Vol. 172, No. 9

Rhizobium meliloti Lipopolysaccharide and Exopolysaccharide CanHave the Same Function in the Plant-Bacterium Interaction

PETER PUTNOKY,1 GYORGY PETROVICS,1 ATTILA KERESZT,1 ERICH GROSSKOPF,lDANG THI CAM HA,1 ZSOFIA BANFALVI,l AND ADAM KONDOROSIl 2*

Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521,H-6701 Szeged, Hungary,' and Institut des Sciences Wgetales, Centre National

de la Recherche Scientifique, F-91198 Gif-sur-Yvette, France2

Received 2 March 1990/Accepted 28 June 1990

A fix region of Rhizobium meliloti 41 involved both in symbiotic nodule development and in the adsorptionof bacteriophage 16-3 was delimited by directed TnS mutagenesis. Mutations in this DNA region were assignedto four complementation units and were mapped close to the pyr-2 and pyr-29 chromosomal markers. Phageinactivation studies with bacterial cell envelope preparations and crude lipopolysaccharides (LPS) as well as

preliminary characterization of LPS in the mutants indicated that these genes are involved in the synthesis ofa strain-specific LPS. Mutations in this DNA region resulted in a Fix- phenotype in AK631, an exopolysac-charide (EPS)-deficient derivative of R. meliloti 41; however, they did not influence the symbiotic efficiency ofthe parent strain. An exo region able to restore the EPS production of AK631 was isolated and shown to behomologous to the exoB region of R. meliloti SU47. By generating double mutants, we demonstrated that exo

and Ips genes determine similar functions in the course of nodule development, suggesting that EPS and LPSmay provide equivalent information for the host plant.

Rhizobia are able to induce nitrogen-fixing nodules on theroots of their host leguminous plants. Products of the bac-terial nodulation (nod) genes are responsible for the earliestevents of the interaction with the plant, including host-specific activation of meristematic cell division and root haircurling. When rhizobia enter the curled root hairs, a polysac-charide tube (the infection thread) forms, grows toward theroot tissue, and branches out into the nodule cells; thisprocess is followed by invasion of bacteria. Bacteria re-leased from the infection thread are wrapped into a mem-brane of plant origin (peribacteroid membrane) and differen-tiate into nitrogen-fixing bacteroids. The development ofsymbiosis is directed also by signals from the environmentand from the symbiotic partner. Constituents of the bacterialsurface are known to be involved in this process, but theirexact role has not been elucidated (for reviews, see refer-ences 14 and 34).The cell envelope of gram-negative bacteria (including

Rhizobium and Bradyrhizobium species) consists of an innercytoplasmic cell membrane and an outer membrane sepa-rated by the periplasmic space and the peptidoglycan layer(35). The extracellular surface of the outer membrane con-sists of abundant channel and specific minor proteins, ex-creted extracellular polysaccharides (EPS), and lipopolysac-charides (LPS). LPS molecules are anchored into themembrane by their lipid A subunits, which carry an oligosac-charide core and an antigenic polysaccharide chain (Oantigen) synthesized from oligosaccharide units (35, 44).

In different Rhizobium species, involvement of either LPSor EPS in the formation of symbiosis has been reported. LPSmutants of Rhizobium leguminosarum bv. phaseoli and bv.viciae unable to synthesize the entire 0 antigen do notinvade host cells; consequently, the nodules are Fix- (11,38, 40). The pss genes involved in EPS synthesis of R.leguminosarum bv. phaseoli are not essential for the effec-

* Corresponding author.

tive noduilation of bean plants, whereas insertions in homol-ogous sequences of R. leguminosarum bv. viciae result in aNod- phenotype on peas (5, 6).

In Rhizobium meliloti SU47, an acidic calcofluor-bindingEPS was shown to be required for effective nodulation.Mutants defective in EPS production form empty noduleslacking infection threads and bacteroids (24, 31, 33). Notonly the absence of EPS but also minor defects in modifica-tion of the polysaccharide chain, such as the lack of succi-nylation or pyruvilation, block the development of symbio-sis, suggesting that EPS takes part in a recognition event (30,37). In contrast, our recent results showed that strainAK631, a derivative of the originally isolated R. meliloti 41(RM41) that had lost the ability to produce calcofluor-binding EPS, was still able to establish effective symbiosiswith alfalfa (41).

In this report, we present the genetic analysis of a chro-mosomal region of RM41 controlling nodule developmentand show that this region carries genes for LPS synthesis.We demonstrate that these genes can replace the symbioticfunction of exo genes when tested in different host plants,suggesting that LPS and EPS can play the same role in theplant-bacterium interaction.

(Part of this work was presented at the 4th InternationalSymposium on Molecular Genetics of Plant-Microbe Inter-actions, Acapulco, Mexico, 1988 [43].)

MATERIALS AND METHODSBacterial strains, bacteriophages, plasmids, and culture

conditions. AK631 is a Fix' Exo- variant of RM41. Deriv-atives of AK631 or RM41 isolated in this study are shown inTable 2, Fig. 2, and Fig. 7. The following Escherichia colistrains were used: HB101 (7) for plasmid propagation, trans-formation and matings; NM512 (supo; obtained from N.Murray, Edinburgh, Scotland) for directed Tn5 mutagenesis;and MC1061 (47) for the marker exchange experiments. Thelysogenic strain PP513 was isolated after infection of NM512by K CIHAl at 30°C. Bacteriophage k CIHAl, coding for a

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thermosensitive repressor, was kindly provided by L. Orosz(Szeged, Hungary). For characterization of R. melilotistrains and mutants, phage 16-3 (39) and phages Ml, M9,M12, and M12hl (17) were used. Bacteriophage X::TnS (4)and pBR322::TnS-CM were used as sources of Tn5 andTnS-CM (48), respectively. Cosmid pVK102 (27) was used toconstruct a genomic library of RM41. pPP428 is a pLAFRicosmid clone harboring thefix-23 region (41). Media, antibi-otic concentrations, and culture conditions were describedpreviously (42). Calcofluor tests were carried out accordingto the method of Leigh et al. (31), using calcofluor brightener28 (Sigma Chemical Co., St. Louis, Mo.). Incubations werecarried out at 30°C unless otherwise stated.

Conjugation, complementation, genetic mapping, and ma-nipulation. Derivatives of cosmids pLAFRi and pVK102were transferred into R. meliloti strains by the method ofFriedman et al. (20). Transconjugants were selected in thepresence of tetracycline and were tested for complementa-tion in a phage test or in a plant test as described earlier (41).Genetic mapping was carried out by using plasmid pJB3JI (8)as described by Forrai et al. (19).The unique Kmr Smr central region of the TnS(109)

insertion was replaced with the Cmr gene of TnS-CM byhomologous recombination between the bordering IS50 se-quences. For this purpose, pEGR214: :TnS(109) and pBR322::TnS-CM were introduced into E. coli MC1061, and recom-binant cosmid derivatives [pEGR214::TnS-CM(109)] wereselected by infecting the transconjugants with A CIHA, andtransducing the in vivo-packaged cosmid population intoPP513. Cmr Tcr transductant colonies were then screenedfor their cosmid content by restriction enzyme analysis.

Directed TnS mutagenesis. Taking advantage of the cosregion, we modified the method of A: :TnS mutagenesis(reviewed in reference 10) as follows. Mutagenesis ofpPP428 and pEGR214 cosmids was carried out in the Xlysogen E. coli strain PPS13. A 1-ml culture of PP513(approximately 109 cells) harboring the cosmid clone to bemutagenized was mixed with A::TnS phage at a multiplicityof infection of 1 to 10 and incubated for 2 h. TnS-containingcells were then selected on LB-tetracycline-chloramphenicolplates (0.2 ml per plate). Colonies were washed off the platesinto S ml of LB, and a sample of the suspension was diluted100 times in LB and incubated with vigorous shaking to anoptical density at 600 nm (OD600) of 0.5 to 0.6. The culturewas transferred into a 45°C water bath for 5 min and thenshaken for 2 to 5 h at 37°C to obtain a clear lysate (0.5 x 1010to 1 x 1010 PFU ml-'). Then 0.2 ml of the lysate was mixedwith 1 ml of PP513 culture. After incubation for 20 min atroom temperature, the cells were shaken for 1 h, and 0.1-mlsamples were plated out onto LB-tetracycline-kanamycinplates. The resulting colonies were tested for cosmid contentby the miniprep method of Ish-Horowicz and Burke (25).The exact positions of the Tn5 insertions were established byrestriction analysis, and these mutations were then recom-bined into the R. meliloti genome by the method of Ruvkunand Ausubel (46), using appropriate cosmid derivatives. Thepositions of TnS insertions in the genome were verified byrestriction enzyme digestions and hybridization.DNA isolation and cloning. For detection of megaplasmids,

we used the method of Eckhardt (16) as described by Forraiet al. (19). Plasmid DNA was isolated as described byIsh-Horowicz and Burke (25). Genomic DNA purificationwas carried out as described by Meade et al. (36). Conditionsfor restriction enzyme digestions and for hybridization ex-periments were described earlier (41). A genomic library ofRM41 was constructed by ligating HindIII-digested cosmid

pVK102 and partially HindIll-digested 20- to 30-kilobase(kb) genomic DNA fragments.

Preparation and characterization of cell envelopes and LPS.Bacterial cell envelopes were isolated by the procedure of deMaagd et al. (12). LPS was extracted by the hot phenol-water method (22, 51). The aqueous phase was exhaustivelydialyzed against water, and low-molecular-weight impuritieswere removed by 4 to 6 h of ultracentrifugation at 100,000 xg. The usual LPS yield was 0.7 to 2.3 mg/liter of log-phasebacterial culture (OD6o0 = 0.4 to 0.5).To monitor the contaminating polysaccharides and the

molecular weight of the LPS, the protocol of Noel et al. (38)was followed. The freeze-dried LPS extract (aqueous phase)was dissolved in 0.1 M EDTA-0.3 M triethylamine, appliedto Sepharose 4B, and eluted with 0.3 M triethylamine.Fractions were assayed for 2-keto-3-deoxyoctulonic acid(KDO) content by the thiobarbiturate procedure (26) and fortotal carbohydrate content by the phenol-sulfuric acid assay(15). To remove proteinase K-sensitive components of theenvelope and protein impurities from the LPS samples,proteinase K digestion was used (1 mg/ml at 60°C for 90min). Proteinase K was inactivated at 100°C for 10 min.1-Glucosidase or P-glucuronidase treatments were applied in0.2 M sodium acetate (pH 5.0) buffer for digestion of thedifferent envelope and ultracentrifuged LPS samples (60min, 37°C). The same treatment in the absence of theenzymes did not affect the inactivation capacity of thesamples. To analyze the content of peak I, lipid impuritiesfrom the freeze-dried material dissolved in water wereextracted in chloroform. Then the water phase was hydro-lyzed by mild acid treatment (1% acetic acid at 100°C for 2h), resulting in a lipid precipitate. The precipitate washydrolyzed further into fatty acids (4 N HCl at 100°C for 6 h),and the released fatty acids were analyzed as describedelsewhere (45). The supernatant was lyophilized and laterfractionated on Sepharose 4B as described above. Phageinactivation experiments were carried out according to themethods of Lindberg (32) and Hancock and Reeves (23). In100 [L of assay solution (50 mM Tris, 100 mM CaCl2, pH7.5), 1 ,ul of envelope or proteinase K-treated envelope or 10p.g of ultracentrifuged LPS samples was incubated with 105phage particles for 1 to 2 h at 30°C. After incubation, thenumber of active phage particles was titered on AK631.

Plant test and microscopic studies. Plant tests were carriedout as described previously (42). Experiments were evalu-ated 6 to 8 weeks after infection when effective symbiosiscould be distinguished by the vitality and lack of chlorosis ofN2-fixing plants. For morphological studies, nodules wereharvested 3 to 4 weeks after inoculation. Samples wereprepared as described elsewhere (41).

RESULTS

Localization of thefix-23 mutation. Strain AK1492 carriesthefix-23 mutation obtained from random TnS mutagenesisof AK631 (Exo- Fix' derivative of RM41) by pSUP1011(28). Recently, a cosmid clone (pPP428) able to complementthe Fix- phenotype of mutant AK1492 has been isolated(41). This clone was used to localize the corresponding fixregion designated thefix-23 region in the R. meliloti genome.In hybridization experiments, pPP428 did not hybridize toAgrobacterium tumefaciens genomic DNA harboring thepRme4lb or pRme4lc megaplasmid of RM41 (ZB717 andZB716; 1), but fix-23 sequences were detected in the chro-mosomal DNA band separated from the megaplasmids of R.meliloti in an Eckhardt gel. When cosmid pPP428 was

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purn-i

narB -15 cys-46met-5

trp-15fix - 23 8 pyr-29, pyr-2

gly-1 pe1pur-4 phe-i5his-2i fur-i

met-2leu-4

FIG. 1. Location of the fix-23 mutation on the RM41 chromo-some, The position of the Kmr gene of thefix-23::Tn5 mutation wasmapped to the markers shown by the method of Forrai et al. (19).The linkage map of RM41 was established by Kondorosi et al. (29),

hybridized to the EcoRI-digested DNAs of AK31 andAK1492, a 12-kb fragment appeared in the sample ofAK1492 instead of the 6.1-kb wild-type fragment, suggestingthat the TnS insertion of strain AK1492 is located on the6.1-kb EcoRI fragment of the fix-23 region (data not shown).The chromosomal location of the fix-23 region was also

demonstrated when the coinheritance of several auxotrophicmarkers withfix-23 was examined by the method of Forrai etal. (19). The Kmr gene of Tn5 showed weak linkage to thecys46 and phe-15 alleles, suggesting that the fix-23::TnSmutation is located between these two markers. This posi-tion was further verified by the use of other auxotrophicmarkers in this chromosomal region. The highest coinher-itance (80 to 90%) was detected to pyr-29 and to pyr-2markers (Fig. 1).

Genetic analysis of the fix-23 region. The EcoRI physicalmap of the fix-23 region carried by plasmid pPP428 wasestablished (Fig. 2). To delimit fix genes in this region,site-directed TnS transposon mutagenesis was applied. First,Tn5 insertions were isolated on the cosmid clone pPP428 bya modified X::TnS mutagenesis method (see Materials andMethods), after which representative mutations were intro-duced into the wild-type (AK631) genome by homologousrecombination. The positions of the insertions were verifiedby hybridizing plasmid pPP428 to genomic DNA preparedfrom the mutant strains. Since the fix-23 mutation showedboth Fix- and phage 16-3-resistant phenotypes, the gener-ated Tn5 mutants were tested for nitrogen fixation ability onalfalfa plants as well as for sensitivity to phage 16-3. In thisway, a 14-kb DNA region involved in symbiosis was delim-ited (Fig. 2). All of the Fix- mutants proved to be phageresistant except for two that carry adjacent insertions[TnS(618) and TnS(608) in Fig. 2]. It was also found that theFix- mutants were sensitive to phages Ml, M9, and M12,

which were not able to infect wild-type RM41 but werespecific for strain SU47. A host range mutant of phage M12(Ml2hl) was able to grow in the fix-23 mutants as well as inthe wild-type strain.To establish the organization offix genes into complemen-

tation units, complementation experiments were carried outby assaying for the restoration of the original phage patternand symbiotic efficiency. pPP428::TnS derivatives were in-troduced into representative homogenotes, and the trans-conjugants were examined in phage and plant tests. Fourcomplementation groups were demonstrated (Table 1 andFig. 2). The largest one spans a region of about 8 kb of DNA,extending from insertion TnS(649) to TnS(678); the secondgroup is represented by Tn5(590) and Tn5(618); the third oneincludes Tn5(608) and TnS(634); and the fourth one containsTnS(633) and Tn5(671).Morphology of nodules induced by mutants of the fix-23

region. The ultrastructure of nodules induced by the wildtype (AK631) and by its derivative carrying the fix-23 muta-tion (AK1492) was described earlier (41). In this study, thesymbiotic phenotypes of the mutants belonging to the dif-ferent complementation units were examined. Mutants car-rying TnS insertions 666, 677, and 575 (for locations, see Fig.2) induced empty nodules lacking infection threads andbacteroids. Tn5 insertions 553, 590, 618, 634, 671 resulted inmixed phenotypes: in addition to the empty nodules, avariable number (20 to 40%) of invaded nodules were de-tected. Invaded nodules contained infection threads, and incontrast to the wild type (Fig. 3a), deteriorated bacteroidswere observed in a few host cells (Fig. 3b). Bacteroidscarrying the TnS(634) mutation differed from the others inmorphology. They harbored large, electron-dense bodies inall cases examined (Fig. 3c).The fix-23 region codes for genes involved in LPS produc-

tion. For assigning a function to the genes located in thefix-23 region, the phage 16-3-resistant phenotype of themutants offered a more convenient approach than theirsymbiotic deficiency. In contrast to the wild-type cells,which inactivated 99.9% of the phages, mutants at the fix-23region were unable to adsorb the phage 16-3 particles,indicating that the reason for phage resistance is an alteredcell surface. The bacterial envelope was isolated from thewild-type and mutant strains, and a phage inactivation assaywas used to identify the surface component altered on themutant cells. The wild-type bacterial envelope adsorbed 99%of the phage particles. No detectable decrease in the phageinactivation activity was observed after proteinase K treat-ment, but P-glucosidase and 3-glucuronidase, which are ableto digest several polysaccharides, destroyed the adsorptionability (data not shown).

16-3 % s s ss s ss RR RRRR R RR RR RRR R s sRRR R R ss s s s s ss sFix +44+ + ++--+++ + + + ++

- .Win UtiemW C 0 4-0Q en -

TnSWWg-Wg. <g eug.UGg..M°e ..

111 1 11 11 11 1 1 1111 lE E E E E EE

a I.

m -_

EE EI a .

E E Ea .

FIG. 2. Physical and genetic map of thefix-23 region of RM41. Vertical bars represent the TnS insertions isolated in the region. Sensitivity(s) or resistance (R) to phage 16-3 and nitrogen fixation ability (Fix) of mutants carrying genomic insertions in an AK631 background areindicated at the top. _, Complementation units (Table 1). Below is shown the EcoRI restriction map of the region.

l~~~~~~

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R. MELILOTI LPS AND EPS IN NODULE DEVELOPMENT 5453

TABLE 1. Complementation groups in the fix-23 region'

Sensitivity

Chromosomal pPP428::TnS derivativemutation None pPP428mutation (649) (674) (678) (590) (618) (608) (551) (634) (633) (671)

A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C

fix-23::Tn5(649) R s - s R + R s - R s -(674) R s - s R + R s - R s - R s -(678) R s- s R + R s - s R +(590) R s - s R + sR + R s - s s - s R + s R +(618) s s- s R + s s s s s R s R(608) s s - s R + s R + s R + sS - s s -(551) R s- s R + s R s R + s s - R s - R s - s R +(634) R s - s R + s R s s R s(633) R s - s R + s R s R s R s R R s R s

apPP428::Tn5 derivatives were introduced into differentfix-23::Tn5 mutants of AK631, and the transconjugants were tested for sensitivity to phage 16-3 (A)and phages Ml, M9, and M12 (B), as well as for symbiotic properties (C). R, Resistant; s, sensitive; +, Fix'; -, Fix-.

Proteinase K-treated cell envelopes can be considered ascrude LPS preparations (11); therefore, LPS purified by theconventional hot phenol method was tested for its phageadsorption ability. Similar to the intact cells and proteinaseK-treated envelopes, wild-type crude LPS preparations in-activated phage 16-3, but the wild-type sample treated with3-glucosidase and the crude LPS of the fix-23 mutantsshowed no activity (Fig. 4). In contrast, samples isolatedeither from the wild-type or from the mutant cells inactivatedthe host range mutant phage M12hl particles (Fig. 4),indicating the specificity of the adsorption. In TY liquidmedium, the mutants exhibited autoagglutination (Fig. 5),similar to the LPS mutants described for R. leguminosarumbv. viciae (40).The LPS was characterized by gel filtration chromatogra-

phy on a Sepharose 4B column (Fig. 6). The fractions were

assayed for total sugar and KDO content. KDO is a charac-teristic component of the oligosaccharide core of all LPS andhas not been detected in any other type of polysaccharide(44). In the case of the mutants, LPS represented by the firstsugar peak was clearly different in its elution characteristicsand KDO content (Fig. 6C and D) from the wild-type LPS(Fig. 6A). To analyze the content of peak I, the freeze-driedmaterial was degraded by weak acid hydrolysis, resulting ina lipid precipitate (see Materials and Methods). The precip-itate was degraded further by an additional hydrolysis ap-propriate to release free fatty acids from lipid A (45).Thin-layer and gas chromatography of the esterified materialdemonstrated the presence of different fatty acids (G. Petro-vics, unpublished results).The water-soluble material from the mild acid treatment

was fractionated on Sepharose 4B. Peak I of the AK631

t =- ~ it,, d : r

F-I~ ~ ~ ~ ~ '

~~~~~~~~~~~~4*~~~~~~~~~~~~~~~~~~~~~7

9 f~ Ig9Sd)~~~~~~~,Se,

.S eh , ,,-

b.?11?.

FIG. 3. Ultrastructure of host cells invaded by the wild-type strain AK631 and its Fix- derivatives. Shown are AK631 (a) and AK631carrying mutations TnS(553) (b) and TnS(634) (c). Bars represent 1 urm. dBD, Deteriorated bacteroid; M, mitochondrion; PBM, peribacteroidmembrane; BD, bacteroid; IT, infection thread; S, starch grain.

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100

w

0.

50~~~~z

w

phage 16-3 16-3 M12hl 16-3 M12hlLPS source - AK 631 - AK631 fix23::TnS

FIG. 4. Phage inactivation ability of LPS samples. LPS of thewild type (AK631) or of a mutant strain [AK631 fix-23: :TnS(553)]was mixed with phage 16-3 or M12hl, and the titer of the remainingactive phage particles was determined. All LPS samples wereprepared by the hot phenol method (see Materials and Methods).Additional treatment is indicated in the column.

crude LPS sample disappeared, and a lower-molecular-weight polysaccharide fraction with KDO content was de-tected (Fig. 6B), indicating the complete hydrolysis of theKDO-containing material into lipid and polysaccharide com-ponents, as described for the LPS of another R. melilotistrain (50). Consequently, peak I contained only LPS. Sim-ilar results were obtained when the KDO-containing frac-tions of a mutant strain were hydrolyzed except that thepolysaccharide part (as expected from the Sepharose chro-matography of the crude LPS) showed elution characteris-tics different from those of the wild type (data not shown).These results provided further support to the hypothesis thatgenes located in the fix-23 region are Ips genes involved inthe synthesis of LPS in strain RM41.

Fix phenotype of the Ips mutations depends on geneticbackground. Strain AK631 is used in our laboratory as awild-type strain (2), although it was isolated from the slimyRM41 and has a compact colony morphology. AK631proved to be as effective in symbiosis with Medicago sativaas the original RM41. Our recent results suggested thatAK631 fails to produce succinoglycan, the calcofluor-bind-ing, main characteristic acidic EPS of R. meliloti; conse-quently, in contrast to the Exo+ parent strain (RM41), it iscalcofluor dark (41). To show the effect of the Exo+ back-ground on the phenotype of fix-23 (Ips) mutations, repre-sentative TnS insertions (649, 666, 668, 677, 644, 553, 590,551, 634, 633, and 671 in Fig. 2) were recombined into theRM41 genome. None of these mutations resulted in a cal-cofluor-dark (Exo-) phenotype, suggesting that Ips genes inthe fix-23 region are not involved in the production of thecalcofluor-binding EPS. The lps::Tn5 mutations exhibitedthe same phage resistance pattern in the Exo+ strain as inthe Exo- AK631 background. Surprisingly, however, allmutants induced effective nodules on alfalfa. These resultssuggested that the Ips region codes for an entity necessaryfor effective symbiosis which is functionally alternative tothat determined by the exo genes.To isolate the exo region defective in AK631, a genomic

library of RM41 (Exo+) was constructed into the cosmidvector pVK102, using partially HindIII-digested total DNA.This library was introduced into the Exo- strain AK631, andtransconjugants were tested for their Exo phenotype on

FIG. 5. Autoagglutination of mutants in the fix-23 region. (A)AK631; (B) AK631 fix-23::TnS(553); (C) AK631 fix-23::TnS(671).Strains were grown in TY liquid medium. After 3 days, the mutantsshowed autoagglutination.

calcofluor-containing plates. A cosmid clone (pEGR214)able to restore the Exo- phenotype of AK631 to Exo+ wasisolated from a calcofluor-bright colony and used in furtherexperiments. pEGR214 showed strong homology to thecosmid clone pD56 (33), which carries the exoB region of thesecond symbiotic megaplasmid (pRmeSU47b) of R. melilotiSU47.To localize the exo gene mutated in AK631 (exo-631) and

to delimit the borders of the exo region, directed Tn5mutagenesis was carried out on pEGR214. The pEGR214::TnS derivatives were introduced into AK631, and theirability to restore the Exo+ phenotype was assayed. Thesame mutations were also introduced into the RM41 (Exo+)genome via homologous recombination, and the mutantswere tested on calcofluor-containing medium as well as inplant tests. All cosmid derivatives except pEGR214:.TnS(109) were able to complement both AK631 and an exoBmutant of strain SU47 (Rm5078; 13) to Exo+. The recom-bined mutations did not affect the Exo+ phenotype in RM41except for the TnS(109) insertion, which resulted in an Exo-Fix' phenotype (Fig. 7).To prove that exo and Ips (fix-23) regions determine

alternative functions required for normal nodule develop-ment, exo Ips double mutants of RM41 were constructed.First, the unique Kmr Smr central region of the TnS(109)insertion was replaced by the Cmr gene of TnS-CM (48)through homologous recombination between the borderingIS50 sequences (see Materials and Methods). Then theappropriate pEGR214::TnS-CM(109) recombinant was intro-duced into RM41 and AK631 and into the lps (fix-23)derivatives of RM41. After recombination of the exo::TnS-CM(109) mutation into these genetic backgrounds, the re-sultant strains were tested for calcofluor-staining, phagesensitivity, and symbiotic properties (Table 2). As expected,none of the single exo or Ips (fix-23) mutants were affected insymbiotic nitrogen fixation ability, whereas all of the doublemutants were Fix-. When the lps region (pPP428) or the exoregion (pEGR214) was introduced into the double mutants,the Fix' phenotype was restored. In contrast to the doublemutants, both lps and exo single mutants were able toestablish effective symbiosis not only with M. sativa but alsowith other Medicago (M. media and M. varia) and Melilotus(M. albus and M. officinalis) species tested.

DISCUSSIONIn this work, we show that RM41 possesses a fix region

(fix-23) involved in LPS synthesis which provides an alter-native function to that of the exo genes during symbiotic

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A , B A Cu

1.2-

1.0-

Q18.

0.6-

0b

0.2-

0

S~~~~~~0~~~~~

X20 30 40

fraction numberFIG. 6. Sepharose 4B chromatograms of hot phenol-water extracts of strain AK631 (A), peak I of the AK631 extract after removal of the

lipid moiety by hydrolysis (B), AK631 fix-23::Tn5(553) (C), and AK631 fix-23::Tn5(671) (D). Extracts from 1 liter of culture (15 mg offreeze-dried powder) were applied to a Sepharose column (1 by 62 cm) in 300 mM triethylamine. Fractions of 1.2 ml were collected, and equalportions were measured at OD548 for KDO (0) and at OD485 for total carbohydrate (0) content as described elsewhere (38). Blue dextranappeared from fraction 18, whereas glucose was eluted from fraction 104 in our system.

nodule development. First, a genetic map of one of the fixregions isolated earlier as nodule developmental regions (41)was established. Genes in this region are clustered on thechromosome close to the pyr-2 and pyr-29 markers andorganized into four complementation units. Since TnScauses polar mutations (3), these units probably representtranscriptional units. The size of unit I (8 kb) and thedifferent phenotypes of TnS insertions of units II and IIIsuggest the existence of more than one cistron within thesecomplementation groups.

Several findings indicate that a surface structure, LPS, isaltered in the mutants: (i) mutants in thisfix region exhibitedan altered phage resistance pattern (they became resistant tophage 16-3 and sensitive to phages Ml, M9, and M12); (ii)mutants showed autoagglutination in TY liquid medium; (iii)polysaccharide-degrading enzymes completely destroyedthe phage 16-3 adsorption ability of the wild-type cell enve-lope and crude LPS preparations; (iv) in contrast to thewild-type sample, crude LPS isolated from the mutants wasnot able to bind phage 16-3; (v) the KDO-containing material(peak I) could be completely hydrolyzed into lipid and

a

polysaccharide moieties; and (vi) LPS of the mutant strainsproved to be different from the wild-type LPS in chromato-graphic properties.These results suggest that genes located in the fix-23

region code for enzymes necessary for the synthesis of astrain-specific LPS. We believe that specific oligosaccharidemotives are necessary for both phage 16-3 adsorption andinvasion of the host plant. These structures may overlap butare not identical, since we could identify two genes indifferent transcription units in which mutations resulted onlyin a Fix-, not in a phage 16-3-resistant, phenotype [TnS(608)and TnS(618) insertions in Fig. 2].The ultrastructure of nodules induced by representative

mutants of thefix-23 region suggests that surface propertiesof the different mutants are not exactly the same, consistentwith their different phage adsorption abilities. Mutants car-rying TnS(666), TnS(575), and TnS(677) are likely to have themost drastic changes on their surfaces, since they induce100% empty nodules, whereas other insertions occasionallyallow bacteria to invade a few host cells but the developmentof symbiosis ceases at the late stage. The inability of

Exo phenotypesc omplementation

of AK631homogenotes

of RM41

TnS

insertions

onb pEGR214 ,p.

H Bg

D _ cCD _ oo

E B E E Bg

+ +

+ +

04

+ + +

_ # t n0D

...I . . .. . . .

E BgEBg B BEBgE E Bg EBgE£ E

kbFIG. 7. Physical and genetic map of the DNA region able to restore the Exo+ phenotype of AK631. (a) EPS production of the

transconjugant AK631 strains harboring pEGR214 cosmids with different Tn5 insertions (vertical bars) and of RM41 homogenotes carryingthese Tn5 insertions. (b) Physical map of the insert of pEGR214. Restriction sites: E, EcoRI; B, BamHI; Bg, BglII; H, HindlIl.

UN'o

C3040

+++ + ++ +-1++ + + + 4++

- - - - CD0 -0---

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TABLE 2. Phenotypes of exo and lps mutations in RM41'

Calcofluor Growth of phage FixStrain Genotype test 16-3 M1 M9 M12 phenotype

RM41 exo+ lps+ Bright s R R R +AK631 exo-631 Ips+ Dark s R R R +PP666h exo-631 lps::TnS(666) Dark R s s sPP59Oh exo-631 lps::TnS(590) Dark R s s sPP699 exo+ lps::TnS(666) Bright R s s s +PP691 exo+ lps::Tn5(590) Bright R s s s +AT22 exo::TnS-CM(109) lps+ Dark s R R R +AT33 exo::TnS-CM(109) lps::TnS(666) Dark R s s sAT31 exo::Tn5-CM(109) lps::Tn5(590) Dark R s s s

a exo-631, exo mutation of AK631; Ips, the fix-23 region. For positions of the mutations, see Fig. 2 and 7. R, Resistant; s, sensitive.

invading bacteria to fix nitrogen suggests that Ips genes maybe involved in at least two stages of symbiotic noduledevelopment.LPS mutants of R. leguminosarum bv. phaseoli or bv.

viciae that fail to produce the entire 0 antigen have beendescribed (38, 40). These mutants are defective in theinvasion process, similar to the EPS (exo) mutants of R.meliloti SU47 (18, 31). Interestingly, LPS mutants of strainSU47 were reported to be Fix' in symbiosis (9).

In contrast to SU47, an Exo- derivative (AK631) ofRM41unable to produce the calcofluor-binding acidic EPS induceseffective nodules on alfalfa. Therefore, we supposed earlierthat other cell surface components may also be responsiblefor the same function during the infection process (41).When fix-23 (Ips) mutations were introduced into an Exo+background, all derivatives remained Fix', indicating thatthe Ips genes can replace the function of the exo genes.Recently, in line with our findings, the locus sxb wassuggested to be involved in the LPS synthesis of RM41which suppressed Exo- mutations (49).To provide evidence for our hypothesis, we characterized

the exo mutation of AK631 (exo-631) and constructed Ipsexo double mutants. The wild-type exo region defectivein AK631 was isolated from RM41 and found to be homol-ogous to the exoB region of R. meliloti SU47. A cosmidclone carrying the exo::TnS(109) mutation was not able torestore the Exo+ phenotype either in AK631 or in an exoBderivative of strain SU47, indicating that TnS(109) hadinserted into the same gene (exoB) or at least into thesame transcriptional unit that is inactivated in AK631. Bothexo and Ips single mutants induced effective nodules onalfalfa, whereas exo Ips double mutants showed a Fix-phenotype, further supporting the suggestion that these tworegions are able to functionally substitute for each other insymbiosis.

Recently, the existence of a second kind of EPS (EPS-II)in R. meliloti SU47 was demonstrated (21). The productionof EPS-Il is repressed in the wild-type strain. EPS-II sup-presses the symbiotic defect of EPS-I-deficient strains on M.sativa but not on four other host plants if a second mutationenables derepression of its synthesis. In contrast to EPS-Ilof strain SU47, LPS and EPS ofRM41 are interchangeable insymbiosis not only with M. sativa but also with other naturalhost plants tested so far.Although several reports on the importance of LPS or EPS

in pathogenic or symbiotic plant-bacterium interactions havebeen published, this is the first evidence that these structurescan substitute each other. It seems that the role of surfacepolysaccharides in plant-microbe interactions is to provide

some signal(s) promoting recognition events irrespective ofthe surface structure containing the polysaccharide or itsoligosaccharide form as signal.

After submission of this report, an article by Williams etal. (52) appeared in which suppression of the symbioticdefect of the exo mutants ofR. meliloti SU47 by introductionof the lpsZ (sxb) gene of RM41 was shown. lpsZ is a singlelocus on the second symbiotic megaplasmid (pRme4lc) inRM41, whereas the fix-23 region described here is a chro-mosomal one and carries a gene cluster. One may speculatethat lpsZ plays a regulatory role in LPS synthesis.

ACKNOWLEDGMENTS

We are grateful to E. R. Signer and L. Orosz for providing phagestrains and to R. Russa, I. Vfgh, and E. Petr6nyi for help with theLPS work. We thank F. Dedk, I. Boros, and M. Nbllenburg foradvice on final preparation of the manuscript, Z. Sarai, I. Kiss, andS. Jenei for technical assistance, and B. Dusha and G. Nbllenburgfor photos.

This work was supported by grant OTKA553 from the HungarianAcademy of Sciences.

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