N and C termini protruding
into the cytoplasm. In addition, we identified a small hydrophobic
domain that resides between helices V and VI (Fig. 5) and postulate
that it is also situated within the cytoplasmic membrane as reported
earlier for KpsM from E. coli K1 (44). This was designated
as the SV-SVI linker. It has been assumed that the transmembrane
domains of ABC transporters form a pore-like structure required for
substrate transport across the cell membrane (45), and it is tempting
to speculate that the SV-SVI linker region of Wzm may also function as
part of a pore for polymer transport.
To our knowledge, no other wzm or wzt homologs in
Rhizobium have been reported. Priefer and Prechel (46)
reported the isolation of two chromosomally encoded ORFs in
Rhizobium leguminosarum bv. viciae strain VF39
with similarities to proteins involved in the export of
polysaccharides, but no sequences were deposited in public data bases.
Chromosome Mapping of wzm and wzt--
We examined the genome
localization of the wzm and wzt genes in R. etli CE3. The physical map of the region identified in this study
is identical to part of the physical map of pCOS109.11 (15). A
hybridization experiment using part of wzm as a DNA probe
indicated that wzm and wzt are located in the
Structural Analysis of FAJ1200--
Several of the previously
mapped mutations were correlated with LPS structural defects to
identify specific biosynthetic functions for particular genetic loci
within the lps PAGE and Immunoblot Analyses--
Fig.
7 shows the PAGE and immunoblot analyses
of FAJ1200 LPS extracted into the phenol and aqueous layers during hot
phenol/water extraction compared with LPS extracted from the parental
strain, CE3. Fig. 7A shows the results of Alcian blue/silver
staining and indicates that FAJ1200 LPS extracted into the aqueous
phase consists only of LPS II (lane 1), whereas LPS
extracted into the phenol layer contains largely LPS II with traces of
LPS I (lane 2). Fig. 7 (B-D) shows the results
of immunoblots using mAbs JIM26, JIM27, and JIM28, which specifically
recognize the O-chain polysaccharide of CE3 LPS (48). As would be
expected, no reaction with any of these mAbs was observed for the
FAJ1200 aqueous extract. However, the trace amount of LPS I found in
the phenol extract of FAJ1200 bound both JIM26 and JIM27, as did
parental CE3 LPS I, but, unlike parental LPS I, was not recognized by
JIM28. Thus, LPS I found in the FAJ1200 phenol extract is structurally
different from parental LPS I in that it failed to bind JIM28. Also, as
may be expected, Fig. 7D shows that for both the FAJ1200
phenol and aqueous extracts and for parental CE3 LPS, the LPS II
fractions all stained with mAb JIM32, which is specific for the inner
core region of R. leguminosarum (and R. etli) LPS
(49). These results indicate that LPS II from FAJ1200, the major LPS
produced by this mutant, has the same core structural features that are
present in parental CE3 LPS.
To prove that the phenotype of mutant FAJ1200 was due to the mutation
in the wzm locus and not the result of polar effects on
putative downstream genes, a complementation experiment was done.
Wild-type wzm and wzt genes were first amplified
by PCR, yielding a PCR fragment of 2.4 kb, and subsequently cloned into the cloning vector pFAJ1708 carrying the nptII promoter
(22), resulting in pFAJ1248. pFAJ1248 was mobilized into the FAJ1200 mutant to determine if supplying the genes in trans could
restore the wild-type phenotype. As shown in Fig.
8, the complemented mutant FAJ1206
carrying pFAJ1248 (containing the PCR-amplified wzm and
wzt genes) was able to synthesize LPS I as the parental strain (lane 3), thus demonstrating that the mutant
phenotype observed was due to wzm mutation. Nevertheless, it
can be noticed that the ratio between LPS I and LPS II in the
complemented mutant differs (more LPS II) from the ratio observed in
the wild-type strain. Interestingly, a similar phenotype (LPS II > LPS I) was observed for two R. etli mutants (CE395 and
CE394) with mutations downstream of the wzt gene (50). Since
we do not know the detailed genetic structure of the wzm/wzt
downstream region, it is still possible that the mutation in
wzm has a slight polar effect on genes farther downstream.
Nevertheless, a clone containing only wzm and wzt
restores the capacity of the FAJ1200 mutant to produce LPS I and
substantiates the finding that wzm and wzt are
indeed responsible for O-antigen export.
HPAEC Analysis of FAJ1200 LPS--
Fig.
9 shows the core HPAEC profiles obtained
from parental CE3 and mutant FAJ1200 purified LPS II. Both the parental
and mutant mild acid hydrolysates contain the expected core tri- and tetrasaccharides. This result is consistent with the immunological data
using JIM32, which show that FAJ1200 LPS II contains those core
structural features found in parental LPS II. In addition, the HPAEC
results for FAJ1200 LPS II also indicate the presence of an additional
peak not found in parental LPS. This peak is monomeric Kdo, which is
also released from FAJ1200 LPS during mild acid hydrolysis. This Kdo
residue is due to the external Kdo in the core region that is attached
to Gal6 of the tetrasaccharide. In parental LPS, the
O-chain is attached to that Kdo residue; and therefore, it is not
observed in the HPAEC profile. However, in the mutant, that Kdo residue
does not have the attached O-chain; and as a result, monomeric Kdo
is liberated during mild acid hydrolysis and is observed in the HPAEC
profile. Finally, FAJ1200 LPS II is contaminated (peak *)
slightly by non-LPS oligosaccharides (see below).
Composition Analysis--
Table
II shows the results of glycosyl
composition analysis of FAJ1200 purified LPS II compared with parental
CE3 LPS. The results show that FAJ1200 LPS II is totally devoid of any
O-chain or outer core glycosyl residues and contains only those
glycosyl residues that should be expected for the inner core region.
The higher level of mannose and the presence of glucose in LPS II of
FAJ1200 are due to a non-LPS contaminant (Fig. 9, peak *)
since this glucose/mannose-containing component does not bind to a
polymyxin B-Sepharose affinity column on re-purification.
We have presented evidence that inactivation of an ABC transporter
in R. etli CE3 drastically affects the structure of LPS in
that it prevents addition of the O-antigenic polysaccharide to the
inner core region. In the ABC transporter-dependent
pathway, the glycosyl residues are added one at a time to the
nonreducing end of the growing polysaccharide, which is attached to the
undecaprenyl phosphate carrier and, once polymerized, is transported to
the periplasm via an ABC transporter and then ligated to the lipid A
inner core region. As mentioned in the Introduction, two other pathways
are known for the polymerization and export of O-antigens (7). The
Wzy-dependent system is the classical pathway first described in Salmonella enterica serogroups A, B, D, and E. Recently, Keenleyside and Whitfield (51) reported a third O-antigen
biosynthetic pathway called the synthase-dependent pathway
for the assembly of the poly-N-acetylmannosamine O-antigen
(factor 54) of S. enterica sv. borreze.
The structure of the O-antigen repeating unit is
strain-dependent. In R. etli CE3, it is a
heteropolymer of a trisaccharide unit consisting of glucuronic acid,
fucose, and 3-O-methyl-6-deoxytalose (2). Therefore, the
isolation of a wzt-homologous gene responsible for the
export of such a heteropolymeric O-antigen in R. etli CE3 is
of interest since most of the bacterial polysaccharide biosynthetic
genes encoding ABC-2 transporters are involved in the biosynthesis of
homopolysaccharides (6). Other known exceptions are the gene cluster of
A. actinomycetemcomitans involved in the biosynthesis of the
heteropolymeric serotype b-specific polysaccharide antigen and
the heteropolymeric type II O-polysaccharide gene cluster of
B. pseudomallei, which also contains the wzm and
wzt genes (43, 52).
Some features of the O-antigen structure of R. etli CE3
further support the operation of an ABC
transporter-dependent pathway, as evidenced in this study.
The CE3 O-chain polysaccharide is characterized by its very discrete
size, i.e. five repeating units, and by the presence of
methylated sugars. Other polysaccharides that are synthesized by
monomeric addition of glycosyl residues to the nonreducing end and that
would utilize the ABC transporter-dependent pathway are
reported to contain methylated capping residues, such as those from
Klebsiella O5 (53) and E. coli O8 (54).
Wzt shows similarity to NodI from R. leguminosarum bv.
viciae, Bradyrhizobium japonicum, S. meliloti, and Rhizobium sp. NGR234. On the basis of
both their sequence similarity to traffic ATPases and their
organization in an operon together with the nodA,
nodB, and nodC genes, the NodI and NodJ proteins
have been implicated in the secretion of
lipochitooligosaccharide molecules. Evidence for such a role has
been obtained by McKay and Djordjevic (55) and Spaink et al.
(56). However, lipochitooligosaccharides are not transported
exclusively via NodIJ because a low level secretion of Nod factors was
observed in the nodJ mutant. An explanation for this could
be the presence of a redundant Nod factor secretory complex. For
example, in S. meliloti, the nolFGHI genes were
proposed to constitute such an alternative transporter (57). However, this hypothesis has not yet been confirmed experimentally (58). A
second possibility is transporter "cross-talk": the idea that transporters such as KpsM and KpsT devoted to secretion of capsular polysaccharides could also secrete Nod factors. However, an E. coli kpsM or kpsT mutant carrying the nodABC
genes of Azorhizobium caulinodans is able to secrete the
produced lipochitooligosaccharide molecules (59). In relation to our
study, it could be speculated that such transporter cross-talk results
in the inefficient transport of O-antigenic polysaccharide, therefore
explaining the trace amounts of LPS I in the phenol phase of the
phenol/water extraction from R. etli FAJ1200. It is not
known why the small amount of LPS I that is made by FAJ1200 is
extracted into the phenol rather than the aqueous phase. However,
recently, another LPS I that fails to bind JIM28 but still binds JIM26
and JIM27 also could not be extracted into the aqueous phase. This LPS
I was present in a CE3 mutant selected for its inability to bind JIM28
and was not extracted into the aqueous phase (60). Interestingly,
also a small amount of O-antigen was detectable by dot immunoblotting from the wzm mutant (formerly called the rfbD
mutant) of Yersinia enterocolitica when whole bacteria were
applied to the filter (61).
The structural analysis of the mutant LPS shows that the mutation in
FAJ1200 gives rise largely to an LPS that does not have the O-chain
polysaccharide. Even though a small amount of LPS I was found, it was
present only in the phenol layer of the hot phenol/water extract and is
structurally different from CE3 LPS I in that it fails to bind mAb
JIM28. The mAb JIM28 epitope disappears when the bacteria are grown in
the presence of low O2, at low pH, or in the presence of an
anthocyanin isolated from seed extract, mimicking the symbiotic
interaction (62). The above results also show that LPS II produced by
the mutant is identical to parental LPS II in its PAGE mobility and in
its binding to mAb JIM32. The HPAEC analysis revealed that mutant LPS
II contains the same core oligosaccharide as that of the parent. The
structure of LPS produced by the FAJ1200 mutant and the fact that the
mutant is affected in a gene encoding an ABC transporter suggest that
the non-repeating unit is also synthesized as part of the O-chain
polysaccharide on the undecaprenyl phosphate carrier. Additionally, the
anomeric configuration of the distal Kdo residue of the inner core
region is phenotype in bean plants.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
region, in which nine complementation groups have been identified,
spanning 17 kilobases of DNA (14).
-region on the
chromosome of R. etli CE3. Furthermore, the LPS produced by
this mutant was structurally analyzed. FAJ1200 LPS is totally devoid of
any O-chain glycosyl residue and contains only those glycosyl residues
that should be expected for the inner core region. This suggests that the non-repeating sequence or O-chain attachment region is also synthesized as part of the O-chain polysaccharide.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Light micrograph of sections of P. vulgaris nodules induced by R. etli CE3
(A), FAJ1200 (B), and CE168
(C). Arrows indicate vascular
bundles.
Global identity scores of ORF1 and ORF2 with similar protein pairs
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Fig. 2.
Physical and genetic map of the region
containing the wzm and wzt
genes. B, BamHI; E,
EcoRI; H, HindIII; P,
PstI; X, XhoI. The
arrowhead indicates the position of the Tn5
insertion in the FAJ1200 mutant.
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Fig. 3.
Multiple protein sequence alignment of Wzt
from R. etli CE3 and similar ATP-binding proteins from
saccharide transport systems. The alignments were generated by
ClustalW (24). Shadings were obtained using the Genedoc
program (25). Black indicates 100% identical or conserved
(D/N, E/Q, S/T, K/R, F/Y/W, L/I/V/M) residues; dark gray
indicates 80% identical or conserved residues; and light
gray indicates 60% identical or conserved residues. Gaps
introduced for optimal alignment are marked by dashes.
Numbers on the right indicate amino acid positions. The
origins of sequences are indicated on the left. Ac, A. actinomycetemcomitans; Ae, A. salmonicida;
Hae, H. influenzae; Nei, N. meningitidis; Sm, S. meliloti;
Ec, E. coli; Kp, Klebsiella
pneumoniae; Bj, B. japonicum; Rl,
R. leguminosarum bv. viciae; Ps,
P. aeruginosa; Re, R. etli;
Sy, Synechocystis; Se, Serratia
marescens; Bu, B. pseudomallei;
My, M. xanthus. For GenBankTM/EBI
accession numbers, see Table I.
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Fig. 4.
Phylogenetic dendrogram showing relative
distances between Wzt-like proteins by the neighbor-joining
method. Numbers represent the bootstrapping score over
100 trials. Only scores above 50 are indicated. The scale at
the top of the tree corresponds to 10% divergence between species. See
the legend to Fig. 3 for species abbreviations.
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Fig. 5.
Multiple protein sequence alignment of Wzm
from R. etli CE3 and similar transmembrane proteins
from polysaccharide transport systems. The alignments were
generated by ClustalW (24). Shadings were obtained using the
Genedoc program (25). Black indicates 100% identical or
conserved (D/N, E/Q, S/T, K/R, F/Y/W, L/I/V/M) residues; dark
gray indicates 80% identical or conserved residues; and
light gray indicates 60% identical or conserved residues.
Gaps introduced for optimal alignment are marked by dashes.
Numbers on the right indicate amino acid positions. The
origins of sequences are indicated on the left. See the legend to Fig.
3 for species abbreviations, and see Table I for
GenBankTM/EBI accession numbers.
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Fig. 6.
Phylogenetic dendrogram showing relative
distances between Wzm-like proteins by the neighbor-joining
method. Numbers represent the bootstrapping score over
100 trials. Only scores above 50 are indicated. The scale at
the top of the tree corresponds to 10% divergence between species. See
the legend to Fig. 3 for species abbreviations.
-region of the chromosome (data not shown). wzm can be
defined by complementation group F, whereas wzt can be
defined by complementation group E, as discussed by Cava et
al. (15). In the study of Cava et al. (15), the two
lps mutations mapped within this DNA region
(lps-22::Tn5 and
lps-183::Tn5) resulted in loss of LPS
I, as attested by SDS-PAGE analysis. The genetic map of wzt
also supports the hypothesis that complementation group E extends into
the adjacent 2.9-kb EcoRI fragment, as mentioned by Cava
et al. (15).
region (47). However, no report was made
concerning mutations lps-22::Tn5 and
lps-183::Tn5. Therefore, we performed a
structural analysis of FAJ1200 LPS.
View larger version (67K):
[in a new window]
Fig. 7.
18% deoxycholate-polyacrylamide gel and
immunoblots of R. etli FAJ1200 and CE3.
Lane 1, LPS from the R. etli FAJ1200 aqueous
layer from phenol/water extraction; lane 2, LPS from the
R. etli FAJ1200 phenol layer from phenol/water extraction;
lane 3, R. etli CE3 LPS. A,
silver-stained 18% deoxycholate-polyacrylamide gel; B,
immunoblot using mAb JIM26; C, immunoblot using mAb JIM27;
D, immunoblot using mAb JIM28; E, immunoblot
using mAb JIM32. The table shows the binding of the specific
antibodies. ++++, strong binding by the antibody; +++ and ++, medium
binding effects; +, weak binding; , no binding.
View larger version (10K):
[in a new window]
Fig. 8.
LPS from R. etli CE3
and derivatives visualized by periodate oxidation and silver staining
after separation by SDS-PAGE using a 15% resolution gel. Crude
LPS extracts were purified as described by Valverde et al.
(63). Lane 1, R. etli CE3 (wild-type); lane
2, FAJ1200 (CE3 wzm::mTn5 mutant);
lane 3, FAJ1206 (FAJ1200 complemented with pFAJ1248).
Arrows indicate the positions of LPS I and LPS II.
View larger version (20K):
[in a new window]
Fig. 9.
Analysis by HPAEC of the core
oligosaccharides released by mild acid hydrolysis of R. etli
CE3 and FAJ1200 LPS. Peak * is contaminating Glc,
which varies in amount in different preparations. Peak 1 is
Kdo, which is derived from the external Kdo residue in the LPS II core.
Peak 2 is GalA. Peak 3 is the tetrasaccharide.
Peaks 4a and 4b are the 4,7- and
4,8-anhydro-Kdo tetrasaccharides, respectively. Peak 5 is
the trisaccharide.
Composition analysis of trimethylsilyl derivatives of LPS from R. etli
CE3 and R. etli FAJ1200 LPS II by gas chromatography-mass spectrometry
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, suggesting that attachment of this residue falls within the domain of the typical core region biosynthetic machinery
(2).
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ACKNOWLEDGEMENTS |
---|
We thank Dale Noel for providing pCOS109.11 and Nick Brewin (John Innes Institute, Norwich, United Kingdom) for the monoclonal antibodies. We thank C. Snoeck for assistance with the plant work and Serge Beullens for technical assistance with the DNA work.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant GM39583 (to R. W. C.) and Department of Energy Grant DE-FG09-93ER20097 (to the Complex Carbohydrate Research Center).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF182824.
§ Supported by "Fonds Wetenschappelijk Onderzoek-Vlaanderen" and the Flemish Government (Geconcerteerde Onderzoeks Actie/98/Vanderleyden).
Postdoctoral Fellow of the Belgian Fund for Scientific Research.
To whom correspondence should be addressed. Tel.: 706-542-4439;
Fax: 706-542-4412; E-mail: RCARLSON@ccrc.uga.edu.
¶ To whom correspondence should be addressed. Tel.: 32-16-32-1631; Fax: 32-16-32-1966; E-mail: Jos.Vanderleyden@agr.kuleuven.ac.be.
Published, JBC Papers in Press, February 9, 2001, DOI 10.1074/jbc.M101129200
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ABBREVIATIONS |
---|
The abbreviations used are: LPS, lipopolysaccharide; ABC, ATP-binding cassette; PCR, polymerase chain reaction; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; HPAEC, high performance anion exchange chromatography; ORF, open reading frame; Kdo, 3-deoxy-D-manno-2-octulosonic acid.
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