Molecular Microbiology (1989) 3(7), 879-889

Characterization of Rhizobium phaseoii Sym plasmidregions involved in nodule morphogenesis and host-range specificity

M. A. Cevallos,^ M. Vazquez/ A. Davalos,^G. Espin,̂ J. Sepulveda^ and C. Quinto^*'Departamenfo de Bioiogia Moiecuiar de Piantas,Centro de Investigacion sobre Fijacion de Nitrogeno,U.N.A.M., Apartado Postal 565-A, Cuernavaca, Morelos,Mexico.^Unidad de Microscopia, Instituto de Fisiologia Celular,UN.A.M., Ciudad Universitaria, Mexico.

Summary

Two nodulation regions from the symbiotic plasmid(pSym) of Rhizobium phaseoii CE-3 were identified.The two regions were contained in overlappingcosmids pSM927 and pSM991. These cosmids, in a Rphaseoii pSym-cured strain background, inducedineffective nodules on Phaseolus vulgaris roots.

Transconjugants of Rhizobium meiiioti harbouringpSM991 induced nodule-like structures on bean roots,suggesting that this cosmid contains host-rangedeterminants.

Analysis of deletions and insertional mutations inthe sequences of pSM991 indicated that the genesresponsible for the induction and development ofnodules in P. vulgaris are organized in two regions20kb apart. One region, located in a 6.8kb EcoRIfragment, includes the common nodABC genes. Theother region, located in a 3.5 kb EcoRI fragment,contains information required for host-range deter-mination.

Introduction

Rhizobium phaseoii induces nitrogen-fix ing nodules oncommon bean roots {Phaseolus vulgaris L.). In R phaseoii,as in other fast-growing Rhizobium species, the genesdetermining both nitrogen fixation and at least the earlysteps of nodulation are tocated on large plasmids calledsymbiotic plasmids or pSym plasmids (Banfatvi ef ai,1981; Beynon et ai, 1980; Hombrecher et ai, 1981;Hooykaase^a/,, 1981; Rosenberg efa/.. 1981), Analysis ofthe CE-3 strain of R. phaseoii has shown the presence ofsix plasmids; one of them, p42d. is approximately 280 kb

Received 24 January, 1989; revised 1 March, 1989, *For correspondence.

in length and confers on bean roots the ability to nodulateand fix nitrogen {Palacios etai, 1985). On this plasmid, thenitrogenase structural genes nifHDK are found in threedifferent locations, in the n//regions a and b. a completeset of these genes is present. The nif region c, however,carries only a single nifH gene (Quinto etai, 1985). It hasbeen shown that when p42d plasmid is transferred into aplasmid-less Agrobacterium tumefaciens strain, thetransconjugants elicit ineffective nodules on bean roots(Brom et ai.. 1988; Palacios ef ai, 1985). With the loss ofplasmid p42d in R phaseoii, the abilities to nodulate andto fix nitrogen are concomitantly lost. Reintroduction ofthe plasmid restores complete nodulation and nitrogen-fixing properties.

Lamb ef ai (1985) have isolated two overlappingcosmids from R. phaseoii strain 8002, capable of restoringnodulation to strains with deletions in part of the symbioticplasmid. These cosmids, however, failed to restore nodu-lation to a pSym-cured strain, indicating that at least twogroups of genes are required for nodule induction anddevelopment.

In this paper we report the Isolation and characterizationof two regions from pSym of R phaseoti. which aresufficient to restore the ability of an R phaseoii pSym-cured strain to induce nodules with infected cells on beanroots.

Results

Isolation of recombinant cosmids carrying R. phaseoii nifand nod genes

A cosmid gene library of R phaseoii strain CE-3 wasconstructed using the pSUP205 cosmid vector, A partialEcoRI totai DNA digestion was size-fractionated in anNaCI gradient; the 30-50kb DNA fractions were pooled,cloned into the EcoRI site of pSUP205 and packaged invitro. The mixture was used to infect K802 Escherichia colistrain. About 1000 Tc'^Cm^ colonies were recovered.

The cosmid library was screened with the pCQ12plasmid containing nifHDK sequences (Quinto ef al.,1982). Eleven positive clones, representing the threedifferent nif regions (a, b and c) were identified by theirEcoRI restriction patterns. A second screening analysis

880 M.A. Cevallos et a\.

c2 3

D

2 3

E

2 3

Fig. 1. Localization of R. phaseoli DNA regions homologous to the common nodABCD genes of R. meliloti by hybridization analysis. Molecular weightsare indicated on the left margin in kb. A. Ethidium bromide-stained EcoRI digestion patterns ot DNA from R. phaseoli pSM82S (lane 1). pSM367 (lane 2),pSM927 (lane 3) and pSM991 {lane 4). Hybridization pattems of the same DNAs against nodAB (B), nodBC (C). nodC (D), and nodD (E). For details onDNA probes see Experimental procedures.

using a probe tbat contains the common nod genes fromft meliloti (pKSKS) (Kondorosi et al., 1984) showed tbatfour of the initial nrfcosmids contained sequences bomo-logous to the nod genes. Two of these cosmids, pSM991and pSM927, contain r)if region a. Cosmid pSM828contains nif region b, and pSM367 contains nif region c.These results confirm a close linkage between the n;/andnod genes in the Sym plasmid of ft phaseoli. as reportedearlier by Lamb et al. (1985).

In order to determine the organization of the sequenceshomologous to the common nod genes of R meliloti, thecosmids containing both nif and nod genes werehybridized against the specific probes for nodAB, nodBC.node or nodD genes (see Experimental procedures). AnEcoRI restriction pattern from total DNA of strain CE-3showed five fragments which hybridized with the specificnod probes. A 6.8kb EcoRI fragment hybndized with theft. meliloti nodABC genes; a 3.3kb EcoRI fragnnentshowed a weak homology to the nodC probe; and threeEcoRI fragments of 5.5,4.9 and 3.4kb hybridized with thenodD probe. The 6.8 kb fragment was present in pSM991,pSM927 and pSM367. The 5.5 kb fragment was present inpSM991 and pSM367. The 4.9kb fragment was foundonly in pSM828 (Fig. 1). The 3.4kb fragment was notcontained In the cosmids described above, but waspresent in the symbiotic plasmJd as indicated by hybridi-zations against total DNA of the wild-type strain and of thepSym-cured derivative (Fig. 2).

Interfunctionality between the common nod genes of R.phaseoli and R. meiiloti

To determine the functionality ol the ft. phaseoli nodABCsequences present in pSM991, this cosmid was intro-duced into three strains of ft. meliloti carrying Tn5insertions in nodA (GMI5382), nodB (GMI5387) and nodC(GMI5383). All transconjugants were able to inducenormal nodules between 15 and 35 days after inoculationon alfalfa seedlings. The ft. meWof/wild-type strain alwaysinduced nitrogen-fixing nodules in less than two weeks.The plants inoculated with the mutant strains did notdevelop nodules (see Table 1).

Identification of nodulation genes by directcomplementation on an R. phaseoli pSym-cured strain

DNA regions involved in the induction and development ofnodules in the common bean were identified by intro-ducing nif-nod cosmids into a pSym-cured ft. phaseolistrain {CFN2001),

Transconjugants containing cointegrates between therecombinant cosmids and the pNC206 plasmid wereinoculated on bean seedlings. Plants were analysed forthe presence of root nodules after 20 days. Only pSM991and pSM927, both containing a complete n/fHDKoperon,were able to Induce nodules (Fig. 3), However, thesenodules exhibited a Fix phenotype.

Table 1 , Symbiotic phenotype of common nodgene mutants ol R. meliloti containing R. pfiaseolirecombinant clones.

Rhizobium phaseoii Sym plasmid regions and nodule morphogenesis 881

strain

RCR2011 RifGMI5382GMI5382 (pNC206:;pSM991)GMI5387GMI5387 (pNC206::pSM991)GMI5383GMI5383 (pNC206::pSM991)

Nodulation onM. sativa

+-

-+•-

N2-fixation onM. sativa

-f-

+-+-+

a. Delayed nodutation.

Definition of the DNA regions involved in nodulation

Cosmids pSM991 and pSM927 only differ by two EcoRIfragments at the end of each insert. Because pSym-curedstrain transconjugants containing either one of these twocosmids are able to induce nodules, the sequencesrequired for nodulation seem to be contained in the FcoRIfragments shared by these cosmids. To characterize themmore precisely, deletions of these fragments weregenerated by religating a partial EcoRI digest of pSM991cosmid DNA at low concentration {20ng ixl"^). The pur-pose was to promote mono-molecular ligations. E. coliHB101 was transformed with 100ng of DNA from theligation mixture. Analysis of the restriction patterns of 50transformants showed the loss of some EcoRI bands. In

Fig, 2. EcoRi hybricJization patiem of total DNA from R. ptiaseoli strainsCE-3 (A) and CFN2001 (B) hybridized against the ft meliloti nodOdetector. At the left margin molecular weights are indicated in kL.

order to identify the phenotype of these deleted cosmids,they were introduced into the pSym-cured strain and thetransconjugants were inoculated onto bean seedlings.Twenty of these derivatives were capable of inducingnodules after 20 days.

A comparison of the Eca Rl restriction patterns betweenpSM927, pSM991 and pSM991 deleted derivatives.which conserve the ability to induce nodules, showed thatthree EcoRI bands are shared by all cosmids: they are a6.8kb fragment, which contains the nodABC genes; a3.5 kb fragment; and a 0.5 kb fragment. AN deletionslacking any of these bands were unable to induce nodules(Fig. 4).

To determine the DNA regions involved in nodulation,pSM991 -25, a deletion derivative which carries the genesnecessary for nodule induction and development, wasmutagenized with Mudll PR13. Thirty five independentinsertions were isolated and their locations determined byrestriction enzyme mapping and Southern blotting, asdescribed in Experimental procedures. The symbioticphenotype of the mutated cosmids was analysed afterbeing introduced into the pSym-cured strain and testedfor nodulation on bean plants. Ten mutants showed aNod" phenotype; seven of these mutants were located inthe 6.8kb EcoRI fragment which contains the nodABCgenes, and three mutations were located in the 3.5 kbEcoRI fragment. The remaining mutations were scatteredall over the cosmid insert (including the 0.5kb EcoRIfragment), and they led to a Nod"^ phenotype (Fig. 5).Insertion mutant 14.1 was introduced into the wild-typestrain by marker exchange, and the resulting strain had aNod" phenotype. This indicates that the 6.8kb EcoRIfragment is essential for nodulation. Thus, only tworegions, separated by approximately 20 kb, were respon-sible for the symbiotic phenotype of pSM991.

Localization of host-range nodulation genes

To localize those sequences involved in the host-rangedetermination, pSM828, pSM367, and pSM991 wereintroduced into R. metiioti, a species that normally nodu-lates alfalfa but not beans. Only the transconjugant

882 M. A. Cevalios et al.

Fig. 3. Twenty-day-old nodules of P. vulgarisinduced by different R. phaseoli strains: CE 3 (A),R. pftaseo//pSym-cured strain (CFN 2001)carrying pSM991 (B), CFN 2001 carrying pSM927(C), and CFN 2001 carrying pSM991-25 (adeletion derivative of pSM991) (D).

containing pSM991 was able to induce a few smallnodule-like structures on bean seedlings. This resultsuggests that host-range genes are located, at least Inpart, in this cosmid (Fig. 6).

Two deletion derivatives of pSM991 were also intro-duced into R. metitoti. One was pSM991-25, whichcontains the two nodulation regions within the 6.8kb and3.5kb EcoRI fragments, and the other was pSM991-32.which only carries the nodulation region contained in the6.8kb EcoRI fragment. The transconjugants wereinoculated on bean seedlings and nodulation was scoredafter 20 days. Plants inoculated with the transconjugantscarrying pSM991-25 developed nodule-like structures,similar to those induced by the complete cosmid {seeTable 2).

Nodute morphotogy

R. phaseoti wild-type strains induce determinate nodules.

After 20 days of development, the nodules are spherical inshape with well-developed vascular bundles. Their exter-nal colour is pink and they fix nitrogen actively. Light-microscopic examination of transverse sections of maturenodules shows a central zone very rich in infected cells.Observation of the infected cells by electron microscopyreveals numerous peribacteroidal membranes enclosinglarge bacteroids with several poly-p-hydroxybutyrategranules. As can be seen in Figs 3,6 and 7, some of thesecharacteristics are present in nodules Induced by strainscarrying nodule-inducing cosmids (Baird and Webster,1982). However, transconjugants of the pSym-curedstrain carrying pSM991 or pSM927 induced green andwhite nodules, but were otherwise similar in size andshape to those induced by the wild-type strain (Fig, 3). Thenodules induced by these transconjugants had a centralzone with infected cells. Within the infected cells, onlya few small and deformed bacteroids, containingless poly-p-hydroxybutyrate granules than wild-type

Table 2, Symbiotic phenotype of R. meliioti

transconjugants containing R. phaseoli

recombinant clones on bean roots.

Rhizobium phaseoli Sym plasmid regions and r)oduie morphogenesis 883

Nodulation on Nj-fixation on

Strain P. vulgaris P. vulgaris

RCR2011 Rif

RCR20n Rif{pNC206::pSM991) +'

RCR2011 Rif(pNC206::pSM828)RCR2011 Rif(pNC206::pSM367)

RCR2011 Rif(pNC206::pSM991-25) +'

RCR2011 Rif(pNC206::pSM991-32)

a. Nodule-like structures.

bacteroids, were found. Bacteria present in intercellularspaces were found more frequently in these nodules (Fig.7, B-D).

Discussion

In this paper we report the isolation and characterization oftwo nodulation (nod) regions of the R p/7aseo//symbioticplasmid. These regions were sufficient to promote noduleInduction, nodule development, bacterial release andhost-range determination in the common bean. These tworegions, separated by approximately 20 kb, were con-tained in recombinant cosmids pSM991 and pSM927.Transconjugants of a pSym-cured R. pfiaseoli strain,containing these cosmids, were able to induce ineffectivenodules on bean roots, similar in size, shape, and time ofappearance to those induced by the wild-type strain.These nodules had a central tissue with infected cells, butbacteroids within them appeared deformed and smallerthan those formed by the wild-type strain.

A possible explanation for why pSM927 and pSM991failed to elicit nitrogen-fixing nodules is that some genesassociated with functions required for an effective noduleare not included in these cosmids. Such is the case for theps/gene (polysaccharide-inhibition). located in an EcoRIfragment next to the one containing nifH of region c(pSM367, data not shown). Strains with a mutation in psigene are unable to fix nitrogen (Borthakur ef at., 1985;Borthakurefa/., 1987).

Comparison of the EcoRI restriction patterns ofpSM927 and pSM991 and the Nod^ pSM991 deletedderivatives, showed that they share only three EcoRIfragments of 6.8kb, 3.5kb and 0.5kb. Deletions lackingany of these bands were incapable of inducing nodules.

PSM991-25, a deletion derivative of pSM991, whichwas able to restore nodulation to the pSym-cured strain,was mutagenized with the Mudll PR13 transposon.Analysis of these mutants showed that insertions whichconferred a Nod phenotype were clustered in the 6.8 kband 3.5 kb EcoRI fragments. This indicates that these two

I iio< D 1 I •»< <>IC

ipSII9e7

PSIII99I-Z5

pSM 991-15

pStl»l-J2

PSM991-36

PSII99I-40

PSII991-44

PSH99I-K

+t

•

+

-----

---------

Fig. 4 . Physical map of n//region a of symbiotic plasmid p42d. Vertical bars represent EcoRI sites. Open boxes show the localization of the EcoRIfragments which hybridized with the common nod genes ot R. meliioti. Regions cloned in cosmids are shown below as solid lines. Sequences deleted mpSM991 derivatives are indicated by broken lines. The table on the right shows the symbiotic phenotype of cosmids in a pSym-cured strain and testedon bean plants. Broken boxes indicate the EcoRI fragments shared by cosmids and deleted derivatives which induced nodules on bear roots in thesame background. Arrows, at the top, indicate the molecular weights, in kb, of two relevant EcoRI fragments (see text). For cosmid clones see Table 3.

884 M. A. Cevallos et ai.

16.2

•.3Q

...Q\TO \

3 4

o o o10 It00

11

14. i14,1

I3.2i13,1 4

It

/L 17.3

le 19O O

24

23.2

\

BE

}2S.3)2S.2

BE

Fig. 5. Restriction endonuclease map andlocation oi Mudll PR13 inseriions within pSM99i-25. Insertion mutants which resulted in a Nodphenotype are indicated by closed circles. Opencircles indicate insertion mutants with a Nod'phenotype. The half-filled circle indicates aNod" phenotype. E, EcoRI; B, SamHI. Arrowsbelow indicate the molecular weights, in kb, oftwo relevant EcoRI fragments.

regions were necessary for production of the symbioticphenotype of pSM991.

DNA regions similar to the common ncdABC geneswere located in the 6.8kb EcoRI fragment of pSM991,which also contains nif region a. corroborating a closelinkage between nif and nod genes. Cosmid pSM991 wasalso able to complement ft. meliloti Nod strains carryingTn5 insertions in the nodA, nodBor nodC genes. However,the complemented strains showed a considerable delay innodulation. This suggests some kind of functionaldivergence between the two gene clusters. Homogeno-tization of insertion 14.1, located in the 6.8kb EcoRIfragment on the CE-3 symbiotic plasmJd, completelyblocks nodulation, indicating that this sequence Is essen-tial for nodulation.

Fig. 6. Roots of P. vuigaris 20 days after inoculation with an R. meliloUstrain carrying pSM991. The photograph shows nodule-like stnjctures(one of them indicated by the triangle).

Transconjugants of wild-type ft. meliloti containingeither pSM991 or pSM991 -25 were able to induce nodule-like structures on bean roots. Furthermore, pSM991 -32, adeletion derivative of pSM991, which contains the nodregion within the 6.8kb EcoRI fragment but lacks the nodregion contained in the 3.5kb EcoRI fragment, failed toinduce nodule-like structures, indicating that host-rangegenes appear to be located, at least in part, on the 3.5 kbEcoRI fragment.

Innes et al. (1987) introduced the host-specific nodu-lation genes (hsn) of ft. trifoliiinto ft. meliloti; nevertheless,they did not observe an extension of the host range of ft.meliloti, indicating a dominance of the ft. meliloti hsngenes. However, in our results described above, thesymbiotic genes of ft. phaseoli did extend the host rangeof ft. meliloti. suggesting that dominance of the host-specificity genes of ft. meliloti might be restricted only tosome species of Rhizobium. Similar results were obtainedwith hsn genes of the broad host-range strain, NGR234(Lewinefa/., 1987).

The ft. phaseoliCE-3 strain contains three pSym EcoRIfragments of 5.5 kb, 4.9 kb, and 3.4 kb which hybridize withthe nodD probe. The first fragment is present in pSM367and in pSM991. The second fragment is contained inpSM828, which contains nif region b, and the thirdfragment is located elsewhere on the symbiotic plasmid,but absent from the cosmids described above. In theftAr/zob/um-legume system, it has been reported that theproduct of nodD, in conjunction with specific plant-factorspresent in the root exudates, regulates the expression ofnodABC {Innes et al.. 1985: Mulligan and Long, 1985;Rossen ef al., 1985). Insertion mutants in ft. legumino-sarum and ft. trifolii nodD completely block nodulation(Debelle efa/.,1986; Djordjevicefa/., 1985). However, in ft.meliloti, in which three copies of nodD have been des-cribed, a triple noc/Dmutant is required to produce a Nodphenotype (Honma and Ausubel, 1987). The absence ofnodD in pSM927 did not impair either nodule developmentor the time of their appearance in a ft. phaseoli pSym-cured strain background. Furthermore, we failed to obtain

Rhizobium phaseoli Sym plasmid regions and nodule morphogenesis 885

Fig. 7. Micrographs from 20-day-old nodules of P. vulgaris induced by R. phaseo//strains: CE 3 (A), CFN 2001 containing pSM991 (B), CFN 2001containing pSM927 and pSM991-25 (C and D, respectively). The upper parts of panels show light micrographs. The bar represents 40^.m. The lowerparts of panels show transmission electron micrographs. The bar represent 7.5 jim. B, bacteroids; I, infected cell; N, nort-infected cells; T, intercellularspace occupied by bacteria.

Nod" insertion mutations in the EcoRI fragment whichcontains nodD in pSM991-25. These results could beexplained in at least two possible ways. First, the nodgenes of pSM927 might be transcribed at a sufficientbasal level to promote nodulation. Second, the transcrip-tion of nodABC is properly regulated not only by nodD, butby other genes that act as negative effectors present in thesymbiotic plasmid but absent from pSM927 and pSM991.These genes may include the two additional nodD genecopies, but the participation of other genes cannot beruled out. In order to determine which genes are impli-cated in the regulation of nodABC, we have constructed aplasmid with a R. phaseoli nodABC-lac fusion and intro-duced it into the pSym-cured strain. We are trying toisolate recombinant cosmids carrying genes that promotetop levels of (i-galactosidase activity or decrease thebasal levels in the presence of root exudates. On the otherhand, the construction of a triple nodD mutant that istested for nodulation on bean plants is required toestablish the role of these genes in symbiosis.

The results described above suggest that only tworegions are sufficient to substitute the pSym of R. phaseolifor nodule development. One region included at least thecommon nodABC genes and the other one containsgene(s} required in the host-range determination. Thesedata are, in part, similar to those obtained for R meiilotiand R. leguminosarum. in which clones containing thecommon nodABCD genes and the host-range genes areable to induce nodules when they are introduced in theirown pSym-cured strain or pSym-deleted background andinoculated onto the proper host (Djordjevic et al., 1986;

Putnoky and Kondorosi, 1986; Schofield et at., 1984).Nevertheless, the lack of a requirement for nodD in noduleinduction is certainly unusual and needs to be elucidated,

Cosmid-complementation experiments on a pSym-cured strain, presented in this work, do not exclude theparticipation of other genes, located in the chromosomeor in other plasmids, in the development of a nitrogen-fixing nodule. In fact, some mutations of the GE-3 chromo-some alter lipopolysaccharide synthesis and normalnodule development (Figurski and Helinski, 1979; Noel etal., 1984; Truchet et at., 1984). Moreover, in the strainCE-3, a mutation which affects nitrogen fixation has beenmapped in a plasmid other than pSym (Morett etat., 1985).

Experimental procedures

Bacterial strains and plasmids

The strains and plasmids used in this work are listed in Table 3.

Media and bacterial growth conditions

R. phaseoti and R. metitoti were grown in PY medium as describedby Noel e/a/. {1984). E. co//was grown as described in Maniatis efat. (1982) or in LBCM (LB medium supplemented with CaCI;. andMgSOrt). Antibiotics were used al the following concentrations.given in (|j.g ml '): Rifampicin (Rf), 50; Tetracycline (Tc), 5;Kanamycin (Km). 30; Streptomycin (Sm), 100; Chloramphenicol(Cm), 30; Speciinomycin (Sp), 100.

886 M. A. Cevalios et al.

Table 3. List of bacterial strains, phage and plasmids used.

Plasmid orbacterial strain

Relevantcharacters

Source orreference

Bacterial strains

R. phaseoliCE-3CFN 2001

R. meiilotiRCR2011 RifGMI5382-GMI5387*GM15383"

£. collHB101K802MC4tOO(Mu C(S)M8820(Mij)

Plasmids

pNC206PSUP205pRK2013PSM367PSM828PSM991pSM927PSM991-25PSM991-15PSM991-32PSM991-36PSM991-82pSM991-40pSM991 -44pCQ12pKSK5

pPHIJI

Phage

Mudll

Str" derivative of wild type strain CFN42, Nod * Fix'Rif" derivative of CFN42 cured of p42a and p42d plasmids

Rif^ derivative of RCR 2011 Nod *,notfA;:Tn5nodB::Tn5nodC::Tn5

F , hsdS20. r0cA13. araU. proA2. lacYI. galK2. rpsL20. xylS, mtli, supE44gal. met. SupEaraD139. i(a/-gF-;ac)U169, rpsU50, relAI, Ube5301. pstF25, dsoCI, (Mucts)araD139, A{araCOIBA-leu)7Q79. SiproAB, argF-laclPOZYA)X\\\ rpSL. (Mu)

IncPl Cb" Km"

K;n''ColE1 TrapSUP205 containing n/7 region CpSUP205 containing nif region b ,pSUP205 containing nrf region a 'pSUP205 containing nif region aDeletion of pSM991Deletion of pSM991Deletion of pSM991Deletion of pSM991pSUP205 containing an EcoH\ fragment of 6.8Kb with nodABCDeletion of pSM991 containing fcoRI fragment of 5.2 kbDeletion ot pSM991 containing fcoRI fragment of 3.5kbpBR328 carrying an EcoRI fragment with nif region bpRK290 can-ying a 6.8 kb Eco RI fragment with the common nod genes of

pRm 41 bincPl.Gm", Km"

lac. Cm"

Noele(aMi984)Palacios el a/. (1983)

Rosenberg et al. (1986)Debelleera/. (1986)Debelleefar. (1986)Debelle era/. (1986)

Boyer era/. (1969)A. PijhlerCasadaban(1976)Casadaban(1975)

A. PiJhlerSimon ef a/. (1983)Figurski and Helinski (1979)This workThis workThis workThis workThis workTTiis workThis workThis workThis workThis workThis workQuinto e( a/. (1985)Kondorosi (1984)

Beringer e( a/. (1978)

Rate! sf a/. (1988)

• RCR 2011 denvatives.

Bacterial crosses

Recipients and donors were grown in the proper liquid medium to

stationary phase, mixed on PY plates and incubated at 3O''C

overnight. Cells were resuspended in sterile water and serial

dilutions were plated on the appropriate selective media.

Nucleic acid procedures

DNA manipulation, transformation. Southern blot and nick-trans-lation procedures were carried out as described by Maniatis etat.(1982).

Nodulation test and acetylene-reduction activities

Surface-sterilized seeds of P. vutgaris L. cv. Negro Jamapa, were

grown in 250ml Erienmeyer flasks with 200ml of a nitrogen-freenutrient medium supplemented with 0.8% agar (Wacek and Brill,1976). Three or four days after germination in the dark, plantlelswere inoculated with 1 ml of a saturated liquid culture of theappropriate Rhizobium strain. After 20 days of incubation in agrowth chamber at 26°C, nodulation was scored and nitrogenaseactivity was determined by the acetylene-reduction assay(Masterson and Murphy, 1980). A similar procedure was usedwith Medicago sativa cv. Peruana, except that seedlings weregrown on slants in 25 x 200 mm tubes.

Re-isolation of bacteria from nodules

Nodules were detached, surface-sterilized with a 20% v/vcommercial bleach solution for 10 min, rinsed in sterile water.crushed, streaked onto PY plates and incubated for 3 days at

Rhizobium phaseoii Sym plasmid regions and nodule morphogenesis 887

30"C. Ten colonies from each nodule were screened in theappropriate selective media for antibiotic-resistance markers.

Microscopic examination of nodules

Twenty-day-old nodules were fixed as described by Truchet et at.(1984). Samples were detiydrated in increasing dilutions ofethanol and embedded in Epon 812. Semi-thin sections of 1.5 p.mwere stained with toluidine blue and observed using a lightmicroscope. Thin sections (0.9 (im) were stained with 2% uranylacetate and 5% lead citrate and observed under a transmissionelectron microscope.

Isolation of specific gene detectors for common nodgenes

The following restriction fragments of pKSK5 plasmid (Kondorosietal.. 1984) were electroeluted from 1 % agarose gels and labelledby nick translation. A 2.2 kb BgtW fragment was used as a nodDprobe, a 1.95kb Ssfll fragment as nodBC probe, a 0.65kb H/ndlllfragment as nodC probe, and a 0.65kb Ssfll-Pvull fragment asnod^Sprobe (Schmidt ef a/., 1984; Tbrbk ef a/., 1984).

Mobilization of cosmids to Rhizobium

Since pSUP205 cosmid is a mobilizable vector but unable toreplicate in Rhizobium, it was necessary to cointegrate it withanother plasmid capable of replication in Rhizobium. To do this,£ coti strain HB101 carrying a broad host-range IncPI-Km"plasmid (pNC206) was transformed with the recombinantcosmid-clones fTc*̂ ). selecting for Km'̂ Tc'̂ colonies. Trans-formants containing both plasmids were conjugated withCFN2001 strain and the Rf"Km"Tc" colonies were isolated.

Cosmid mutagenesis

Cosmid pSM991-25 was mutagenized with the transposablebacleriophage, Mudll PR13 (Ratet ef al.. 1988) as follows. Thecosmid was introduced in E. co//MC4100 (Mucfs) (Mudll PR13)by triparental mating using pRK2013 as helper plasmid andselecting for Cm"Tc"Km^ colonies. The resulting strain wasthermo-induced to obtain a lysate which was used to infect E. coliM8820(Mu). Colonies were selected by their Cm"Tc" phenotype.The localizations of the Mudll PR13 insertions were determinedby EcoRI and SamH! restriction mapping. The mutated cosmidswere introduced as cointegrates with pNC206 plasmid into R.phaseoti CFN2001 as described above. To check the stability ofthe insertions, Southern blots of the Rhizobium transconjugantswere performed using pSM991-25 plasmld as probe.

Preparation and use of Mu tysates

An overnight culture of E. coti MC4100(Mucfs)(MudllPR13)(pSM991-25), grown at 3O''C in LB medium supplementedwith Cm-Tc, was diluted 1;100 in lOmi of LBCM medium. Thisculture was grown at 30°C to mid-exponential phase. The culturewas then shifted to 37°C for 20 min with shaking and incubatedlater at 42°C In the same agitation conditions until lysis was

observed. Chloroform. MgSOa and CaCl2 were added to thelysate to a final concentration of 1%, 2mM, and 20mM.respectively; cell debris was removed by centrifugation. Simul-taneously, the receptor strain M8820 (Mu) was grown to mid-exponential phase and concentrated 10-fold in LBCM medium.Infections were carried out by mixing 0.1 ml of the M8820(Mu)concentrated culture with 0.1ml of the lysate. Adsorption wasperformed at 30°C for 30 min without shaking. After the addition of2 ml of LB, the mixture was incubated for 45 min with agitation at30°C. Cells were washed with 10mM NaCI before being plated inLB plus 5 mM CaCI? and chloramphenicol as selecting antibiotic.

Site-directed mutagenesis

Site-directed mutagenesis was performed essentially as des-cribed by Ruvkun and Ausubel (1981). but using the incPlplasmid pPHlJI as the incoming plasmid.

Acknowledgements

The work was supported in part by the Consejo Nacional deTecnologia (CONACyT) grant, by the US National Academy ofSciences/National Research Council by means of grant BNF-MX-68777 from the Agency for international Development, by EECgrant CI1-0105-MEX, and by Fondo de Estudios 'Ricardo J.Zevada'. We thank P. Leon, J. E. Padilla. L. Segovia and F.Sanchez for critical reading of the manuscript, J. Denarie, E.Kondorosi and A. Puhler for providing plasmlds and strains, J. L.ZItlalpopoca and A. PIchardo for technical assistance; and M. D.Cuellar for secretarial support. M.A.C. was supported by aCONACyT fellowship.

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