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Journal of Bacteriology, October 2000, p. 5317-5324, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Department of Biology, Marquette University, Milwaukee, Wisconsin,1 and Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia2
Received 7 April 2000/Accepted 9 July 2000
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ABSTRACT |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Judged by migration of its lipopolysaccharide (LPS) in gel
electrophoresis, the O antigen of Rhizobium etli mutant
strain CE166 was apparently of normal size. However, its LPS sugar
composition and staining of the LPS bands after electrophoresis
indicated that the proportion of its LPS molecules that possessed O
antigen was only 40% of the wild-type value. Its LPS also differed
from the wild type by lacking quinovosamine
(2-amino-2,6-dideoxyglucose). Both of these defects were due to a
single genetic locus carrying a Tn5 insertion. The
deficiency in O-antigen amount, but not the absence of quinovosamine,
was suppressed by transferring into this strain recombinant plasmids
that shared a 7.8-kb stretch of the R. etli CE3
lps genetic region 
, even though this suppressing DNA
did not carry the genetic region mutated in strain CE166. Strain CE166
gave rise to pseudonodules on legume host Phaseolus vulgaris, whereas the mutant suppressed by DNA from
lps region elicited nitrogen-fixing nodules. However,
the nodules in the latter case developed slowly and were widely
dispersed. Two other R. etli mutants that had one-half or
less of the normal amount of O antigen also gave rise to pseudonodules
on P. vulgaris. The latter strains were mutated in
lps region and could be restored to normal LPS content
and normal symbiosis by complementation with wild-type DNA from this
region. Hence, the symbiotic role of LPS requires near-normal abundance
of O antigen and may require a structural feature conferred by quinovosamine.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Rhizobium etli bacteria induce bean plants to form root nodules in which they fix nitrogen. As nodules develop, the bacteria penetrate to interior plant cell layers by means of an infection thread, in which a linear bacterial colony is surrounded by a tubular wall and plant plasma membrane that separates the bacteria from plant cytoplasm (24).
Mutants of R. etli that lack the O-antigen portion of the lipopolysaccharide (LPS) are defective in infection (6, 10, 30, 35). Fewer nodules are elicited by such mutants per region of susceptible root, and in the nodules that are formed, very few bacteria can be recovered. Microscopic examination reveals that the infection threads in these nodules are distended and cease development within the root hair or the subjacent cell layer (35). Closely resembling the development of nodules elicited by mutants that do not infect at all (46), the resulting nodule structure lacks most features of a normal mature nodule and has been described as a pseudonodule (27, 35). O-antigen-deficient mutants of Rhizobium leguminosarum and Bradyrhizobium japonicum also exhibit defects in infection and cause aberrant development of nodules on their respective host legumes (4, 12, 22, 23, 37, 38, 39, 44).
Although these studies demonstrate that the presence of O antigen is
important in the symbiosis, the specific structural features that are
required are still not established (30). However, there is
evidence that addresses the question of how much O antigen each
bacterium must possess in order to be symbiotically proficient. In
R. etli mutant strain CE166 the proportion of LPS molecules that possess O antigen is less than normal (10). Strain
CE166 is as defective in symbiosis as mutants that lack O antigen
completely. Based on these properties of strain CE166, it has been
argued that R. etli must have more O antigen than this
strain in order to infect bean plants proficiently. However, work to be
presented shows that the LPS of strain CE166 also lacks quinovosamine
(QuiN) (2-amino-2,6-dideoxyglucose), which is contained in wild-type O-antigen-containing LPS as a single residue (6, 16, 17). Therefore, by itself, the phenotype of strain CE166 allows the possibility that either of these LPS features
the relative content of
O-antigen-containing LPS or the presence of QuiNis crucial in the symbiosis.
In previous work it was reported that the O-antigen deficiency and the symbiotic defects of this mutant fortuitously were suppressed by the introduction of lps DNA that does not contain the genetic locus that is mutated in CE166 (10). The biochemical and genetic basis for this suppression was examined further in the work described below. In particular, the suppressed strain was examined for QuiN content in its LPS. The symbiotic phenotype resulting from this genetic suppression was scrutinized more completely as well.
Two other mutants that synthesize less O-antigen-containing LPS than normal were isolated. They exhibited the same symbiotic phenotype as CE166.
The results strengthen the argument that the relative amount of O antigen is an important symbiotic determinant. At the same time, some of the findings are consistent with previous suggestions (12, 20) that specific LPS residues may be important for infection and/or nodule development. In addition, consideration of the proposed structure of the LPS and the absence of QuiN in CE166 raise intriguing questions about the fidelity of complex polysaccharide biosynthesis.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Growth of bacteria.
Bacteria were grown to stationary phase
in TY brotha medium composed of 0.5% tryptone, 0.3% yeast extract,
and 10 mM calcium chloride (3, 41)at 30°C in Fernbach
flasks (for LPS chemical analysis) or glass tubes (for plant
inoculation) shaken at 200 rpm.
Isolation of mutants CE394 and CE395.
Wild-type strain CE3
(Table 1) was subjected to transposon
mutagenesis with Tn5 suicide plasmid pSUP2021 (10,
42). Kanamycin-resistant colonies were screened for binding of
monoclonal antibody JIM28 under conditions in which the LPS I of strain
CE3 changes so that the antibody no longer binds (32). The
details of the screening procedure will be described in a forthcoming
report (V. J. Neumann, D. M. Duelli, and K. D. Noel,
unpublished data). Certain properties of these strains have been
reported previously (31).
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Construction of strain CE451. The 11-kb EcoRI fragment of strain CE166 that contains the Tn5 insertion was cloned into the EcoRI site of pBluescript and subcloned into plasmid pJQ200 (40) by ApaI and XbaI digestion of vector and source DNA. This construct (pEQ166) (Table 1) was transferred into strain CE3, and substitution of the lps-166 mutant locus for the corresponding wild-type locus was selected by plating on TY agar containing kanamycin (30 mg/ml) and 5% sucrose (40). Strain CE451 was one of the colonies arising on this plate.
SDS-polyacrylamide gel electrophoresis (PAGE). Sodium dodecyl sulfate (SDS) extracts of washed cells from fully grown cultures were subjected to electrophoresis in 18% polyacrylamide, and the gels were stained by the periodate-silver procedure as described previously (10). Relative amounts of LPS in different bands were assessed by analysis of the silver staining with an Ambis optical imaging system (32).
LPS chemical analysis. LPS was extracted by the hot phenol-water method (6) and purified by either Sepharose 4B chromatography (8, 10) or by polymyxin-agarose affinity chromatography (17). Neutral sugars and aminosugars were quantified by gas chromatography on SP2330 columns (Supelco) as alditol acetate derivatives after hydrolysis in 2 M trifluoroacetic acid at 121°C for 2 h (1), whereas the content of acidic sugars was determined by gas chromatography on DB-1 (J & W Scientific) and SPB-1 (Supelco) columns as trimethylsilyl (TMS) methyl glycoside derivatives after methanolysis in methanolic 1 M HCl (17). The main GC peak contributed by glucuronic acid (GlcA) during TMS methyl glycoside formation coincides with a peak contributed by C14 hydroxy fatty acids; therefore, some samples were first treated at 100°C with 1% acetic acid to remove lipid A before subjecting the soluble material to derivatization and GC analysis. High-performance anion exchange chromatography (HPAEC) on a Carbo Pac PA-1 column (Dionex), eluted with a sodium acetate gradient in 100 mM sodium hydroxide, was carried out after treatment of the purified LPS with 1% acetic acid at 100°C as previously specified (7).
Complementation or suppression of mutants with cloned wild-type DNA. Cloned DNA in Escherichia coli was transferred to R. etli recipients by conjugation as described previously (10, 11, 13), using plasmid pRK2013 or its derivative pRK600 (15) to mobilize the plasmids listed in Table 1.
Nodulation tests. R. etli strains were tested for nodulation of Phaseolus vulgaris L. cv. Midnight Black Turtle Soup in growth pouches as previously described (35). Nitrogenase activity was measured by the acetylene reduction catalyzed by intact nodulated roots (19, 34), after which the nodules were stripped off the roots, counted, and weighed. Bacterial CFU were determined from crushed, surface-sterilized nodules (35).
Infection by strains labelled with gusA (Table 1) was determined after making longitudinal hand sections from 1-cm-long segments of nodulated roots with fresh portions of double-edged razor blades. The slices were placed immediately in 1 ml of
-glucuronidase (GUS) staining solution (50 mM sodium phosphate [pH 7.0], 1 mM EDTA,
0.1% Sarkosyl, 0.1% Triton X-100, 1 mM ferricyanide, 1 mM ferrocyanide, and 500 µg of 5-bromo-4-chloro-3-indonyl--glucuronic acid [X-GlcA] [from Biosynth] per ml) in a loosely capped
13-by-100-mm glass tube and incubated for 30 min to 24 h at 30°C
with shaking in the dark (47). Staining was arrested by
removing the staining solution and replacing it with the same solution
without X-GlcA or ferricyanide or ferrocyanide. Sharply defined blue
infection traces were observed in wet mounts of the slices by light microscopy.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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O-antigen structure and content of CE166.
The SDS-PAGE gel in
Fig. 1 compares the LPS profiles of
strain CE166 and wild-type strain CE3. The slower-migrating bands labeled as LPS I carry the polysaccharide portion designated as O
antigen, whereas faster-migrating bands labeled as LPS II do not
(6). The relative deficit of LPS I in CE166 is apparent. From image analysis of the silver staining of similar gels, the mutant
appeared to harbor 34% as much LPS I as the wild-type strain (relative
to the same amount of LPS II). However, the migration in SDS-PAGE
suggested that the mutant LPS I was approximately the same size as the
wild-type LPS I.
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Linkage of two LPS defects to same genetic locus. Strain CE451 was selected as a recombinant in which the 11-kb EcoRI fragment of CE166 carrying the Tn5 insertion had replaced the corresponding wild-type DNA of strain CE3. This strain exhibited the sugar composition of strain CE166, specifically the absence of QuiN and the deficiency in O-antigen sugars. On SDS-PAGE strain CE451 exhibited the LPS I deficiency of CE166, having about one-third the LPS I content of wild-type CE3 relative to LPS II (data not shown).
Suppression by DNA from R. etli lps region .
Recombinant plasmid pCOS109.11 carries 30 kb of R. etli DNA
(Fig. 2). The genes within this DNA that affect LPS are known collectively as lps region (29). In previous
work (10) this plasmid was transferred into mutant CE166 to
create strain CE166 (Table 1). In strain CE166 the abnormal ratio
of LPS I and LPS II of strain CE166 is suppressed (Fig. 1). By image
analysis of LPS staining on SDS PAGE (e.g., Fig. 1), the LPS I content
of strain CE166 was at least 85% of the wild-type value relative to
LPS II. This conclusion also was supported by the contents of O-antigen
sugars, which averaged about 80% of the wild-type values (Table 2).
Although the mean values of the molar ratios of O-antigen sugars in
CE166 LPS were uniformly lower than those of the wild type (Table
2), the differences were only marginally statistically significant at
the 90% confidence level for Fuc and Man contents, and not significant
even at that level for differences in the O-methyl-deoxyhexoses. On the
other hand, the differences between CE166 and CE166 in contents of
O-antigen sugars were statistically significant at the 99% confidence
level in a t test.
, the LPS
of this strain clearly retained one deficiency of CE166 LPS. It lacked
QuiN (Table 2).
Using SDS-PAGE as the assay, shorter portions of this lps
genetic region (11) were tested for suppression of CE166.
Plasmids pDEL2, pDEL2-3, and pDEL14 (Fig.
2) also suppressed the LPS I/LPS II
deficiency, whereas pDEL21 did not (data not shown). Therefore, the
suppression appears to require one or more genes in the 7.8-kb region
common to both pDEL2-3 and pDEL14 (Fig. 2).
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Other R. etli mutants deficient in LPS I amount.
Like strain CE166, mutant strains CE394 and CE395 (Table 1) also made
LPS I in lower amounts than the wild type (Fig.
3). The purified LPS from CE395, the
strain with the higher LPS I content, had the normal content of QuiN
(Table 2). Aside from having lower ratios of all O-antigen sugars, it
specifically lacked the 2-O-methyl-6-deoxyhexose found in the wild type
(Table 2).
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region (Fig. 2), as shown by DNA-DNA hybridization. Southern blots of
EcoRI digests of the DNA from strains CE394, CE395, and CE3
(wild type) were probed with labeled pCOS109.11. A wild-type 0.93-kb
band was replaced with a 6.5-kb band in CE394, and an 8.5-kb band was
present in CE395 instead of the 2.9-kb wild-type band. These size
increases are consistent with mutation by insertion of the 5.8-kb
transposon. In this type of analysis, CE166 DNA gave the same
hybridization pattern with pCOS109.11 as wild-type DNA.
Plasmid pCOS109.11 restored the normal LPS I/LPS II ratio when
transferred into these mutants (CE394 and CE395 [Fig. 3]). As
expected from the location of the insertion mutations, plasmids pDEL2
and pDEL2-3 (Fig. 2) also restored the normal LPS I amount, whereas
plasmids pDEL14 and pDEL21 did not (data not shown). The genetic region
in which these mutations map and which complements these mutants,
therefore, is distinct from the locus that suppresses CE166.
Symbiotic phenotypes.
Mutant CE166 gave rise to the
pseudonodulation typical of certain auxotrophs and mutants that lack
LPS I (10, 27); the (pseudo)nodules developed in a slow,
stunted fashion, were highly dispersed along the root, and never
exhibited nitrogen-fixing activity (Table
3). Infections were examined by using a
derivative of strain CE166 carrying gusA (strain CE434
[Table 1]) and viewing the staining of GUS activity in hand sections
of (pseudo)nodules by light microscopy. The results supported previous
observations (10) that infections by CE166 stopped within
the infected root hairs or at most penetrated one to two cell layers
beyond. Infections by a gusA-containing derivative of mutant
CE109 (strain CE433, Table 1), which lacks O antigen completely
(6), gave results indistinguishable from those of CE166.
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gave rise to true nodule
development, although many fewer nodules appeared and their development lagged at least 4 days behind the development incited by the wild type.
For instance, at 14 days after inoculation (d.a.i.), these nodules were
smaller and much less active than those incited by the wild type (Table
3). However, although never as many nodules developed, at 20 d.a.i. the nodules were larger and nearly as active as those of the
wild type (Table 3). Whereas nodules inoculated by CE166 averaged
104 CFU/nodule, those inoculated with either CE3 or
CE166 averaged 2 × 107 CFU/nodule at this time. At
26 d.a.i. the nodules induced by the suppressed mutant averaged
twice the mass of the wild-type nodules, although the specific
nitrogenase activity was still less than wild type (Table 3).
Similarly, infections by CE166 (viewed by GUS staining of
gusA-containing derivative CE346 [Table 1]) were greatly
behind those of the wild type at 16 d.a.i. but were comparable to
those of the wild type by 26 d.a.i. Bacterial isolates from the
eventually well-developed nodules incited by CE166 gave the same
delay in nodule development as CE166 (CE166N of Table 3).
CE166 transconjugants carrying pDEL2-3 or pDEL14 gave results similar
to those of CE166 (data not shown).
Mutants CE394 and CE395 exhibited the same symbiotic block as CE166.
However, when strains CE394 and CE395 were complemented with wild-type
alleles carried on plasmids pCOS109.11, pDEL2, or pDEL2-3, their
symbiotic properties were similar to those of the wild type and clearly
superior to CE166 suppressed by carrying these same plasmids (Table 3).
In summary, the tested strains fell into three groups according to
development of the symbiosis. Group I (strains CE3, CE394, and
CE395) had normal or near-normal proficiency. Group II consisted of
strains CE166 and CE374, the latter being a previously documented strain that appears to have very subtle alterations in LPS I structure (32). These strains elicited markedly fewer nodules, whose
development was obviously slower (at least 4 days behind that of the
wild type) but eventually had near-normal specific nitrogen-fixing activity and greater masses than normal. Group III (strains CE166, CE394, and CE395) elicited sparsely distributed pseudonodules that
contained very low numbers of bacteria and lacked nitrogen-fixing activity.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Structural alterations of CE166 LPS. Clearly, there are at least two alterations: the absence of QuiN and the reduced amount of O antigen in the LPS population. In other respects the structure of strain CE166 LPS I appears to be similar to that of the wild type, as revealed by antigenic reactions, sugar ratios, and SDS-PAGE migration.
The absence of QuiN is intriguing. Structural analysis indicates that QuiN (as its N-acetyl derivative) is present as a 1,3-linked residue in the wild-type LPS structure at the junction between the core oligosaccharide and O-antigen repeating unit (Fig. 4) (16, 17), such that its elimination should truncate the LPS at that point. One possibility is that it is substituted by another residue to allow for LPS I production in strain CE166. The sugar compositions of CE166 and CE166 LPS do
not reveal an obvious candidate for this substituted residue, however.
In particular, glucosamine does not appear to be substituted for QuiN
(Table 2).
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Genetic and biochemical basis of the effect of lps DNA on CE166.
Genetic analysis indicates that the effect of
pCOS109.11 is one of extragenic suppression. The cloned
EcoRI fragment of CE166 that contains the Tn5
insertion is not of a size expected of an insertion in any of the
EcoRI fragments of pCOS109.11, nor does it hybridize with
the DNA of pCOS109.11 in Southern blots (9, 10). Likewise,
the pattern of EcoRI bands revealed by hybridization with
pCOS109.11 as a probe is the same in both CE166 and CE3.
) suppresses only the LPS I deficiency.
Very recent single-pass nucleotide sequencing outward from the
Tn5 insertion of CE166 indicates that it is situated in an open reading frame specifying a protein of 325 amino acids that is
homologous to a family of enzymes involved in deoxysugar synthesis (K. D. Noel, unpublished data). The highest matches are to two proteins (WbfT and WbpV, with 50 and 47% identity, respectively) believed to be involved in the second step of the synthesis of UDP-QuiNAc from UDP-GlcNAc (2).
The DNA responsible for the suppression apparently lies within the
7.8-kb EcoRI fragment common to plasmids pDEL2-3 and pDEL14. This DNA exists already in CE166, presumably with wild-type
functionality if the mutant phenotypes are conferred by the
Tn5 insertion mapping elsewhere. Therefore, it is likely
that the suppression is due to the multicopy dosage of a gene or genes
in this 7.8-kb EcoRI fragment.
There are several reasonable hypotheses for the biochemical mechanism
of suppression, with the basic premise being that the gene encodes a
transferase, O-antigen exporter, or LPS ligase with lax specificity or
that it specifies a regulatory factor, and that multiple genetic copies
lead to higher concentration of the encoded protein. Variations of this
idea are worth pursuing as a means of producing wild-type amounts of
other polysaccharides having an altered or absent residue, so that the
biological importance of the residue itself can be tested. A case in
point is the 2-O-methylation of fucose that is absent from mutant CE395
and whose absence probably is the cause of the lower O-antigen
synthesis or attachment to lipid A.
Implications regarding requirements of LPS structure for
symbiosis.
Comparisons of the phenotypes of strains CE166,
CE166, and CE3 indicate that LPS I must be more abundant than LPS II
in order that a strain be successful in the symbiosis between R. etli and bean roots. The structure conferred by a particular
residue, QuiN, may also be important, but the argument based on the
data of this study for this LPS I feature is equivocal.
infections yielded well-developed nodules with nitrogen-fixing activity. An
obvious inference is that relative abundance of LPS I is crucial in
symbiosis. This conclusion is supported also by the phenotypes of
strains CE394 and CE395. In fact, there are no exceptions to this idea
among the approximately 30 R. etli mutants altered in LPS
structure. Strain CE395 has the most LPS I of any mutant obviously deficient in LPS I; this mutant and all strains carrying less LPS I
fail at an early stage in infection. It should be noted that all extant
strains having a lower LPS I/LPS II ratio exhibit additional
abnormalities in LPS structure, e.g., the abnormal LPS I banding of
CE394 and CE395 (Fig. 3), the methylated sugar missing from strain
CE395, and the absence of QuiN from mutant CE166. Nevertheless, the
only obvious feature common to CE166, CE394, and CE395 is deficiency in
the relative amount of LPS I.
Although near restoration of O-antigen amount in CE166 overcomes the
absolute block in infection suffered by mutant 166, nodule development
by CE166 is slower and sparser than normal. The difference in
symbiosis between CE166 and wild type might be due to either of two
deficiencies in LPS structure in strain CE166. According to
SDS-PAGE, strain CE166 consistently had a lower LPS I/LPS II ratio
than the wild type, and its content of O-antigen sugars was lower
(although without high statistical confidence). Hence, that its
symbiotic phenotype was intermediate between those of CE166 and the
wild type could be because LPS I content in CE166 is not restored
completely to wild-type levels. On the other hand, the LPS of strain
CE166 also differed from wild-type LPS in lacking QuiN. It may be
that this portion of the LPS structure is specifically involved in
promoting nodule development (perhaps by promoting an interaction
required in infection thread development).
Another possibility is that CE166 suffers problems caused by
multiple copies of plasmid pCOS109.11 or its possible instability (36). However, mutants CE394 and CE395 complemented with
pCOS109.11 behave in symbiosis much more like wild type than CE166
(Table 3); therefore, this possibility seems less likely.
Comparison with mutant CE374 and LPS mutants of other species.
The LPS and symbiotic phenotypes of CE166 are reminiscent of the
phenotypes of R. etli mutant strain CE374, whose properties also have suggested that specific LPS structure plays a role in the
frequency of nodule formation and in the rate of infection. Strain
CE374 appears to have wild-type amounts of LPS I, but its LPS I
displays subtle antigenic differences and minor differences in SDS-PAGE
banding compared with the wild type (32, 45). Although the
symbiotic deficiencies of strain CE374 are very similar to those of
CE166, strain CE374 is not deficient in QuiN. In fact, analysis of
sugar composition has not revealed an obvious difference between the
LPSs of mutant CE374 and the wild type. The difference may be in
acid-labile "decorations" of LPS I residues.
and CE374, however, await more definitive information about the LPS structures of all three mutants.
Possible biological roles of these LPS I structural features. Some of the inferences from this work can be summed up in the following hypotheses. Insufficient O antigen leads to complete blockage of infection soon after infection starts (perhaps due to plant toxins that abundant O antigen can prevent from penetrating to targets on the rhizobial cell [14]). This role may be relatively independent of particular structure. However, certain other aspects of the infection process are greatly facilitated by specific structural features of the LPS, possibly including a feature contributed by the QuiN residue. Changes in these LPS features slow infection and, thereby, nodule development and also lead to lower incidence of nodules per infectible region of the root.
That subtle alterations in LPS structure slow infection suggests, as one hypothesis, that infection requires structurally specific interactions between the bacterial LPS and plant receptors on the plasma membrane. Analogous interactions with animal cell receptors have been proposed as part of the activity of LPS in certain bacterial pathogens (21). In developing nodules these interactions might direct the targeting of machinery for membrane growth to the sites at which the bacteria impinge directly on the plant plasma membrane, including the site at which the infection thread is initiated (5) and, later, the tip of the infection thread (30). Similar suggestions have been made previously (24, 44). The trifoliin A protein of clover roots has been proposed as a receptor of the LPS of R. leguminosarum bv. trifolii strains (12, 20). Interestingly, trifoliin A binds at much higher affinity to LPS containing QuiN and the binding is inhibited by QuiN (20). It would be of interest to investigate whether there are LPS-binding proteins from bean that interact preferentially with R. etli CE3 LPS containing QuiN and whether such proteins are associated with infection threads.| |
ACKNOWLEDGMENTS |
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K.D.N. acknowledges the technical assistance of Valerie Neumann, Tina Thorp, Kevin Barleben, and Jodie Box.
This work was supported by grants DE-FG02-98ER20307 from the U.S. Department of Energy (to K.D.N.) and GM39583 from the National Institutes of Health (to R.W.C.) as well as DOE grant DE-FG09-93ER20097 to the CCRC.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biology, Marquette University, P.O. Box 1881, Milwaukee, WI 53201-1881. Phone: (414) 288-1475. Fax: (414) 288-7357. E-mail: dale.noel{at}marquette.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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