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Arch Microbiol (1985) 143:225- 232
9 Springer-Verlag 1985
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
I nodO nOdAlnOdlB node i
Materials and methods
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.
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).
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_
nif KDH
//
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
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
227
Table 1. Bacterial strains a
Strain
Plasmid
Relevant characteristics
Reference
248
LBA937
1064
1830
LPR5039
pRLIJI
R772
pRmSL26
pJB4JI
-
R. leguminosarum w.t
A. tumefaciens R772
E. coli HB101 pRmSL26
E. eoIipro,met pPH1JI : :Mu: :Tn5
R. trifolii RCR5 str cured of its
Joscy et al. 1979
Hille et al. 1982
Long et al. 1982
Beringer et al. 1978
Hooykaas et al. 1981
RBL5502
RBL5505
RBL5506
RBL5515
RBL5516
RBL5523
RBL5559
pRL1JI
pRL650
pRmSL26
sym plasmid pRtr5a
LPR5039 pRL1JI
LPR5039 rif,spc
LPR5039 cam
LPR5039 r/f
LPR5039 spc
RBL5515 pRL1 ::Tn1831
RBL5515 pRmSL26
Priem et al.
Priem et al.
Priem et al.
Priem et al.
Priem et al.
Priem et al.
This work
RBL601
RBL602
RBL603
RBL604
RBL605
RBL606
RBL607
RBL609
RBL610
RBL611
RBL613
RBL615
RBL618
RBL621
RBL622
RBL631
RBL632
pRL601
pRL602
pRL603
pRL604
pRL605
pRL606
pRL607
pRL609
pRL610
pRL611
pRL613
pRL615
pRL618
pRL621
pRL622
pRL631
pRL632
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
1984
1984
1984
1984
1984
1984
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
I
RBL602,
RBL610,
RBL631,
RBL611
RBL601,
RBL605,
RBL621,
II
III
RBL607, RBL609
RBL613, RBL615
RBL632
RBL603, RBL604
RBL606,RBL618
RBL622
Root hair
curling
Tsr
phenotype
Infection
thread
formation
'Delayed
modulation'
Complemenration
by pRmSL26
Hac-
-
-
-
+
Had +
-
-
-
+
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
x~X/
e~
9 60-
8 40C
"~ 2o$
/.
x
.a
0-
/_./
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
e7
days after inooulation
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.
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.
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-
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.
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
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
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