Vol. 174, No. 16JOURNAL OF BACTERIOLOGY, Aug. 1992, p. 5183-51890021-9193/92/165183-07$02.00/0Copyright © 1992, American Society for Microbiology

Different Plasmids of Rhizobium leguminosarum bv. phaseoliAre Required for Optimal Symbiotic Performance

SUSANA BROM,* ALEJANDRO GARCIA DE LOS SANTOS, TOMASZ STEPKOWSKY,MARGARITA FLORES, GUILLERMO DAVILA, DAVID ROMERO,

AND RAFAEL PALACIOSDepartamento de Genetica Molecular, Centro de Investigacion sobre Fijacion de Nitr6geno,

Universidad Nacional Aut6noma de Mexico, Apartado Postal 565-A,Cuemavaca, Morelos, Mexico

Received 18 March 1992/Accepted 11 June 1992

Rhizobium leguminosarum bv. phaseoli CFN42 contains six plasmids (pa to pf), and pd has been shown to bethe symbiotic plasmid. To determine the participation of the other plasmids in cellular functions, we used apositive selection scheme to isolate derivatives cured of each plasmid. These were obtained for all except one(pe), ofwhich only deleted derivatives were recovered. In regard to symbiosis, we found that in addition to pd,pb is also indispensable for nodulation, partly owing to the presence of genes involved in lipopolysaccharidesynthesis. The positive contribution of pb, pc, pe, and pf to the symbiotic capacity of the strain was revealedin competition experiments. The strains that were cured (or deleted for pe) were significantly less competitivethan the wild type. Analysis of the growth capacity of the cured strains showed the participation of the plasmidsin free-living conditions: the pf strain was unable to grow on minimal medium, while strains cured of anyother plasmid had significantly reduced growth capacity in this medium. Even on rich medium, strains lackingpb or pc or deleted for pe had a diminished growth rate compared with the wild type. Complementation of thecured strains with the corresponding wild-type plasmid restored their original phenotypes, thus confirming thatthe effects seen were due only to loss of plasmids. The results indicate global participation of the Rhizobiumgenome in symbiotic and firee-living functions.

Bacteria of the genus Rhizobium have the capacity tointeract with the roots of leguminous plants, forming nitro-gen-fixing nodules. In many Rhizobium species, symbiotic(pSym) plasmids have been defined as the plasmids thatcarry the essential genes for nodule formation and nitrogenfixation on the appropriate host plants (18, 26, 34). Geneticinformation present on the chromosome (15, 31, 32, 35) andother extrachromosomal elements (20-22, 24) has also beenshown to participate in the symbiotic process, contributingto early or late nodule development, nitrogen fixation, andcompetitiveness (8, 12, 19, 20, 22, 27, 41, 43, 44). Rhizobiumleguminosarum bv. phaseoli CFN42 contains six plasmids(pa to pf) ranging in size from 150 to 600 kb, and pd has beenidentified as the symbiotic plasmid, as it carries nod and nifgenes (36, 37) and confers on anAgrobacterium tumefaciensstrain the capacity to nodulate beans (5). The completephysical map of this plasmid has been determined (16). Thestructural complexity of the R. leguminosarum bv. phaseoligenome has been evidenced by its high content of reiteratedsequences (13, 16) and the occurrence of frequent genomicrearrangements (4, 14, 40).

Concerning pSym unlinked genes, Noel et al. (30, 33) haveshown that in R leguminosarum bv. phaseoli CFN42, se-quences involved in lipopolysaccharide (LPS) productionare required for formation of completely differentiated nod-ules.As an approach to study the functional relationship of the

different genomic elements, we decided to analyze theparticipation of each one of the plasmids of CFN42 in thesymbiotic and free-living cellular functions. We selectedderivatives cured of each plasmid through a strategy that

* Corresponding author.

allows positive selection for insertion as well as for loss offunction. A similar approach was used by Hynes and Mc-Gregor in a comprehensive study of the plasmids of R.leguminosarum bv. viciae VF39 (20).The strategy employed allowed us to obtain derivatives

cured of each of five plasmids and deleted for one of them.The data show that in addition to the physical complexity ofthe R. leguminosarum bv. phaseoli genome, a functionalcomplexity may also exist, as all of the plasmids affectsymbiotic and/or growth functions.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The differentstrains used are listed in Table 1. R. leguminosarum bv.phaseoli strains were grown at 30°C in PY (32) or minimalM9 (1) medium with 10 mM ammonium chloride and succi-nate as nitrogen and carbon sources, respectively. Esche-nchia coli and Agrobacterium strains were grown on LBmedium (29) at 37 and 30°C, respectively. For growth ratemeasurements, overnight cultures of the strains were grownon PY medium and the cells were centrifuged, suspended insterile water (twice), and diluted in 250-ml Erlenmeyer flaskswith 100 ml of either minimal or PY medium. The flasks wereincubated at 30°C with shaking (100 rpm). Growth wasmonitored by measurement of turbidity in a Klett-Summer-son photoelectric colorimeter, with a 500- to 570-nm rangefilter. For the pb- strain, growth was determined by seriallydiluting the cultures in 10 mM MgSO4-0.01% (vol/vol)Tween 40, plating them on PY, and counting CFU. Mediacontaining sucrose at a final concentration of 12.5% (wt/vol)were prepared as previously described (40). Antibiotics wereadded in the following concentrations: nalidixic acid, 20 (toselect against E. coli) or 100 (to select against A. tume-

5183

5184 BROM ET AL. J. BACTERIOL.

TABLE 1. Bacterial strains

Strain Relevant characteristic(s) Source or reference

R. leguminosarum bv. phaseoliCFN42 Wild type 36CFNX182 CFN42 cured of pa This workCFNX183 CFN42 cured of pb This workCFNX184 CFN42 cured of pc This workCFNX89 CFN42 cured of pd This workCFNX185 CFN42 deleted for pe This workCFNX186 CFN42 cured of pf This workCFNX187 CFNX182 complemented with pa::TnS-mob This workCFNX188 CFNX183 complemented with pDel27 This workCFNX189 CFNX183 complemented with pCosl26 This workCFNX190 CFNX183 complemented with pb::TnS-mob This workCFNX191 CFNX184 complemented with pc::TnS-mob This workCFNX192 CFNX89 complemented with pd::TnS-mob This workCFNX193 CFNX185 complemented with pe::TnS-mob This workCFNX194 CFNX186 complemented with pf::TnS-mob This workCFNX180 Chromosomal TnS-mob insertion (referred as CFN42-18 in This work

reference 27)CFNX181 Source of pe::TnS-mob of CFN42 This workCFN2001 CFN42 cured of pa and pd 23

Agrobacteiium tumefaciensGM19023/pa::TnS-mob Source of pa::TnS-mob of CFN42 13GM19023/pb::TnS-mob Source of pb::TnS-mob of CFN42 13GM19023/pc::TnS-mob Source of pc::TnS-mob of CFN42 13GM19023/pd::TnS-mob Source of pd::Tn5-mob of CFN42 13GM19023/pf::TnS-mob Source of pf::TnS-mob of CFN42 13

Escherichia coliS17/pDR21 Source of TnS-GDYN1 39HB101/pDel27 Source of pDel27 3HB101/pCosl26 Source of pCosl26 6HB101/pRK2013 Conjugation helper 11

faciens) jg ml-'; rifampin, 100 jig ml-1; neomycin, 60 ,ugml-'; spectinomycin, 75 ,ug ml-'; tetracycline, 10 ,g ml-';streptomycin, 100 ,g ml-'.

Plasmid profiles and hybridization analysis. Plasmid pat-terns were visualized by the procedure of Eckhardt (9),blotted onto nitrocellulose, and hybridized as describedpreviously (13). Genomic DNA was digested with EcoRI,subjected to electrophoresis in 1% agarose gels, blotted ontonitrocellulose, and hybridized as described above. The plas-mids used as probes were purified from Agrobacteniumtransconjugants harboring each one of the plasmids (13) bythe method of Hirsch et al. (17) and labelled by nicktranslation (38).

Genetic manipulations. Transposon TnS-GDYN1 containsthe GDYN1 cassette, which carries kanamycin-gentamicinand spectinomycin-streptomycin resistance markers andsacR sacB genes (40) flanked by" the IS50 insertion se-quences of TnS (39). CFN42 derivatives carrying TnS-GDYN1 insertions in each plasmid were obtained by matingE. coli S-17 containing the transposon with CFN42 andselecting Nalr 5pr transconjugants. TnS-GDYN1 was local-ized by hybridization of'plasmid profiles from the transcon-jugants by using the transposon as a probe.

Plasmid curing was carried out by plating overnight cul-tures of the transposon-labelled strains on PY plates con-taining 12.5% sucrose. Sucrose-resistant colonies were se-lected and verified as SpS. Plasmid profiles of such colonieswere analyzed by ethidium bromide staining and hybridiza-tion to the purified plasmids of CFN42.

The derivatives cured of pa, pb, pc, pd, and pf werecomplemented with the corresponding TnS-mob-labelledplasmid by conjugation with Agrobacterium strains thatcontain these plasmids (13) by using pRK2013 (11) as ahelper. Transconjugants were selected on PY with nalidixicacid and neomycin. Strain CFN2001 is a derivative ofCFN42 which lacks pd and pa (23). The pe of this strain waslabelled with Tn5-mob, and this strain was used as a donor tointroduce pe into a derivative deleted for this plasmid, withpRK2013 as a helper and selection for Strr Nmr Nalr trans-conjugants.

Complementation of CFN42 derivatives lacking pb withpCosl26 and pDel27 (3, 6) was done by mating E. coli HB101containing each clone with the Rhizobium strain andpRK2013 as a helper and selecting for Nalr Tcr transconju-gants.

Nodulation and nitrogen fixation assay. Overnight cultureswere used to inoculate surface-sterilized Phaseolus vulgariscv. Negro jamapa seeds. Plants were grown in 250-mlErlenmeyer flasks with Fahraeus agar medium (10), withoutadded nitrogen, at 28°C. At day 15, nodulation was scoredand acetylene reduction was assayed as previously de-scribed (25). Surface-sterilized nodules were crushed on PYplates, and the plasmid pattern of single colonies waschecked by ethidium bromide staining and hybridization tothe corresponding plasmids.

Competition assays. Seedlings were inoculated with mix-tures of parental and derived strains in an approximatelyequal ratio. One of the strains in the mixture always carried

R. PHA4SEOLI PLASMIDS REQUIRED FOR SYMBIOSIS 5185

FIG. 1. CFN42 derivatives with Tn5-GDYN1 insertions in eachplasmid. Odd-numbered lanes are ethidium bromide-stained plasmidprofiles. Even-numbered lanes are Southern blots of each plasmidprofile probed with the transposon. Lanes: 1 and 2, CFN42; 3 to 14,CFN42 derivatives with TnS-GDYN1 insertions in pa (lanes 3 and4), pb (lanes 5 and 6), pc (lanes 7 and 8), pd (lanes 9 and 10), pe(lanes 11 and 12), and pf (lanes 13 and 14). The letters on the leftmark the positions of the different plasmids.

TnS-mob, either on the chromosome or on one of theplasmids, while the other did not. Bacteria for inocula weregrown overnight on PY, centrifuged, and diluted in sterilewater. Inoculum concentrations were determined by A600.Cell numbers were adjusted in suspensions to formulate themixtures of inoculum strains. The final cell numbers wereverified by serially diluting the inocula in 10 mM MgSO4-0.01% (vol/vol) Tween 40, plating them on appropriate mediafor identification of each strain, and counting CFU. Todetermine which strain was responsible for nodule forma-tion, surface-sterilized nodules were crushed on PY platesand single colonies were picked and tested for growth onselective media. The plasmid pattern of some colonies wasalso checked.LPS production. Washed cells of R. leguminosarum bv.

phaseoli strains were examined for LPS production bysodium dodecyl sulfate (SDS)-polyacrylamide gel electro-phoresis (PAGE) by the method of Cava et al. (6). Gels werefixed and stained by using a Bio-Rad silver staining kit aspreviously described (6).

RESULTS

Isolation of cured derivatives and complementation withwild-type plasmids. The six different plasmids of R. legumi-nosarum bv. phaseoli CFN42 were labelled with transposonTnS-GDYN1 as explained in Materials and Methods. The

plasmid pattern and hybridization to the transposon ofderivatives with insertions in each plasmid are shown in Fig.1. These derivatives were plated on PY containing sucrose toselect for loss of plasmids. Some sucrose-resistant coloniesretained the antibiotic resistance markers, indicating thatthey had acquired sucrose resistance by a mechanism thatdid not involve loss of the Tn5-GDYN1 element. Sucrose-resistant colonies that had also lost the antibiotic resistancemarkers were screened for loss of plasmids in Eckhardt-typegels stained with ethidium bromide and hybridized to thepurified plasmids. The results show that derivatives lackingplasmids pa, pb, pc, pd, and pf were obtained, as they didnot present the plasmid band, either by direct visualization(Fig. 2, lanes 1, 5, 13, 17, and 25) or by hybridization to thecorresponding plasmid (Fig. 2, lanes 2, 6, 14, 18, and 26).Also, some derivatives with deletions of pc or pd wereobtained (data not shown). In the case of pe, only strainswith deletions were recovered (peA) (Fig. 2, lanes 21 and 22).We were unable to cure pe, although we screened sucrose-resistant derivatives obtained from six different strains withTnS-GDYN1 insertions in pe and varied the growth medium(PY, MY, or PY supplemented with Casamino Acids) ortemperature (30 or 37°C). We also selected sucrose-resistantderivatives of a strain that had the transposon inserted inpeA&. All of the derivatives obtained still contained thisplasmid.To ensure that the phenotypic traits of the derivatives (see

below) were due only to loss of plasmids, each strain wascomplemented with the corresponding TnS-mob-labelledplasmid as described in Materials and Methods. The ethid-ium bromide-stained plasmid profile (Fig. 2, lanes 3, 11, 15,19, 23, and 27) and the hybridization against the purifiedplasmids (Fig. 2, lanes 4, 12, 16, 20, 24, and 28) of theseconstructions are shown. It should be noted that the strainthat had peA lost it when the complete plasmid was intro-duced.The pb-cured strain was also complemented with clones

pCosl26 and pDel27 (3, 6), which carry genes required forLPS production. Their plasmid pattern is shown in Fig. 2,lanes 7 and 9; both clones hybridized with pb (Fig. 2, lanes8 and 10). These clones complement phenotypic propertiesof the pb-cured strain (see below).

Saprophytic characteristics of cured strains. In Table 2,phenotypic traits of the plasmid-cured strains are presented.It can be seen that the pb- strain has an opaque appearance

5 6 7 8 9 1011 12 13141516 17181920

i5000 LU38' ;: ' ' lt : '?C

FIG. 2. Plasmid-cured, deleted, and complemented derivatives of CFN42. Odd-numbered lanes are ethidium bromide-stained plasmidprofiles. Even-numbered lanes are Southern blots of each plasmid profile probed with the following CFN42 plasmids: pa, lanes 2 and 4; pb,lanes 6, 8, 10, and 12; pc, lanes 14 and 16; pd, lanes 18 and 20; pe, lanes 22 and 24; pf, lanes 26 and 28. Strains: CFNX182 (lanes 1 and 2),CFNX187 (lanes 3 and 4), CFNX183 (lanes 5 and 6), CFNX188 (lanes 7 and 8), CFNX189 (lanes 9 and 10), CFNX190 (lanes 11 and 12),CFNX184 (lanes 13 and 14), CFNX191 (lanes 15 and 16), CFNX89 (lanes 17 and 18), CFNX192 (lanes 19 and 20), CFNX185 (lanes 21 and22), CFNX193 (lanes 23 and 24), CFNX186 (lanes 25 and 26), and CFNX194 (lanes 27 and 28). The asterisk on the left of each panel indicatesthe cured or deleted plasmid.

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5186 BROM ET AL.

TABLE 2. Characteristics of plasmid-cured derivatives of CFN42

Strain Relevant Colony Flocculationa Motilityb Melanin' Nodulation Acetylenegenotype morphology reduction

CFN42 Wild type Glossy - + + + +CFNX182 pa- Glossy - + + + +CFNX183 Pb- Opaque + - +CFNX184 pC- Glossy - + + + +CFNX89 pd- Glossy - + - -CFNX185 peA Glossy - + + + +CFNX186 pf- Glossy - + + + +CFNX188 pb-/pDel27 Glossy - + + +CFNX189 pb-/PCosl26 Glossy - + + +CFNX190 pb-/pb+ Glossy - + + +CFNX192 pd-/pd+ Glossy - + + +

a Checked in liquid minimal or PY medium.b Checked in PY with 0.3% agar.c Checked in PY with tyrosine (50 p,g/ml) and CuS04 (20 ,g/ml).

on solid medium, flocculates in liquid medium, and lacksmotility. These characteristics have been associated with anLPS-impaired phenotype (6, 20, 35). The pb or either of theLPS clones restored the phenotype of the pb- strain (Table2). All of the other strains were similar to the wild type,except for the pd- strain, which showed no melanin produc-tion. The presence of genes required for melanin synthesison this plasmid has been previously reported (2).The growth capacities of the different strains in minimal

and rich media are presented in Fig. 3. The absence ofplasmids pa, pb, pc, pd, and pf or deletion of pe significantlydiminished the growth of the strains on minimal medium,and the most dramatic effect was presented by the pf- strain,which was unable to grow in this medium. The addition of amixture of amino acids, vitamins, or Casamino Acids did notfulfil the growth requirement of the pf- strain (data notshown). Even in rich media, loss of pb or pc or deletion of pesignificantly reduced the growth rates of the strains. Thegrowth rates of some plasmid-complemented strains (pa-/

pa+, pb-/Del27, pb-/pCosl26, pb-/pb+, and pf-/pf+) inminimal medium were measured. In all cases, they wereindistinguishable from that of the wild-type strain (data notshown).

Symbiotic properties of plasmid-cured strains: nodulationand competitiveness. In regard to symbiosis, the absence ofnodulation in the pd- strain was confirmed. Surprisingly,strains lacking pb were not capable of inducing noduleformation (Table 2); inoculation of P. vulgaris roots with thepb- strain resulted only in the formation of a few (zero tothree) bumps per plant. The nodulation capacity was re-stored when the plasmidless strain was complemented eitherwith wild-type pb, pCosl26, or pDel27 (Table 2 and Fig. 4).A direct effect on nodulation or nitrogen fixation of

derivatives cured of the other plasmids was not observed(Table 2), but when we tested their competitiveness incomparison with that of the wild-type strain (Table 3), it wassignificantly (99% level) diminished for pc-, pf-, peA, andpb- strains complemented with pCosl26 or pDel27. On the

A

1*

7

1 2 3 4 5 6 7

B

* *p 1,!* *A

1 2 3 4 5 6 7

FIG. 3. Growth of plasmid-cured or deleted derivatives ofCFN42 on PY (A) and minimal (B) media. Growth is expressed as

the specific growth rate compared with that of the wild-type strain.Strains: CFN42 (lanes 1), CFNX182 (lanes 2), CFNX183 (lanes 3),CFNX184 (lanes 4), CFNX89 (lanes 5), CFNX185 (lanes 6), andCFNX186 (lanes 7). An asterisk indicates a significant differencefrom the wild-type strain at a 90% confidence level for PY mediumand at a 95% confidence level for minimal medium, determined by a

Neuman Keuls test. The specific growth rate is defined as the ratioof the increase in turbidity or cell number to time. Each growthmeasurement was repeated at least three times.

C D

FIG. 4. P. vulgaris roots inoculated with R leguminosarum bv.phaseoli CFNX183 (panel A), CFNX188 (panel B), CFNX189 (panelC), and CFNX190 (panel D) at 15 days after inoculation.

0

i, 50-C. _

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R. PH4SEOLI PLASMIDS REQUIRED FOR SYMBIOSIS 5187

TABLE 3. Competition of CFN42 and its plasmid-cured and complemented derivatives for nodule occupancy in P. vulgarisa

Strains in inoculum No. of nodules observed (expected) showingoccupancy by:

Parent" Derivative Ratio (parent/ Parent Derivative Bothderivative)

CFNX180c CFNX182 (pa-) 1:1 5 (9) 13 (9) 10CFN42 CFNX187 (pa-/pa+) 1:0.8 9 (8) 6 (7) 9CFN42 CFNX188 (pb-/pDel27) 1:0.76 26 (15) 0 (11) 2"CFN42 CFNX189 (pb-/pCosl26) 1:0.76 23 (14) 1 (10) 6dCFN42 CFNX190 (pb-/pb+) 0.8:1 8 (9) 13 (12) 9CFNX180 CFNX184 (pc-) 0.74:1 22 (11) 3 (14) od

CFN42 CFNX191 (pc-/pc-) 1:1 11 (11) 11 (11) 8CFNX180 CFNX185 (peA) 0.85:1 34 (17) 3 (20) id

CFN42 CFNX193 (peA&/pe') 0.8:1 16 (14) 16 (18) 6CFNX180 CFNX186 (pf-) 1:0.8 25 (14) 1 (12) 3dCFN42 CFNX194 (pf-/pf+) 1:1 8 (9) 10 (9) 7

a Approximately 30 nodules from three different plants were analyzed.b Wild type.c TnS-mob-labelled CFN42 retains its original competitiveness (27).d The derivative was significantly different from the parental strain at a 99% confidence level in a x2 test.

other hand, the pa- strain seemed to be more competitivethan strain CFN42, although this difference was significantonly at the 90% level. In all cases, the complementation ofeach strain with its corresponding plasmid restored its wild-type competitive capacity (Table 3).

Localization of LPS production genes. The genes fromCFN42 required for LPS production, reported by Cava et al.(6), which are cloned in pCosl26 and subcloned (7.5-kbEcoRI fragment) on pDel27, complement the morphologiccharacteristics and the nodulation phenotype of the pb-strain. The plasmid localization of the LPS genes wasdetermined by Southern hybridization of restriction enzyme-digested DNA of pb- and wild-type cells by using pCosl26as a probe. The results (Fig. 5) showed no hybridizationsignal in the pb- strain, while the wild-type or comple-mented strains presented several bands. It has already beenmentioned that when pb was used as a probe on Eckhardt-type gels, it hybridized with both cosmids (Fig. 2, lanes 8 and10).Furthermore, LPS production was determined in the

1 2 3 4

FIG. 5. Localization of LPS-coding sequences on pb of CFN42.Autoradiograph of EcoRI digested DNA probed with pCos126.Strains: CFN42 (lane 1), CFNX183 (lane 2), CFNX188 (lane 3), andCFNX189 (lane 4).

different strains by using silver-stained SDS-PAGE gels asdescribed in Materials and Methods. Figure 6 shows that thepb- strain does not contain an LPS I band, which corre-sponds to the complete LPS molecule, having only the LPSII band that corresponds to the LPS core (6), while thewild-type and complemented strains present both bands.The increase of LPS II in extracts of strains that lack LPS Ihas already been shown (3, 6, 35).

DISCUSSIONIn this work, the contribution to symbiotic and free-living

functions of all of the different plasmids present in an R.leguminosarum bv. phaseoli strain was determined. Hynesand McGregor (20) have reported the systematic curing of allplasmids present in a strain of R. leguminosarum bv. viciae.The analysis of the cured derivatives allowed them to show

LPS I-~

LPS I1- II

FIG. 6. LPS profiles on SDS-PAGE gels. Strains: CFN42 (lane1), CFNX183 (lane 2), CFNX188 (lane 3), CFNX189 (lane 4), andCFNX190 (lane 5).

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5188 BROM ET AL.

that the genes necessary for development of efficient nodulesare present on two plasmids in addition to pSym. One ofthese plasmids carries genes involved in LPS production,and the other seems to be important for free-living growth,as well as for nitrogen fixation.Here we report for the first time that a plasmid (pb) distinct

from the one that carries nod and nifgenes is indispensable fornodule formation on P. vulgaris roots by R leguminosarumbv. phaseoli. We show that previously reported sequencesinvolved in LPS production (6, 30) are localized on thisplasmid. Sequences that are functionally similar to these LPSgenes are present in the genome of R. legwminosarum bv.trifolii ANU843 and in a plasmid of R. leguminosarum bv.viciae VF39. This was shown by complementation of LPSmutants of both strains for LPS production and the symbioticphenotype with R. leguminosarum bv. phaseoli LPS clones(3, 20). Previous reports indicate that R. leguminosarum bv.phaseoli LPS mutants give rise to abortive infection threads,resulting in empty nodules (6, 33). Our results show thatnodule formation is completely impaired when pb is lost. Thissuggests that pb carries other sequences involved in noduleformation, in addition to the LPS genes described by Noel etal. (33). This is further supported by the fact that the symbi-otic properties of pb-cured strains were not completely re-stored by the LPS clones, since the competitive capacity ofthe complemented derivatives was significantly diminishedcompared with that of the wild-type strain.We have previously reported that A. tumefaciens

GM19023 carrying pd of R leguminosarum bv. phaseoliCFN42 is capable of eliciting the formation of differentiatednodules on P. vulgaris roots (5). Taking into account theresults presented here concerning the requirement of pb fornodulation by CFN42, it might be inferred that Agrobacte-rium sp. contains chromosomal genes that are functionallyequivalent to those present in pb of CFN42. A. tumefacienscontaining the Ti plasmid induces formation of tumors on

dicotyledonous plants (42). LPS purified from Agrobacte-rium sp. has been shown to inhibit tumor induction byvirulent bacteria (45). Additionally, chromosomal mutationsin Agrobacterium sp. have been obtained which cause thecells to be avirulent, defective in attachment to plant cells,and altered in cell surface properties (7, 28), thus suggestinga relationship between LPS and virulence. It would beinteresting to know whether the same LPS sequences areneeded for tumor induction and for nodulation by Agrobac-terium sp. carrying either the Ti or a symbiotic plasmid.An approach used in this work for analysis of symbiotic

properties was determination of the competitive capacitiesof the different strains. This approach revealed featureswhich could not be detected solely by analysis of theindividual nodulation capacity of each cured strain. Previousstudies have correlated the competitive behaviors of differ-ent Rhizobium and Bradyrhizobium strains with specificcharacteristics, such as hydrogen oxidation, bacteriocinproduction, speed of infection, dicarboxylic acid transport,nitrogen metabolism regulators (nifA and nodD), and growthcapacity (19, 43). Our results suggest that a complex inter-action of genomic sequences affects the symbiotic propertiesof a strain, viewed as its competitive capacity.The diminished competitiveness of the plasmid-cured de-

rivatives of CFN42 cannot be ascribed solely to lessergrowth capacity. Our results show that equivalent decreasesin competitiveness can occur in strains that have dissimilargrowth decreases (pc- and pf- strains) and that restorationof wild-type growth capacity in a pb- strain complemented

with pDel27 or pCosl26 does not result in wild4type com-

petitiveness.Plasmid preservation is also important for free-living cells,

as demonstrated by the altered growth capacities of plasmid-cured derivatives in different media. Furthermore, essentialgenes present in pe may account for the fact that derivativescompletely lacking this plasmid could not be obtained,notwithstanding all of the strategies employed to find suchstrains. Genes present on pf also have a fundamental role forthe cell, as seen by the inability to grow on minimal medium;this inability seems to be due to a complex metabolicfunction which could not be reversed by addition of aminoacids and vitamins. This somewhat resembles the situationfound with a strain cured of pe ofR leguminosarum VF39,which is also unable to grow on minimal medium, but in thiscase addition of Casamino Acids partially restored growth(20).

This report shows that when the appropriate traits are

surveyed, the participation in symbiosis and/or growth func-tions of all of the plasmids from a strain may be evidenced.The experiments presented in this work were done underlaboratory conditions. The survival and symbiotic capacitiesof plasmid-cured strains in the field should be studied to gaininsight into the functional importance of the different plas-mids under these conditions.

ACKNOWLEDGMENTS

We are grateful to Osvaldo L6pez for technical assistance and toDale Noel for providing LPS probes.

Partial financial support for this research was provided by grant936-5542.01-523-8.600 from the U.S. Agency for International De-velopment.

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