Arch Microbiol (1985) 143:225- 232

Microbiology
9 Springer-Verlag 1985

Archives of

Genetic and functional analysis of the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI
Carel A. Wijffelman*, Elly Pees, Anton A. N. van Brussel, Rob d. H. Okker, and Ben J. J. Lugtenberg University of Leiden, Department of Plant Molecular Biology, Nonnensteeg 3, NL-2311 VJ Leiden, The Netherlands

Abstract. Thirty Tn5- or Tn1831-induced nodulation (nod) mutants of Rhizobium leguminosarum were examined for their genetic and symbiotic properties. Thirteen mutants contained a deletion in Sym plasmid pRL1JI. These deletions cover the whole nod region and are 50 kb in size. All remaining seventeen mutations are located in a 6.6 kb EcoRI nod fragment of the Sym plasmid. Mutations in a 3.5 kb part on the right hand side of this 6.6 kb fragment completely prevent nodulation on Vicia sativa. All mutants in this 3.5 kb area are unable to induce naarked root hair curling and thick and short roots. Mutations in a 1.5 kb area on the left hand side of the 6.6 kb nod fragment generate other symbiotic defects in that nodules are only rarely formed and only so after a delay of several days. Moreover, infection thread formation is delayed and root hair curling is more excessive than that caused by the parental strain. Their ability to induce thick and short roots is unaltered. Mutations in this 1.5 kb region are not complemented by pRmSL26, which carries nod genes of R. meliloti, whereas mutations in the 3.5 kb region are all complemented by pRmSL26. Key words: Rhizobium leguminosarum - Nodulation mutants - Hair curling - Infection thread - Sym plasmid - Complementation

Bacteria belonging to the genus Rhizobium are capable of inducing nitrogen-fixing root nodules on leguminous plants. This symbiosis between bacterium and plant is a complex process in which genes of both bacterial and plant origin play an essential role (Vincent 1980). During the past few years it has become evident that a number of symbiotic functions is located on large plasmids, so-called Sym plasmids (Johnston et al. 1978; Banfalvi et al. 1981; Hombrecher et al. 1981; Hooykaas et al. 1981; Rosenberg et al. 1981; Lamb et al. 1982; Djordjevic et al. 1982). Among these symbiotic functions are root hair curling Offprint requests to: C. A. Wijffelman Abbreviations. Rps, repression of production of small bacteriocin; Mep, medium bacteriocin production; Nod, nodulation; Fix, fixation; Tsr, thick and short roots; Hac, root hair curling; Hsp, host specificity; Had, root hair deformation; Tc, tetracycline; Kin, kanamycin; Cm, chloramphenicol; Sp, spectinomycin; Sin, streptomycin; R, resistant

(Hac), host specificity (Hsp) and nitrogen fixation (Fix). It has been shown in our laboratory that the Sym plasmid is also required for the ability of R. leguminosarum to induce thick and short roots (Tsr) on Vicia sativa (Van Brussel et al. 1982). Recent results have indicated that nod genes are required for the induction of the Tsr phenotype (Van Brussel et al. 1984). In R. Ieguminosarum strain 248 the symbiotic functions are located on the self-transmissible plasmid p R L I J I , which is 200 kb in size (Hirsch et al. 1980). When this plasmid is transferred to other Rhizobium species or to Agrobacterium tumefaciens, the recipient strain gains the capacity to induce nodules on plants of the pea cross-inoculation group (Johnston et al. 1978; Van Brussel et al. 1982). Only a small region of this plasmid is essential for nodulation as it was found that either of two cosmid clones, containing only 10 kb of cloned pRL1JI D N A in common, can restore the nodulation ability of strains of Rhizobium which had been cured from their own Sym plasmid (Downie et al. 1983 a, b). This 10 kb nod region consists of two EcoRI fragments of 6.6 kb and 3.2 kb. Some of the Sym plasmid-encoded nod genes of R. meliloti can be substituted by the nod genes of pRL1JI. These genes are therefore designated as 'common' nod genes (Kondorosi et al. 1984). Djordjevic et al. (1985) demonstrated that some nod genes are common in R. leguminosarum, R. trifolii, R. melitoti and a Rhizobium strain able to nodulate cowpea and the non-legume Parasponia. As other Sym plasmid-encoded nod genes of R. meliloti cannot be replaced by genes of p R L I J I , the latter R. meliloti genes are assumed to play a role in host-specific steps of the nodulation process (Kondorosi et al. 1984). Recently D N A of some common nod genes of R. leguminosarum and R. meliloti has been sequenced (Rossen et al. 1984; T6r6k et al. 1984; Jacobs et al. 1985). Three nod genes were identified as open reading frames e.g. nodA, B and C. The D N A has a highly conserved nucleotide sequence and the existence of three genes has been confirmed in an E. coli transcription/translation system, in which three polypeptides with molecular weights of respectively 23,000, 28,500 and 44,000 d are formed (Schmidt et al. 1984). In this paper we describe the genetic and physiological analysis of thirty transposoninduced nod mutants of pRL1JI. This analysis allowed us to divide the nod region of p R L 1JI in two areas, one containing common nod genes involved in root hair curling and thick and short root formation, and another one involved in efficiency and/or rate of nodulation.

226
Materials and methods
I nodO nOdAlnOdlB node i
nif KDH
//

Bacterial strains and plasmids

The bacterial strains and plasmids used are listed in Table 1. For the construction of strain RBL5559, plasmid R772 was transferred from Agrobaeterium tumefaciens strain LBA937 into Escherichia coli strain 1064 which harbours pRmSL26, a plasmid containing a number of nod genes of R. meliloti (Long et al. 1982). The resulting exconjugant harbours both pRmSL26 (TcR) and R772 (Km~). From this strain pRmSL26 was mobilized into RBL5506, selecting for TcR Cm ~ colonies. The latter exconjugants were tested for the absence of R772. About 50% of these exconjugants were Km s, lacked a plasmid of the size of R772 and contained only one additional plasmid of the size of pRmSL26.

i~

!! !&,<,!

~ ,~ ~ HSaSa SSaHBo ~ El ,i >i !r, ' 1 ' 2 a 6 '

,/

nifA
kb.

,t ,!\

31

//

Fig. 1. Restriction enzyme map of the 6.6 kb EeoRI nod fragment of pRL1JI. Restriction sites are indicated as follows. E, EcoRI; Sa, SalI; S, SmaI; B, BamHI; H, HindIII. The higher horizontal line indicates the position of the nodA, B, C and D genes. The lower horizontal lines indicate the regions of DNA which are deleted in strains RBL607 (nod7) and RBL631 (nod31). The lower verical lines indicate the position of the transposon. The mutation number is given at the bottom of this line

Media and microbiological techniques

Media and mating conditions were as described previously (Wijffelman et al. 1983). The following antibiotics were used at the indicated concentrations (rag/l): kanamycin (200), rifampycin (20), spectinomycin (100), chloramphenicol (10), streptomycin (10) and tetracycline (5). Transductions were performed as described by Buchanan-Wollaston (1979) using phage RL38JI. The presence of the pRL1JI markers Mep (medium bacteriocin production) and Rps (repression of production of small bacteriocin) was investigated as described by Priem et al. (1984), using strains 248 and RBLI01 respectively as indicator bacteria. Methods used for the isolation of plasmid DNA and for the calculation of the molecular weights of plasmids were as described before (Wijffelman et al. 1983).

labeled DNA probes, prepared by nick-translation, were as described by Maniatis et al. (1982). Localization of the transposon insertions was accomplished by analyzing restriction enzyme digests of mutant DNA. The enzymes used were EcoRI, BamHI, HindIII, SalI and SmaI. The orientation of the 6.6 kb EcoRI nod fragment to the nifHDK genes was determined by hybridization of HpaI digests of the mutants to the nifHDKY probe, pSA30.
Results

Genetic analysis of transposon-induced nodulation mutants

Insertion mutants of Tn5 in the R. leguminosarum Sym plasmid pR1JI were isolated as described by Van Brussel et al. (1984), using the suicide plasmid pJB4JI (Beringer et al. 1978). 27 mutants were Nod- or nodulated Vicia sativa very poorly. These mutants were analyzed further. In addition three nod mutants of pRL1JI, which were induced by Tn1831 (Sp R, Sm R, HgR), were analyzed. The procedure used for the isolation of the latter mutants will be described elsewhere. Based on the size of the plasmid the mutants could be divided into two separate groups. The first group, containing 17 strains, harboured a pRL1JI plasmid of an apparently normal size (200 kb) whereas a second group, containing 12 mutants induced by Tn5 and one induced by Tn1831, did not contain a plasmid of the size of pRLIJI but harboured an additional, smaller, plasmid of approximately 150 kb. The latter plasmids are presumably deleted derivatives of pRLIJI, since (i) they were, like pRL1JI itself, self-transmissible with a frequency of 10- 2 to 10-- 3 (ii) they contained the pRL1JI markers Mep and Rps, (iii) the Tn5-marked plasmids were incompatible with pRLI ::Tn1831. The group of nodulation mutants which contain a pRL1JI plasmid of an apparently normal size was analyzed further (Table 1). Fifteen of the mutants carried Tn5 and two of them Tn1831. The site of the transposon insertion in the nodulation mutants was determined by Southern blotting, as described in Material and methods. All Tn5induced mutants, with the exception of RBL607 and RBL621 carry a transposon insertion in the 6.6 kb EeoRI nod fragment (Fig. 1). To determine whether the Tn5 insertions in strains RBL607 and RBL621 were coupled to the Nod- phenotype, phage RL38 lysates of these strains were used to transduce cells of strain RBL5502, harbouring a wild type pRL1JI, to Km R. From each transduction 20 transductants were tested

Nodulation tests and root hair examinations

Nodulation tests on agar slants containing the plants Vicia sativa L. var nigra and Melilotus albus L., reisolation of bacteria from root nodules and nitrogen fixation were carried out as described by Van Brussel et al. (1982). In order to inspect root systems by bright field microscopy, plants were removed from the tubes, thoroughly rinsed with sterile water and mounted between a slide and a coverslip. Marked curling was defined as a curling of root hairs that is frequently more than 360 ~ Infection threads were judged using magnification of 100 and 200 times. The number of infection threads of plants at different times after inoculation was determined by inspecting the number of infection threads along the main root of five plants.

DNA probes and hybridization

Plasmid pNP520 (Pannekoek et al. 1978) was used as a Tn5 probe and pSA30 (Cannon et al. 1979) as a nifHDKYprobe. T h e nod probe used was pMP40, a plasmid which contains the 6.6 kb EcoRI nod fragment of pRL1JI with the nod3 : :Tn5 insertion of pRL603 cloned in the unique EcoRI site of pBR322. DNA of small plasmids was prepared by the method of Birnboim and Doly (1979) and purified using a buyant density centrifugation in a cesium chloride gradient. Extraction of Rhizobium DNA and digestions with restriction endonucleases were carried out as described by Maniatis et al. (1982). DNA fragments were transferred from agarose gels to nitrocellulose filters using the methods of Southern and the conditions for hybridization with 32p_

227
Table 1. Bacterial strains a

Strain 248 LBA937 1064 1830 LPR5039 RBL5502 RBL5505 RBL5506 RBL5515 RBL5516 RBL5523 RBL5559 RBL601 RBL602 RBL603 RBL604 RBL605 RBL606 RBL607 RBL609 RBL610 RBL611 RBL613 RBL615 RBL618 RBL621 RBL622 RBL631 RBL632

Plasmid pRLIJI R772 pRmSL26 pJB4JI

-

Relevant characteristics
R. leguminosarum w.t A. tumefaciens R772 E. coli HB101 pRmSL26 E. eoIipro,met pPH1JI : :Mu: :Tn5 R. trifolii RCR5 str cured of its

Reference Joscy et al. 1979 Hille et al. 1982 Long et al. 1982 Beringer et al. 1978 Hooykaas et al. 1981 Priem et al. Priem et al. Priem et al. Priem et al. Priem et al. Priem et al. This work 1984 1984 1984 1984 1984 1984

pRL1JI pRL650 pRmSL26 pRL601 pRL602 pRL603 pRL604 pRL605 pRL606 pRL607 pRL609 pRL610 pRL611 pRL613 pRL615 pRL618 pRL621 pRL622 pRL631 pRL632

sym plasmid pRtr5a LPR5039 pRL1JI LPR5039 rif,spc LPR5039 cam LPR5039 r/f LPR5039 spc RBL5515 pRL1 ::Tn1831 RBL5515 pRmSL26 RBL5505 pRLl,nodl ::Tn5 RBL5505 pRLl,nodD2 ::Tn5 RBL5505 pRLl,nod3 ::Tn5 RBL5505 pRLl,nod4::Tn5 RBL5039 pRLl,nod5::Tn5 RBL5505 pRLl,nod6 ::Tn5 RBL5505 pRL1 ::Tn5,nodC7 RBL5505 pRLl,nodC9: :Tn5 RBL5505 pRLl,nodA 10 ::Tn5 RBL5505 pRLl,nodB11 ::Tn5 RBL5516 pRLI ,nodC13::Tn5 RBL5516 pRL1 ,nodC15 : :Tn5 RBL5505 pRLl,nodl 8 : :Tn5 RBL5505 pRL1 ::Tn5,nod21 RBL5505 pRLl,nod22: :Tn5 RBL5515 pRLl,nod31 ::Tn1831 RBL5515 pRLl,nodA32: :Tn1831

Independent Nod- mutants of pRL1JI

" str streptomycin, r/frifampycin, spc spectinomycin, cam chloramphenicol, nodnodulation

for nodulation on V. sativa. Eighteen transductants from RBL607 and three from RBL621 were Nod +, indicating that neither in strain RBL607 nor in strain RBL621 the nodulation defect was caused by the insertion of Tn5. The 6.6 kb EcoRI nod fragment was not intact (in RBL607 and RBL621). A 300 bp deletion in the 600 bp BamHI fragment was detected in strain RBL607, whereas in strain RBL621 an insertion element of 2.5 kb, normally present in the chromosome of strain LPR5039 (unpublished results), was found to be inserted in the left EcoRI-HindIII fragment. Finally we analyzed strains RBL631 and RBL632, the nod mutants induced by Tn1831. In strain RBL632, Tn1831 was found to be inserted in the same HindIII-SalI fragment as Tn5 in strain RBL610. The precize location of ThiS31 within this small fragment has not been determined. In RBL631, Tn1831 is inserted in the internal 1.7 kb HindIII fragment. In addition, the strain carries a deletion of 12 kb, starting from the transposon and extending to the right-handside, removing a 3.2 kb EcoRI nod fragment and part of the nifA area (Fig. 1).
Root hair curling

The symbiotic behaviour of the nodulation mutants was studied and compared with that of the wild type strain RBL5502 and the Sym plasmid-less strain RBL5515. The morphology of the root hairs of V. sativa 8 days after inoculation with cells of strains RBL5502, RBL5515 or of one of the mutant strains is shown in Fig. 2. Based on the root

hair morphology induced by the mutant strains, they could be classified in three different groups (see Table 2). Class I strains are Hac- and no difference in root hair curling was observed between these mutants and the Sym plasmid-less strain RBL5515 (Fig. 2A). Also the mutants with the 50 kb deletion in the sym plasmid showed this root hair morphology. Class II has only one representative, strain RBL611. This strain causes severe root hair deformation compared with class I strains (Fig. 2 C), but marked curling like caused by the parent strain (Fig. 2 B) was not observed. The phenotype of this mutant is indicated as Had +. Class III mutants cause the first visible root hair curling 24 h after inoculation, a behaviour comparable to that of the wild type strain RBL5502 (Fig. 2B). However at day 4 and later the root hair curling caused by class III mutant strains was much stronger than that caused by the wild type strain in that more root hairs were curled and also in that the individual root hairs were curled more strongly (Fig. 2D). The difference with the root hair curling caused by the wild type strain is the most striking at the lower part of the main root and at the lateral roots. The wild type strain induces hardly any root hair curling at these sites, whereas class lII mutant strains induce very strong curling of almost all root hairs. This phenotype is indicated as Hac + + (Table 2).
Thick and short roots (Tsr)

The root system of V. sativa inoculated with wild type R. legurninosarum strains shows the Tsr + phenotype, i.e. the

228 Table 2. Properties of nodulation mutants Class Strain Root hair curling Tsr phenotype Infection thread formation 'Delayed modulation' Complemenration by pRmSL26

II III

RBL602, RBL610, RBL631, RBL611 RBL601, RBL605, RBL621,

RBL607, RBL609 RBL613, RBL615 RBL632 RBL603, RBL604 RBL606,RBL618 RBL622

HacHad + Hac + +

+ + -

Fig. 2. Root hairs of Vicia sativa infected with the sym plasmid cured strain RBL5515 A, the wild type strain RBL5502 B, strain RBL611, as a class II mutant C and strain RBL601, a representative of class III mutants D. All class I mutants yield a picture indistinguishablefrom A. The bar represents 50 gm

roots are shorter and thicker than those of uninfected plants. Class I and II m u t a n t s are T s r - , like RBL5515. All class III mutants induce the same Tsr + phenotype as the wild type strain.

Infection threads
In plants infected with class I or class II m u t a n t s infection threads were not observed. In contrast, all V. sativa plants inoculated with class III m u t a n t s contained infection threads. However, these showed several differences with the

infection threads induced by wild type strains. The most striking difference is the time at which the first infection threads were observed, which was 60 h after inoculation with the wild type strain RBL5502 and as long as 7 days when a class III m u t a n t was used. The n u m b e r of infection threads along the main root at different times after infection with the wild type strain RBL5502 and with a representative of class III, strain RBL60I, is given in Fig. 3. Although the time of appearance of the first infection threads was much later with strain RBL601, the total n u m b e r of infection threads at later times was comparable

229

80T
e~

9 60-

8 40C

"~ 2o$
.a

0-

/_./

/.
e7

x~X/
days after inooulation

for the two strains. A third difference between infection threads induced by strain RBL5502 and by the class III mutants is their morphology. Most of the infection threads induced by RBL5502 were straight (Fig. 4A), whereas those induced by class III mutants were often twisted (Fig. 4 B). In addition, more than 80 % of the infection threads induced by class III mutants were abortive or apically directed (Fig. 4 C , D ) as compared to less than 10% for those induced by strain RBL5502.
Nodulation

Fig. 3. Kinetics of infection thread formation on Vicia sativa by the wild type Sym plasmid containing strain RBL5502 ( x ) and by the class III mutant strain RBL601 (O). Each point in the figure represents a mean number of the infection threads of 5 plants

Mutant strains belonging to Class I and class II were not able to induce nodules at all. All class III mutants were able to induce some nodules and their nodulation behavior on V. sativa, designated as 'delayed nodulation', can be summarized as follows. (i) The first nodules are visible as late as day 15 as compared to day 7 for the wild type strain. (ii) Nodules were formed on only 1 0 - 30% of the inoculated plants. This percentage is higher than 98% for the wild type strain. (iii) The number of nodules per nodulating plant was

Fig. 4. Infection threads induced on Vicia sativa by the wild type strain RBL5502 A and by the class III mutant strain RBL601 B, C, D. The bar represents 50 pm

230 low, in most cases one, whereas with the wild type strain the average number was three to four. (iv) The site of the nodules is on the lower part of the main root or on one of the lateral roots, whereas with the wild type strain the nodules are present at the upper part of the main root. (v) The nodules induced by the mutant strains contain bacteroids and fix nitrogen at the wild type level. After surface sterilization of nodules induced by strains RBL601, RBL606 and RBL621, bacteria were reisolated out of these nodules. They all appeared to be Km R and their nodulation behaviour was indistinguishable from that of the original mutant strains, namely 'delayed nodulation'. Therefore nodulation induced by the mutant strains is not caused by revertants, but it is an intrinsic property of the mutant strains. Common nodulation genes All mutants were tested to check whether the inability to nodulate could be complemented by the R. meliloti nodulation genes present on pRmSL26 (Long et al. 1982; Hirsch et al. 1984). For this purpose pRmSL26 was introduced in the Sym plasmid-less R. trifolii strains RBL5506. This strain, RBL5559, which neither induces nodules on Medicago sativa nor on Vicia sativa, was used for further complementation studies. The pRLIJI Sym plasmids of the mutant strains were transferred to this strain, using selection for the transfer of Km R, and the resulting exconjugants were tested for their ability to nodulate V. sativa. For all class I and class II mutations the nodulation on V. sativa was restored by the presence of pRmSL26 (Table 2). The first visible nodules appeared 6 days after inoculation and the nodules appeared to be effective with the exception of those induced by nod 31 (RBL63 I), which were ineffective. In contrast, the defects of class III mutations were not complemented by the presence of pRmSL26. The nodulation behaviour of the latter exconjugants was indistinguishable from that of the mutant strains lacking pRmSL26, i.e. 'delayed nodulation'. In conclusion, plasmid pRmSL26 contains nodulation genes that are able to replace the impaired genes in class I and class II R. leguminosarum mutants functionally. From results with cosmid clones it was concluded that a 9.8 kb region of pRLl JI, consisting of two EcoRI fragments of 6.6 and 3.2 kb is required for nodulation on pea (Downie et al. 1983 a, b). As the insertion sites of all transposons are in the 6.6 kb nod fragment (Fig. 1), our results confirm the above-mentioned conclusion of Downie et al. (1983 a, b). From the comparison of the data on the physical localization of the mutations (Fig. l) with recent sequence data of the right hand side of the 6.6 kb nod fragment (Rossen et al. 1984), it can be concluded that strains RBL610 and RBL632 carry the transposon in gene nodA, strain RBL611 in nodB and strains RBL609, RBL613 and RBL615 in gene nodC. Based on the phenotypes induced on Vieia sativa (Figs. 2 and 4, Table 2) the mutants can be divided into three classes, which correspond precisely with the positions of the insertions in the DNA (Fig. I). Class I strains, representing all mutants in genes nodA and nodC, are Hac- and Tsrand their symbiotic behaviour is indistinguishable from that of the Sym plasmid-less derivative of the parental strains. The only class II mutant is strain RBL611, which is also the only nodB mutant. This mutant behaves exactly like class I mutants except that it causes significant root hair deformation (Fig. 2C). Rossen et al. (1984) have reported that genes nodA, B and C are sufficient to cause root hair curling. Nevertheless we found a Hac- mutant, strain RBL602, which carries a Tn5 insertion at the left hand side of the nodA, B and C region. This region is mentioned nodD by Downie et al. (1985). It thus appears that a cluster of at least four genes, localized at the right hand part of the 6.6 kb nod fragment, is directly or indirectly involved in root hair curling. As all these genes also seem to be involved in the formation of thick and short roots (Table 2) it seems possible that the factor(s) causing Hac and Tsr are similar or even identical. The genes nodA through nodD are likely to be expressed in all mutants in which the transposon insertion is located at the left hand side of the nod2 mutation in the nodD gene (Fig. 1) as they all induce root hair curling and thick and short roots (Table 2). The root hair curling induced by these class III mutants differs considerably from that induced by the wild type strain (compare Fig. 2B and D). (i) After inoculation, the degree of root hair curling induced by the wild type strain decreases with the time whereas the curling caused by the class III mutants seems to increase with time to a degree, designated as Hac + +, never observed when the wild type strain was used. (ii) The infection threads induced by class III mutants differ from those induced by the parental strain with respect to both time of appearance (Fig. 3) as well as their morphology (Fig. 4). (iii) The appearance of nodules induced by class III mutants is delayed, the number of nodules is decreased and they appear at unusual sites on the roots. Recently Downie et al. (1985) described also the n odulation behaviour of nodulation mutants of pRLIJI on pea. Because we have used V. sativa as the test plant, the symbiotic phenotype of the nod mutants is not necessarily the same for both plants as appears from a comparison of the symbiotic properties of the nod mutants with mutations located on similar places on the physical map of the nod region (Fig. 1). Nod mutants on the left hand side of the 6.6 kb EcoRI nod fragment (left to nodD) exhibit delayed nodulation in both studies, but supercurling of root hairs is only reported for V. sativa. The nodD mutant does not in-

Discussion

Based on the size of the plasmid the 30 nodulation mutants induced by an insertion of either Tn5 or Tn1831 in R. leguminosarum Sym plasmid pRL1JI can be divided in two groups. Whereas the plasmids of seventeen mutants have a size comparable to that of the parental plasmid, those of the remaining thirteen mutants carry a deletion of approximately 50 kb. The so-called 'symbiotic area' of pRL1JI, which comprises a nodulation region flanked by two areas involved in nitrogen fixation (Ma et al. 1982; Downie et al. 1983a), is approximately 45 kb in size. The nodulation negative phenotype of the 13 deletion mutants together with the negative outcome of hybridization experiments in which besides the nod probe also nifHDK and nifA probes were used as markers for the two fixation areas (unpublished results), show that in all mutants the deletions extent over the whole symbiotic area. Both this result and the high frequency with which these deletion events occur suggest that specific deletions, possibly generated by the transposition processes of Tn5 and Tn1831, are involved.

231 duce r o o t hair d e f o r m a t i o n on V. sativa, whereas Downie et al. (1985) reported delayed r o o t hair deformation for one o f their nodD m u t a n t s on peas. N o nodA mutants have been reported by Downie et al. (1985). nodB mutants were H a d + according to b o t h studies but reversion to the N o d + phenotype occurs only on peas. M u t a n t s m u t a t e d in nodC exhibit in b o t h studies a phenotype c o m p a r a b l e to that o f Sym plasmid-cured strains. We did not find mutants in the 3.2 kb EcoRI nod fragment, presumably since these mutants, which give a delayed n o d u l a t i o n with only a slightly reduced number o f nodules on pea plants (Downie et al. 1985) were scored N o d + in our screening procedure with V. sativa. K o n d o r o s i et al. (1984) p r o p o s e to designate nodulation genes, which are able to functionally replace nod genes from bacteria from other cross-inoculation groups, as c o m m o n nodulation genes. Using p R m S L 2 6 we have determined which genes o f R. leguminosarum can be replaced by the presence o f this plasmid, p R m S L 2 6 seems to carry at least the nodA, B and C genes o f R. meliloti, as the presence o f this plasmid in A. tumefaciens causes .root hair curling on Medicago (Hirsch et al. 1984). As the defects o f all class I and class II m u t a n t s are complemented by the presence o f p R m S L 2 6 (Table 2) the genes nodA, nodB, nodC and nodD must be c o m m o n nodulation genes. This conclusion is consistent with the high degree o f conservation observed between the nodA, B and C genes o f R . leguminosarum (Rossen et al. 1984) and R. meliloti (Schmidt et al. 1984; T 6 r 6 k et al. 1984; Jacobs et al. 1985). The defects caused by our class III mutants could not be complemented by p R m S L 2 6 (Table 2). As it is not k n o w n yet whether all c o m m o n nod genes are present on p R m S L 2 6 it cannot be concluded that the genes localized at the left h a n d part o f the 6.6 kb nod fragment are involved in host-specific nodulation functions. F u t u r e research should clarify this p r o b l e m as well as the possible role o f genes localized in the 3.2 kb nod fragment in c o m m o n or host-specific steps o f the nodulation process. Downie JA, Ma QS, Knight CD, Hombrecher G, Johnston AWB (1983a) Cloning of the symbiotic region of Rhizobium leguminosarum: the nodulation genes are between the nitrogenase and a n/fA-like gene. EMBO J 2 : 9 4 7 - 952 Downie JA, Hombrecher G, Ma QS, Knight CD, Wells B, Johnston AWB (1983b) Cloned nodulation genes of Rhizobium leguminosarum determine host-range specificity. Mol Gen Genet 190:359-365 Downie JA, Knight CD, Johnston AWB, Rossen L (1985) Identification of genes and gene products involved in the nodulation of peas by Rhizobium leguminosarum. Mol Gen Genet 198: 255 - 262 Hille J, Klasen I, Schilperoort R (1982) Construction and application of R prime plasmids carrying different segments of an octopine Ti plasmid from Agrobacterium tumefaciens, for complementation of Vir genes. Plasmid 7:107-118 Hirsch AM, Wilson KJ, Jones JDG, Bang M, Walker VV, Ausubel FM (1984) Rhizobium meliloti nodulation genes allow Agrobacterium tumefaciens and Eseheriehia coli to form pseudonodules on Alfalfa. J Bacteriol 158:1133-1143 Hirsch PR, van Montagu M, Johnston AWB, Brewin NJ, ScheU J (1980) Physical identification of bacteriocinogenic nodulation and other plasmids in strains of Rhizobium leguminosarum. J Gen Microbiol 120:403-412 Hombrecher G, Brewin N J, Johnston AWB (1981) Linkage of genes for nitrogenase and nodulation ability on plasmids in Rhizobium teguminosarum and R. phaseoli. Mol Gen Genet 182:133-136 Hooykaas PJJ, van Brussel AAN, den Dulk-Ras H, van Slogteren GMS, Schilperoort RA (1981) Sym plasmid ofRhizobium trifolii expressed in different rhizobial species and Agrobacterium tumefaeiens. Nature (Lond) 291:351 -353 Jacobs TW, EgelhoffTT, Long SR (1985) Physical and genetic map of a Rhizobium meliloti nodulation gene region and nucleotide sequence of nodC. J Bacteriol 162: 4 6 9 - 476 Johnston AWB, Beynon JL, Buchanan-Wollaston AV, Setchell SM, Hirsch PR, Beringer JE (1978) High frequency transfer of nodulating ability between strains and species of Rhizobium. Nature (Lond) 276:635- 636 Josey DP, Beynon JL, Johnston AWB, Beringer JB (1979) Strain identification in Rhizobium using intrinsic antibiotic resistance. J Appl Microbiol 46:343-350 Kondorosi E, Banfalvi Z, Kondorosi A (1984) Physical and genetic analysis of a symbiotic region of Rhizobium meliloti: Identification of nodulation genes. Mol Gen Genet 193:445-452 Lamb JWG, Hombrecher G, Johnston AWB (1982) Plasmid-determined nodulation and nitrogen-fixation abilities in Rhizobium phaseoli. Mol Gen. Genet 186 : 449 - 452 Long SR, Buikema W J, Ausubel RM (1982) Cloning of Rhizobium meliloti nodulation genes by direct complementation of nodmutants. Nature (Lond) 298: 4 8 5 - 488 Ma QS, Johnston AWB, Hombrecher G, Downie JA (1982) Molecular genetics of mutants of Rhizobium leguminosarum which fail to fix nitrogen. Mol Gen Genet 187 : 166-171 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Lab, New York Pannekoek H, Noordermeer IA, v Sluis C, v d Putte P (1978) Expression of the uvrB gene of Escherichia coli: in vitro construction of a pMB9 uvrB plasmid. J Bacteriology 133:884- 890 Priem WJE, Wijffelman CA (1984) Selection of strains cured of the Rhizobium leguminosarum Sym-plasmid pRLIJI by using small bacteriocin. FEMS 25, 247-251 Rosenberg C, Boistard P, Denarie J, Casse -Delhart F (1981) Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti. Mol Gen Genet 184:326-333 Rossen LR, Johnston AWB, Downie JA (1984) DNA sequence of the Rhizobium leguminosarum nodulation genes nodA, B and C required for root hair curling. Nucl Acid Res 12:9497-9508 T6r6k I, Kondorosi E, Stepkowski T, P6sfai J, Kondorosi A (1984) Nucleotide sequence of Rhizobium meliloti nodulation genes. Nucl Acid Res 12: 9509 -- 9524

Acknowledgements. We thank I. Mulders and T. Tak for their skilful technical assistance, W. Priem and B. Zaat for stimulating discussions and S. Long for pRmSL26.

References

Banfalvi Z, Sakanyan V, Koncz C, Kiss A, Dusha I, Kondorosi A (1981) Localization of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti. Mol Gen Genet 184:318-325 Beringer JE, Beynon JL, Buchanon-Woltaston AV, Johnston AWB (1978) Transfer of the drug resistance transposon Tn5 to Rhizobium. Nature (Lond) 276:633-634 Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acid Res 7:1513-1524 Buchanan-Wollaston AV (1979) Generalized transduction in Rhizobium leguminosarum. J Gen Microbiol 112:135 - 142 Cannon FC, Riedel GE, Ausubel FM (1979) Overlapping sequences of Klebsiella pneumoniae nil DNA cloned and characterized. Mol Gen Genet 174:59-66 Djordjevic MA, Zurkowski W, Rolfe BG (1982) Plasmids and stability of symbiotic properties of Rhizobium trifolii. J Bacteriol 151:560-568 Djordjevic MA, Schofield PR, Ridge RW, Morrison NA, Bassam BJ, Plazinski J, Watson JM, Rolfe BG (1985) Rhizobium nodulation genes involved in root hair curling (Hac) are funtionally conserved. Plant Molecular Biology 4:147 - 1 6 0

232 Schmidt J, John M, Kondorosi E, Kondorosi A, Wieneke U, Schr6der G, Schell J (1984) Mapping of the protein-coding region of Rhizobium meliloti common nodulation genes. EMBO J 3:1705-1711 Van Brussel AAN, Tak T, Pees E, Wijffelman CA (1982) Small leguminosae as test plants for nodulation of Rhizobium leguminosarum and other Rhizobia and Agrobacteria harbouring a leguminosarium plasmid. Plant Sci Lett 27: 3 1 7 - 325 Van Brussel AAN, Pees E, Wijffelman C, Tak T (1984) Funktional analysis of Tn5 insertion modulation mutants of pRL1JI, a R. leguminosarum Sym plasmid. In: Veeger C, Newton WE (eds) Advances in nitrogen fixation research, p 724 Vincent JM (1980) Factors controlling the Legume-Rhizobium symbiosis. In: Newton WE, Johnson-Orme WH (eds) Nitrogen fixation, vol II. Univ. Park Press, Baltimore, pp t 0 3 - 1 2 9 Wijffelman CA, Pees E, van Brussel AAN, Hooijkaas PJJ (1983) Repression of small bacteriocin excretion in Rhizobium leguminosarum and Rhizobium trifolii by transmissible plasmids. Mol Gen Genet 192:171 - 176

Received June 12, 1985/Accepted July 12, 1985