2-O-Methylation of Fucosyl Residues of a Rhizobial Lipopolysaccharide Is Increased in Response to Host Exudate and Is Eliminated in a Symbiotically Defective Mutant

When Rhizobium etli CE3 was grown in the presence of Phaseolusvulgaris seed extracts containing anthocyanins, its lipopolysaccharide(LPS) sugar composition was changed in two ways: greatly decreasedcontent of what is normally the terminal residue of the LPS,di-O-methylfucose, and a doubling of the 2-O-methylation ofother fucose residues in the LPS O antigen. R. etli strain CE395was isolated after Tn5 mutagenesis of strain CE3 by screeningfor mutant colonies that did not change antigenically in thepresence of seed extract. The LPS of this strain completelylacked 2-O-methylfucose, regardless of whether anthocyaninswere present during growth. The mutant gave only pseudonodulesin association with P. vulgaris. Interpretation of this phenotypewas complicated by a second LPS defect exhibited by the mutant:its LPS population had only about 50% of the normal amount ofO-antigen-containing LPS (LPS I). The latter defect could besuppressed genetically such that the resulting strain (CE395 ${alpha}$ 395)synthesized the normal amount of an LPS I that still lacked2-O-methylfucose residues. Strain CE395 ${alpha}$ 395 did not elicit pseudonodulesbut resulted in significantly slower nodule development, fewernodules, and less nitrogenase activity than lps⁺ strains. Therelative symbiotic deficiency was more severe when seeds wereplanted and inoculated with bacteria before they germinated.These results support previous conclusions that the relativeamount of LPS I on the bacterial surface is crucial in symbiosis,but LPS structural features, such as 2-O-methylation of fucose,also may facilitate symbiotic interactions.

INTRODUCTION

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Introduction

Bacteria that enter into intimate associations with plants andanimals, whether pathogenic or mutualistic, respond to signalsthat are thought to indicate the presence of the host. In thesymbiosis between legumes and the bacteria collectively knownas rhizobia, one example is the production of Nod factors (lipochitooligosaccharides)by the bacteria in response to flavonoid compounds exuded bythe plant (17, 27). Another potential example is the alterationof the lipopolysaccharide (LPS) of Rhizobium etli when grownin the presence of the most potent nod inducers from Phaseolusvulgaris (17), the anthocyanins exuded by germinating seeds(10, 22, 23). These induced LPS alterations have been detectedby the lack of recognition of the altered LPS by certain monoclonalantibodies (23). Modifications of the LPS structure in strainsof this and other rhizobial species also have been reportedto occur during nodule development and in culture in responseto low pH and low oxygen, as well as nod inducers (1, 16, 18,19, 29, 31). However, in no case have precise chemical changesbeen determined, nor is it known whether these induced LPS modificationsare important in the development or functioning of the symbiosis.

Although little is known of the biological roles of specificstructural features of the rhizobial LPS, the importance ofwild-type LPS in symbiosis is well established. Studies withmutants of a wide variety of rhizobial species have indicatedthat defects in LPS structure severely impair infection andnodule development on numerous legume hosts (20, 22). Generally,conclusions are based on phenotypes of mutants in which largeportions of the LPS molecules are missing. From studies withmutants derived from R. etli CE3, it has been concluded thatthe LPS O antigen must be present in normal amounts in orderfor R. etli to infect and incite normal root nodule developmenton P. vulgaris (22, 24). The cellular basis for this requirementhas not been determined, but it is likely that the LPS playsseveral biological roles that are important in symbiosis andthat a different combination of structural features is requiredin each role (22).

The structure of the LPS of R. etli CE3 grown in tryptone-yeastextract medium has been almost completely determined (Fig. 1)(2, 14, 15, 28). This accomplishment affords the opportunity,not currently possible with other rhizobia, of correlating biologicalroles with specific structural features of the molecule. Oneexample is a mutant LPS whose N-acetylquinovosamine (QuiNAc)residue is replaced by a QuiNAc precursor. The mutant O-antigenstructure is normal otherwise and confers normal sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) mobilityand antigenic properties (15a, 24). Even when the mutant O antigenis present in nearly normal amounts, greatly reduced nodulenumbers and retarded nodule development result (24). Hence,there may be a specific requirement for QuiNAc or some otherstructural feature that depends on QuiNAc, as had been suggestedby studies of differences in the LPS of another rhizobial strainat different growth stages (8).

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FIG. 1. Overview of R. etli LPS I structure and the effects of seed extract. The terminal Fuc has been reported to be either 2,3-di-O-methylated (DOMFuc, as shown above) or tri-O-methylated (11, 14). However, in this study, it was almost exclusively present as DOMFuc. On average, only about one of the other six Fuc residues per LPS molecule is methylated after growth in the absence of seed extract or seed exudate, and the exact distribution of the methylation is unknown. Hence, the residues are depicted as unmethylated, while the small arrows indicate the sites at which the 2-O-methylation occurs (14). Anthocyanins are the components of seed extract or exudate to which the bacteria respond by changing LPS antigenicity (10). In this study it was found that seed extracts induce decreased DOMFuc content and increased 2-O-methylation of Fucs (depicted as the - and + effects, respectively, on the diagram). It is not known which Fuc(s) is up-methylated. Other abbreviations: 3M6dTal, 3-O-methyl-6-deoxytalose; Fuc, fucose; Man, mannose; Gal, galactose; QuiNAc, N-acetylquinovosamine; MeGlcA, methylglucuronate; GalA, galacturonic acid; GlcN, glucosamine; Kdo, 3-deoxy-D-manno-2-octulonic acid; GlcN, glucosamine; GlcON, 2-aminogluconate; R, fatty acyl groups.

In work to be described, changes in LPS sugar composition inducedby growth in host anthocyanins were examined. One of the LPSalterations was a doubling in the proportion of fucose (Fuc)that is 2-O-methylated. Mutation lps-395::Tn5 prevented 2-O-methylationof Fuc under inducing and basal conditions. This mutation providedan approach for testing whether this methylation is requiredin symbiosis.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains, plasmids, and culture conditions.
The bacterial strains and plasmids used in this study are listedin Table 1. For induction of LPS changes with seed extracts,R. etli cultures were grown by gyratory shaking at 200 rpm at30°C in YEC liquid (0.5% yeast extract, 10 mM CaCl₂ [pH6.0]) (9). For genetic manipulations, R. etli was grown in TY(0.5% tryptone, 0.3% yeast extract, 10 mM CaCl₂) solidifiedwith 1.5% agar and supplemented with the appropriate antibiotic(streptomycin [STR], 250 µg/ml; nalidixic acid [NAL],30 µg/ml; kanamycin [KAN], 30 µg/ml; or tetracycline[TET], 5 µg/ml). Escherichia coli strains were grown at37°C on solid or liquid Luria-Bertani media supplementedwith the appropriate antibiotic (STR, 250 µg/ml; NAL,30 µg/ml; KAN, 30 µg/ml; TET, 15 µg/ml; orchloramphenicol, 30 µg/ml).

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TABLE 1. Bacterial strains and plasmids used in this study

Seed extract.
To obtain crude preparations of anthocyanins for induction ofLPS modifications, black P. vulgaris (cv. Midnight Black TurtleSoup) (Idaho Seed Bean, Twin Falls, Idaho) seeds were extractedas described previously (9). The seed extract was collectedfrom 720 g of seeds that were thoroughly rinsed in deionizedwater and surface sterilized with 95% ethanol for 2 min. Onehundred twenty grams of each of the treated seeds was addedseparately to Fernbach flasks containing 250 ml of sterile 0.1%HCl. The flasks were then incubated at 30°C on a shakerat 200 rpm for 16 h. The liquid phase was centrifuged at 700x g for 20 min at 4°C. The supernatant was filtered over2 sterile Whatman #2 filter papers. The total volume was adjustedto 900 ml with sterile deionized water. To obtain bacteria withinduced LPS changes, 450 ml of this extract was added to a 2.8-literFernbach flask with 550 ml of 1.8x YEC (0.91% yeast extract,18 mM CaCl₂), resulting in a final pH of 5.9, and 20 ml of R.etli fully grown in YEC was added. The control cultures weregrown at the same time in the same type of flask with 1.0 litersof YEC lacking seed extract. Bacteria were harvested when fullgrowth was indicated by the appearance of a copious cell pellicleon the side of the flask (generally at 24 h).

SDS-PAGE and analysis of LPS antigenicity.
Bacterial cells from 1 ml of fully grown cultures were washedin 1 ml of deionized water two times, pelleted, and extractedin 0.1 ml of SDS-PAGE sample buffer (6) at 100°C for 3 min.Samples of purified LPS or crude phenol-water extracts wereprepared by adding 1 mg of the lyophilized material in 1 mlof SDS sample buffer and heating at 100°C for 3 min. Aftercentrifugation to remove insoluble material, the SDS extractswere subjected to electrophoresis in discontinuous SDS-PAGE(6) with 18% (wt/vol) acrylamide resolving gels. LPS in thegels was stained by modification of a previously described periodate-silvermethod (6). The gels were shaken in six changes of fixative(10% glacial acetic acid, 40% methanol) for at least 1 h. Afterincubation in 0.7% sodium metaperiodate for 10 min, the gelswere rinsed in six changes of glass-distilled water for 35 min,followed by a 10-min incubation in 0.2% silver nitrate and a1.0-min (no longer) rinse in glass-distilled water, and thendeveloped for 5 to 6 min in Bio-Rad silver stain developer.After SDS-PAGE, some gels were electrotransferred to nitrocellulosebefore periodate-silver staining of the residual gel contents.The dried nitrocellulose blots were immunostained by using monoclonalantibody (MAb) Jim 28 (a generous gift of N. J. Brewin, Norwich,United Kingdom) and anti-rat immunoglobulin M conjugated withalkaline phosphatase (Sigma Chemical Co.) as previously described(23).

LPS sugar compositions.
LPS was extracted from washed bacterial cell pellets by thehot phenol-water method (4) and purified by Sepharose 4B chromatography(5, 26). After complete hydrolysis of LPS sugar linkages in2 M trifluoroacetic acid, reduction with NaBD₄, and acetylation,alditol acetate derivatives of the LPS were analyzed for neutraland amino sugar compositions by gas chromatography on a 60-mby 0.53-mm by 0.20-µ fused silica capillary SP2330 column(Supelco) (14). The oven temperature was raised 1°C min^-1from 150 to 185°C, 10°C min^-1 from 185° to 245°C,and then maintained at 245°C for 35 min. With this protocol,2-methylfucose was eluted at 25.8 min, and its peak returnedcompletely to baseline before 3-methyl-6-deoxytalose emergedat 26.4 min. 2,3-di-O-methylfucose emerged at 21.7 min, wellseparated from all other peaks. Acidic sugar compositions weredetermined on an SPB-1 column (Supelco) as trimethylsilyl-methylglycosidederivatives after methanolysis in methanolic 1 M HCl (14). Identificationof tri-O-methylfucose, di-O-methylfucoses (DOMFuc), and 2-O-methylfucose(2MeFuc) was done by comparison of elution times with thoseof a standard mixture of O-methylated Fucs that varied in positionand number of methyl groups, kindly provided by L. Scott Forsberg.Coupled GC and electron impact mass-spectral analyses confirmedthe positions of the methyl groups on the 6-deoxyhexose moiety.Where standard deviations are given in the tables that follow,they are based on values from LPS preparations from at leastthree separately grown cultures. The standard deviations arebased on the deviations in the molar ratios before the datawere normalized.

Isolation of mutant strain CE395.
This mutant was isolated after Tn5 mutagenesis by immunoblotscreening of colonies replicated onto agar amended with seedexudate. The exudate was prepared by aqueous acetone extractionof material exuded into water during germination of P. vulgaris(cv. Midnight Black Turtle Soup) seeds (procedure B in reference23). After evaporation of the acetone and lyophilization ofresidual water, the dry material was dissolved in water andadded to TY agar plates containing NAL, STR, and KAN, such thatthe agar contained the exudate from 1.0 seed per ml. Such plates(SE plates) were used within 2 days of preparation and alwayswere protected from light. Colonies on TY agar plates containingNAL, STR, and KAN after Tn5 mutagenesis with pSUP2021 (6) werereplicated (using velvet) onto SE plates. After 2 days at 30°C,colonies were lifted onto a dry nitrocellulose sheet (BA85;Schleicher & Schuell) by letting the sheet contact a smallarea of the plate and, as it became wetted by capillary action,letting it slowly contact the rest of the plate. After beingin contact with the colonies for 5 min, the sheet was allowedto dry fully in air and stained with MAb JIM28 by the procedureabove used for gel immunoblots. Strain CE395 was a solitarycolony that stained darkly among approximately 100 lightly stainingcolonies.

Genetic suppression.
pLPS ${alpha}$ 395 was isolated by exploiting in vivo homologous recombinationin R. etli strain CE395 ${alpha}$ that replaced the corresponding wild-typeallele on the lps region ${alpha}$ in pLPS ${alpha}$ with the mutant allele lps-395::Tn5.Such recombinant plasmids were identified by selecting theirtransfer into E. coli strain HB101. On a TY plate, freshly grownbroth cultures of CE395 ${alpha}$ , HB101, and MT616 were spread togetherin a 4:1:1 ratio of culture volumes and incubated at 37°Covernight. Bacteria from the plate then were streaked or spreadafter dilution onto a Luria-Bertani plate containing KAN andTET and incubated at 37°C overnight. Plasmids were isolatedfrom purified colonies that had arisen on this plate. EcoRIdigests of each plasmid preparation verified that allele replacementhad occurred (absence of the 2.9-kb band from pLPS ${alpha}$ and the appearanceof a new 8.5-kb band). One such colony (HB101/pLPS ${alpha}$ 395) was matedwith CE395 by means of helper strain MT616 in the same manneras the first mating above. Purified transconjugants from thismating that grew on TY containing STR, NAL, KAN, and TET weredeemed to be strain CE395 carrying pLPS ${alpha}$ 395 and designated CE395 ${alpha}$ 395.

Nodulation tests.
P. vulgaris cv. Midnight Black Turtle Soup seeds disinfectedwith 5% hypochlorite were pregerminated on water-saturated filterpaper, planted, and inoculated in 50-ml serum vials wrappedin aluminum foil containing plant nutrient solidified with 0.7%agar or in plastic growth pouches (Mega International, Minneapolis,Minn.) as previously described (25). Alternatively, disinfectedseeds were transferred into the above vials without pregermination,inoculated with bacteria, and put directly into a growth chamber.Regardless of procedure, inoculated plants received 0.25 mlof the respective bacterial culture grown in TY broth. Plantswere grown at 27°C with a 14/10-h light/dark cycle. Nitrogenaseactivity of the acetylene reduction catalyzed by intact nodulatedroots was measured by gas chromatography on a Porapak N column(25). Following the acetylene reduction assay, the nodules werestripped off the roots, counted, and weighed. Occupancy of particularstrains in nodules was measured by counting CFU on agar platescontaining strain-selective antibiotics. The plates were spreadwith aliquots from the contents of crushed, surface-sterilizednodules suspended in TY broth (26).

Statistical analysis.
The probabilities (P) of the null hypothesis (no differencebetween data sets) were calculated using unpaired t tests andone-way analysis of variance with Sigma Stat software, version2.03.

RESULTS

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Results

Alterations in wild-type LPS after growth in anthocyanins (seed extract).
As monitored by the extent of binding of MAb JIM28, the LPSalteration in response to anthocyanins is dose dependent (10,23). LPS was purified from a culture of wild-type strain CE3(Table 1) that had been exposed to sufficient seed extract sothat the synthesized LPS bound very weakly to MAb JIM28 (Fig.2). The sugar composition of the purified LPS from bacteriagrown under these conditions revealed increased content of 2MeFuc,a corresponding decrease in Fuc content, and the near absenceof what is normally the terminal sugar of the LPS, DOMFuc (Table2). The differences in DOMFuc and 2MeFuc after growth in anthocyaninswere highly significant (P < 0.001). The differences in Fucalso were significant (P < 0.05). The other values were notsignificantly different between the two conditions of growth(P $>=$ 0.40). Table 2 gives only the compositionsof neutral and basic sugars, which were determined as alditolacetates. Acidic sugars Kdo, GlcA, and GalA were determinedas trimethylsilyl-methylglycosides; their contents did not differsignificantly after growth in anthocyanins (data not shown).

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FIG. 2. The effect of seed extracts on the antigenicity of LPS. (A) Purified wild-type LPS preparations used to obtain the data in Table 2. LPS was purified after growth in the presence or absence of seed extracts containing anthocyanins, and the LPS was subjected to SDS-PAGE. (B) SDS-PAGE comparison of the LPS in crude phenol-water extracts from strain CE395 and the wild type that had been grown in parallel and, where noted, exposed to the same seed extract. Lanes 1 and 2 in both panels are LPS from wild-type strain CE3. All lanes 3 and 4 are from strain CE395. For all lanes 2 and 4, the bacteria were grown in the presence of seed extract. In each panel the image on the left is of gel lanes stained with silver. (In panel A the gel was stained after being elecroblotted, resulting in barely visible LPS II bands.) On the right in each panel is the blot of these lanes reacted with monoclonal antibody JIM28.

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TABLE 2. Neutral and basic sugar composition of CE3 LPS after growth in seed extract^d

Mutant strain CE395.
Strain CE395 was isolated after Tn5 mutagenesis by virtue ofits not losing reactivity with MAb JIM28 after growth in thepresence of anthocyanins (or crude P. vulgaris seed extract)(Fig. 2B). Figures 2B and 3A illustrate three other propertiesof this mutant strain. First, the amount of O-antigen-containingLPS (LPS I) relative to a form not carrying the O antigen (LPSII) was about 60% of the wild-type ratio, as determined by integratingthe intensities of staining from several gels. Second, the stainingof the mutant LPS I by JIM28 in immunoblots was reproduciblymore intense than the staining of the wild-type LPS I even inthe absence of anthocyanins. The mutant LPS I apparently isa better antigen than the wild type, and this antigenicity isnot substantially decreased after growth in anthocyanins (Fig.2B and 3B). Third, LPS I was split into two almost equally prominentbands. The faster-migrating band comigrated with a very minorband in the wild-type LPS profile.

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FIG. 3. SDS-PAGE and immunoblot analysis showing the effects of recombinant plasmid pLPS ${alpha}$ and mutant plasmid pLPS ${alpha}$ 395. After growth in YEC, with or without seed extract, washed bacteria were processed for SDS-PAGE. (A) Strains CE3 (lanes 1), CE395 (lanes 2), and CE395 ${alpha}$ 395 (lanes 3) grown in YEC without seed extract are compared. (B) Strains carrying pLPS ${alpha}$ (CE3 ${alpha}$ and CE395 ${alpha}$ ) are included; the effects of seed extracts are shown. The analyzed strains were CE3 (lanes 1 and 2), CE395 (lanes 3 and 4), CE3 ${alpha}$ (lanes 5 and 6), CE395 ${alpha}$ (lanes 7 and 8), and CE395 ${alpha}$ 395 (lanes 9 and 10). In lanes 2, 4, 6, 8, and 10 of panel B, the strains had been grown in the presence of seed extract, which is responsible for the streaking above LPS I in the silver-stained gel. In each panel the image on the left is of gel lanes stained with silver. (In panel B the gel was stained after being elecroblotted, resulting in relatively less-intense staining of LPS II bands.) On the right in each panel is the blot of these lanes reacted with monoclonal antibody JIM28.

The overall content of O-antigen sugars in the purified LPSof strain CE395 was lower than that of the wild type (Table3), consistent with the lower content of LPS I revealed by SDS-PAGE.In fact, it is consistent with the mutant LPS population havingabout 50% of the O-antigen content found in the wild type. Inaddition, the mutant LPS completely lacked 2MeFuc (Table 3).2MeFuc could not be detected even by searching for its signatureby mass spectral analysis (R. W. Carlson and K. D. Noel, unpublisheddata). Relative to the other O-antigen sugars, the mean contentof Fuc was higher in the mutant, to an extent that roughly correlatedwith the absence of 2MeFuc (Table 3). Given the lower totalO-antigen content in the mutant, the relative proportions ofthe O-antigen sugars other than 2MeFuc and Fuc were approximatelythe same in the mutant and the wild type.

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TABLE 3. LPS sugar compositions of 395 and 395 ${alpha}$ 395 grown with or without seed extract

Although the wild type showed an increased relative amount of2MeFuc in its LPS after growth in seed extract, the mutant LPSstill lacked 2MeFuc under this condition (Table 3). The otherchange in wild-type LPS sugar composition induced by seed extractdoes appear to occur in mutant CE395; DOMFuc was greatly decreasedin the mutant by growth in the presence of seed extract (Table3).

Suppression of the deficiency in LPS I concentration by harboring lps region ${alpha}$ carrying mutation lps-395::Tn5.
Previously reported restriction analysis (24) indicated thatthe Tn5 insertion of CE395 had occurred in a 2.9-kb EcoRI fragmentof the R. etli CE3 lps region ${alpha}$ (7). lps region ${alpha}$ is operationallydefined as the long stretch (at least 18 kb) of lps genes thatare contained within the 30-kb insert of recombinant plasmidpLPS ${alpha}$ . This plasmid complemented mutation lps-395 such that normalamounts of LPS I were synthesized (Fig. 3). Production of 2MeFucwas also restored in CE395 ${alpha}$ (strain CE395 carrying pLPS ${alpha}$ ). Infact, as in the wild type harboring pLPS ${alpha}$ (CE3 ${alpha}$ ), the amount of2MeFuc in CE395 ${alpha}$ was elevated relative to the content in strainCE3 (Table 4). At the same time, the binding of MAb JIM28 toLPS I was noticeably weaker in strains CE395 ${alpha}$ and CE3 ${alpha}$ than inCE3 (Fig. 3). Nevertheless, strains CE395 ${alpha}$ and CE3 ${alpha}$ respondedto anthocyanin induction; this relatively weak antigenicitywas lost after these strains were cultured in the presence ofanthocyanins or seed extract (Fig. 3).

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TABLE 4. Effects of pLPS ${alpha}$ (lps region ${alpha}$ ) on 2MeFuc contents^a

Based on a previously reported means of suppressing the deficitin LPS I for strain CE166, it was reasoned that multiple copiesof DNA from lps region ${alpha}$ outside of the mutant lps gene mightsuppress the deficiency in the amount of LPS I for CE395. Totest this idea, the corresponding wild-type allele on pLPS ${alpha}$ wasreplaced by lps-395::Tn5 through recombination in vivo, andthis plasmid (pLPS ${alpha}$ 395) was mobilized into CE395. For the resultingstrain (CE395 ${alpha}$ 395), an approximately normal LPS I/II ratio onSDS-PAGE (Fig. 3) and a normal ratio of core versus O-antigensugars (Table 3) were restored. However, like CE395, this strainhad no detectable 2MeFuc (Table 3). Strain CE395 ${alpha}$ 395 made itpossible to separate the biological consequences of the deficiencyin LPS I abundance in CE395 from the consequences of other LPSproperties conferred by lps-395, including the inability to2-O-methylate Fuc.

Plasmid pLPS ${alpha}$ 395 also was used to test the possibility that theLPS I deficiency in CE395 was due to a separate mutation thatmay have existed in CE395 in addition to lps-395::Tn5. If thiswere the explanation, then pLPS ${alpha}$ 395 apparently did not carrythis second mutation; otherwise it should not suppress thisdeficiency. Therefore, pLPS ${alpha}$ 395 was introduced into CE3 as anagent to reconstruct a mutant that would carry only lps-395::Tn5.This mutant, designated CE471, was identical to CE395 in SDS-PAGEand immunoblot analysis.

Symbiosis.
Regardless of the method of testing nodulation, the mutant strainCE395 elicited pseudonodules that resembled in every way thepseudonodules elicited by R. etli mutants that completely lackO antigen (6, 26). P. vulgaris pseudonodules have a characteristicincomplete nodule development that in past studies has beenshown to result from nodulation in the absence of bacterialinfection or with bacterial infection that is limited to roothairs before it stops (21, 26, 32). Because there are no bacteroids,pseudonodules lack nitrogenase activity. On the other hand,CE395 ${alpha}$ 395 exhibited only about a 50% deficiency relative to thewild type in total plant nitrogenase activity when inoculatedon seedlings that had been pregerminated on filter paper andplanted in growth pouches (data not shown). When mixed withthe wild type in the inoculum at equal viable cell density,strain CE395 ${alpha}$ 395 was recovered from nodules of plants grown thisway at a frequency less than 5% of the wild type's. Therefore,although the difference in symbiotic performance was small whenassayed in pouches, it had a measurable impact on the abilityto compete for nodule occupancy. A greater deficiency in nodulationand total plant nitrogenase activity was exhibited when symbiosiswas assayed by growing the inoculated plants in vials containingagar. The greatest deficiency was observed when seeds were plantedwithout pregermination and inoculated with bacteria at the timethey were planted on the agar (Table 5). Fewer, smaller, andless-active nodules resulted from the mutant inoculation. Table5 presents the results of a representative experiment assayedat 20 days after inoculation. At 17 days after inoculation,the differences between CE395 ${alpha}$ 395 and the wild type were greater,a reflection of the noticeably slower development of the mutantnodules. CE395 ${alpha}$ behaved like the wild type under each methodof testing symbiosis.

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TABLE 5. Nodulation and nitrogenase activity of CE395 and CE395 ${alpha}$ 395ⁱ

DISCUSSION

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Discussion

The LPS sugar composition after growth of wild-type R. etliin anthocyanins is consistent with the following hypotheses:(i) Anthocyanins somehow inhibit or repress the transfer ofDOMFuc to the O-antigen terminus. (ii) At the same time, theseseed compounds promote an increase in 2-O-methylation of internalFuc residues. Both hypotheses are supported by the observeddecrease in Fuc content that roughly matches the increase in2MeFuc. If, instead, the change at the O-antigen terminus wereto add a Fuc that was unmethylated or the increase in 2-O-MeFucwere due to the terminal Fuc not having the 3-O-methyl group,the Fuc content would not decrease. Moreover, the phenotypeof CE395, including its sugar compositions after growth withand without anthocyanins, strongly supports the idea that thedecrease in DOMFuc and the increase in 2MeFuc are two separateeffects.

The methods of this study cannot answer the intriguing questionof whether the additional methyl group is going to one particularFuc residue in the O antigen. Indeed, the same question holdswith regard to the average of one 2MeFuc per LPS in the wildtype grown without anthocyanin. Once the experimental approachto answer this latter question is established with that LPS,which is much easier to obtain in large quantities, the issueshould be revisited with regard to the LPS structure of bacteriainduced by anthocyanins.

MAb JIM28 has been very useful in tracking the alteration ofLPS induced by anthocyanin, low pH, and other environmentalconditions (10, 23, 31). Analyses of the LPSs of mutants CE367(11) and CE395 lead to the following conclusions regarding theepitope of antibody JIM28. (i) As 2-O-methylation of Fuc increases,binding of the antibody decreases. This correlation is illustratedby inspecting the sugar compositions (Table 4) and the immunoblots(Fig. 3) of CE3 and CE395 versus CE3 ${alpha}$ and CE395 ${alpha}$ . The greatestantibody-binding affinity is obtained when the O antigen lacks2MeFuc altogether (as demonstrated by CE395 and CE395 ${alpha}$ 395 [Fig.1 and 2]). (ii) If DOMFuc is absent and the LPS is changed inno other way (e.g., in mutant CE367), binding of this antibodyis eliminated (11). The anthocyanin effect, therefore, reducesbinding in at least two ways, by increasing the incidence of2MeFuc and greatly decreasing the content of the terminal O-antigenresidue. These results suggest that the epitope may be at theterminus of the O antigen and that DOMFuc and the 2-hydroxylportion of a Fuc residue are points of contact with the antibodybinding site (Fig. 4). Interestingly, analysis of CE395 LPSafter growth in anthocyanin indicates that the enhanced bindingconferred by the absence of 2MeFuc overrides the loss of DOMFuc.

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FIG. 4. A model depicting hypotheses that three structural features of the R. etli CE3 O antigen affect symbiotic proficiency and/or binding to antibody JIM28.

As noted in the introduction, it is well established that absenceof the O antigen in Lps mutants causes severe breakdowns inthe infection process during the development of root nodulesymbioses. However, it has not been determined at a cellular-biochemicallevel why the O antigen is required, nor is there much informationregarding any specificity in this structural requirement. 2-O-methylationof Fuc residues, as affected by the lps-395 mutation, providesthe second illustration of a specific LPS structural featurewhose alteration may affect the symbiosis of R. etli with P.vulgaris. The first such illustration was the absence and/oralteration of the quinovosamine residue (Fig. 1) caused by thelpsQ166 mutation (15a, 24). In all other reported cases of Lpsmutants affected in symbiosis, either several residues are missingfrom the LPS (generally at least the entire O antigen) or thespecific structural effect is not known (20, 22). Although themutants carrying mutation lpsQ166 or lps-395 (CE166 and CE395)are superior in this respect, they have complex LPS phenotypesthat preclude simple interpretations of the biochemical causesof the biological effects. Nevertheless, both are subject toone approach that has helped in this regard: genetic suppression.

In wild-type R. etli CE3 bacteria, the LPS molecules that carryO antigen (LPS I) predominate over those that lack O antigen(LPS II) both in planta and ex planta, although the ratio ofLPS I to LPS II varies in unexplained ways with culture ageand conditions. When compared at a particular growth condition,strains CE166 and CE395 have about 40 and 50%, respectively,of the wild-type amount of LPS I relative to LPS II (24; Fig.2 and 3 and Tables 3 and 4). This deficiency in LPS I can besuppressed extragenically in each case by introducing multiplecopies of DNA from the R. etli CE3 lps region ${alpha}$ . Since lpsQ liesoutside of lps region ${alpha}$ , the extragenic suppression of mutationlpsQ166 was achieved with the wild-type pLPS ${alpha}$ , giving the straindesignated CE166 ${alpha}$ . In the case of lps-395, however, it was necessaryto introduce the lps-395 mutation on pLPS ${alpha}$ to bring about extragenicsuppression (in CE395 ${alpha}$ 395) rather than complementation with wild-typealleles of the mutated gene (in CE395 ${alpha}$ ). Each suppressed strainstill has the same LPS sugar defect as the unsuppressed mutant:CE166 ${alpha}$ still lacks QuiN (24), and CE395 ${alpha}$ 395 still lacks 2MeFuc.

Comparing these suppressed and unsuppressed mutants leads tothe inference that the amount of LPS I relative to that of LPSII is crucial in the symbiosis. Even though CE166 and CE395have about half the normal LPS I content, the consequence insymbiosis is the same as having no O antigen (no LPS I) at all.Pseudonodules rather than nodules result, and the rate of appearanceof these pseudonodules is much lower than the rate of nodulationelicited by the wild type (i.e., the pseudonodules are relativelydispersed and continue to appear on lower roots, which in wild-typeinoculations do not receive nodules). Although infection byCE395 has not been studied by microscopy, infection by CE166was shown to be blocked at an early point, apparently the samepoint as that for mutants completely lacking the O antigen (24,26). Ongoing experiments, in which R. etli is engineered toproduce the O antigen of an R. leguminosarum strain in placeof its normal O antigen, suggest that overcoming this blockdoes not require a specific O-antigen structure (although, likeCE166 ${alpha}$ and CE395 ${alpha}$ 395, these hybrid strains infect more slowly)(L. Benziger, E. L. Kannenberg, and K. D. Noel, unpublisheddata). One hypothesis consistent with these results is thatR. etli mutants lacking near-normal content of the O antigenare unable to cope with host defenses and/or compounds thatbecome inhibitory to such mutants, and the inhibition becomesinsurmountable very early in infection (12).

Having near-normal LPS I content, the suppressed strains donot suffer this block in infection. However, both strains elicitslower nodule development, lower specific nitrogenase activity,and fewer (more dispersed) nodules. Impaired infection is probablythe basic defect; infection seems to be slowed from the verystart and throughout nodule development. It is tempting to suggestthat the symbiotic deficiencies of CE166 ${alpha}$ and CE395 ${alpha}$ 395 are dueto the specific LPS structural features they lack. However,neither of these strains is in all other respects exactly likethe wild type. In the case of CE395 ${alpha}$ 395, there is a good possibilitythat the Tn5 insertion has polar effects on genes that may affectthe LPS in ways other than solely 2-O-methylation of Fuc. Nevertheless,its phenotype is consistent with the idea that specific O-antigenstructures facilitate infection. This idea is also supportedby the R. etli/R. leguminosarum hybrids mentioned above. Thesehybrids also lead to slower development of (more dispersed)nodules and nitrogenase activity. As one possibility discussedmore fully elsewhere (22, 24), these hints of structural specificityin at least one role of the LPS could indicate that R. etliLPS interacts with a plant receptor that somehow promotes infectionthread development. More specifically, the phenotypes of CE166 ${alpha}$ and CE395 ${alpha}$ 395 could be explained if interaction with the hypotheticalreceptor were enhanced by features conferred by QuiNAc and a2MeFuc residue (Fig. 4).

ACKNOWLEDGMENTS

We thank Russell Carlson for helpful discussions and corroboratingdata, Scott Forsberg for advice and the gift of a series ofmethylated Fucs, and Stephanie Rebone and Andrew Zychowicz forresearch assistance.

This work was funded by grant DE-FG02-98ER20307 from the U.S.Department of Energy.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biological Sciences, P.O. Box 1881, Marquette University, Milwaukee, WI 53201. Phone: (414) 288-1475. Fax: (414) 288-7357. E-mail: dale.noel{at}mu.edu.

REFERENCES

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