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FEMS Microbiology Letters 110 (1993) 269-274 © 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00 Published by Elsevier
In vivo formation of R-prime plasmids harbouring nod genes of Rhizobium "hedysari" F. Javier Ollero, M. Angeles Valverde, M. Rosario Espuny and R a m 6 n
Departarnento de Microbiologfa, Facultad de BiologEa, Seville, Spain (Received 1 April 1993; accepted 6 April 1993)
Abstract." The symbiotic plasmid pRHclJ and the helper plasmid pJB3Jl were transferred from Rhizobium "hedysari" strain RJ77 to Agrobacterium turnefaeiens strain GMI9023. Transconjugants harboured recombinant plasmids (R-prime plasmids) consisting of pJB3JI carrying DNA fragments, of different sizes, surrounding the TnSmob insert in pRHclJ. Two of these R-prime plasmids (pR1 and pR2) carried nod genes and were able to restore the Nod + phenotype of pSym- derivatives of R. "hedysari". The R. "hedysari" nod genes harboured by both R-primes were expressed in R. legurninosarum biovar trifolii wild-type but not in a pSymderivative.
Key words: Rhizobium "hedysari"; R-prime plasmid; nod Gene
Some plasmids belonging to the incompatibility group IncP can mediate transfer of the host chromosome. Chromosome mobilization allows the recovery of R-primes when a divergency at DNA level exists between donor and recipient strains or when the recipient strain is Rec-. In vivo constructed R-prime plasmids, mediated by the P1 type plasmid R68.45, carrying Rhizobium chromosomal region have been reported [9,10]. Banfalvi et al.  demonstrated that it is possible to obtain R-primes harbouring a
Correspondence to: R.A. Bellogln, Departamento de Microbiologla, Facultad de Biologla, Apdo. 1095, 41080 Sevilla, Spain.
symbiotic region from the megaplasmid of R. meliloti by transferring the kanamycin resistance, encoded by transposon Tn5 inserts in the Sym plasmid, to a R e c - Escherichia coli background, mediated by the plasmid pJB3JI (kanamycin-sensitive derivated from R68.45). Rhizobium " hedysari" nodulates on Hedysarum coronarium (sulla), a plant used as a forage crop in different calcareous or clay soils of some mediterranean countries. Previously the behaviour of its pSym in different Rhizobium species has been studied . This paper describes the in vivo formation of different R-primes from Rhizobium "hedysari" by pJB3JI-mediated transfer of the Tn5mob-tagged Sym plasmid to A. tumefaciens. Their expression and stability in different strains have been studied.
Materials and Methods
Bacterial strains, plasmids and culture conditions Bacterial strains and plasmids are shown in Table 1. Rhizobia were grown at 28°C on complete (TY) and minimal (SY) media as defined by Beringer . Agrobacterium tumefaciens was grown in Lb medium . When required, the media were supplemented with the appropriate antibiotic (rifampicin, 50/xg ml-1; kanamycin, 60 /zg ml- 1; streptomycin, 400/zg ml- t; tetracycline, 10 ~g ml-~; chloramphenicol, 50/~g ml-~). Media were solidified by the addition of 1.8% (w/v) Bacto-agar. Bacterial matings Conditions for plasmid transfer by conjugation were as described by Buchanan-Wollaston et al. [161.
Electrophoresis of plasmid DNA Plasmid profiles of bacterial strains were visualized according to the agarose gel electrophoresis method described previously . Plasmid stability in culture To test culture stability of plasmids pR1, pR2, pRHclJ and pJB3JI, strains harbouring the different plasmids were subcultured three times on TY without antibiotics. Then 100 colonies of each strain were checked for plasmid-encoded antibiotic resistance; kanamycin and tetracycline for pR1 and pR2, kanamycin for p R H c l J and tetracycline for pJB3JI. Nodulation test Trifolium repens, T. pratense, T. alexandrinum and Hedysarum coronarium seeds were sterilized and germinated and plants were grown and inoculated as described previously . Nitrogen fixa-
Table 1 Bacterial strains and plasmids Strain or plasmid
Wild-type Spontaneous Sm r derivative of IS123 Spontaneous Rif r derivative of IS123 Nod- derivative of RB16 cured of its symbiotic plasmid RJ68(pRHclJ, pJB3JI) Nod- derivative of RB19 cured of its symbiotic plasmid
Wild-type Spontaneous Sm ~ derivative of RS169 Nod- derivative of RS169 cured of its symbiotic plasmid, Smr
 [21 M.R. Espuny
Rif r, Sm r, pTi-cured derivative of A. tumefaciens C58 Em r, Cm r, pTi-cured derivative of A. tumefaciens C58 Transconjugants from cross RJ77 × GMI9023
Tra ÷, Tc r, Ap r IS123 Sym plasmid tagged with Tn5 mob R-primes obtained from cross R J77 × GMI9023 harbouring about 60, 45 and 30 Md, respectively of DNA from pRHclJ
Rhizobium "hedysari" IS123 RB16 RB19 R J68 R J77 RJ243
[81  [131 
R. leguminosarurn biovar trifolii RS169 RS169S14 RS169NA4
Agrobacterium tumefaciens GMI9023 C58C1 R J l l 3 to RJ122
pJB3JI pRHclJ pR1, pR2 and pR3
Abbreviations: Sm r, resistance to streptomycin; Rif r, resistance to rifampicin; Km r, resistance to kanamycin; Tc r, resistance to tetracycline, Ap r, resistance to ampicillin; Em r, resistance to erytbromycin; Cm r, resistance to chloramphenicol; Tra +, conjugative.
tion was measured by testing acetylene reduction activity. Bacteria were isolated from squashed single nodules as described previously .
3-Ketolactose test This test was used to distinguish A. tumefaciens strains GMI9023 and C58C1 (3-ketolactose-positive) from Rhizobium strains (3-ketolactose-negative). According to Bernaerts and De Ley , bacteria were grown on LY medium in a small heap. After incubation for 3 days, 50/xl of Benedict's reagent was added to each heap. The test was positive when a yellow ring of cuprous oxide was formed around the heap.
Results and Discussion
The symbiotic plasmid pRHclJ was transferred from Rhizobium "hedysari" strain RJ77, that also harbours the helper plasmid pJB3JI, to Agrobacterium tumefaciens GMI9023 at a frequency of 3 x 10-7/recipient cell. Transconjugants, selected on TY supplemented with kanamycin, were tetracycline-resistant. Ten of them (termed RJll3 to RJ122) were chosen for further studies. They were 3-ketolactose-positive like the recipient strain GMI9023. Figure 1 shows the plasmid profiles of eight of these transconjugants. They harbour a plasmid with an electrophoretic mobility between the plasmids pRHclJ (230 MDa) and pJB3JI (40 MDa). None of these transconjugants nodulated
on sulla except one that induced nodule-like structures, from which it was not possible to isolated rhizobia. These results show that it is possible to obtain R-prime plasmids between the symbiotic plasmid pRHclJ and pJB3JI by a simple transfer for both plasmids to A. tumefaciens GMI9023 selecting for kanamycin resistance. In order to check if some of these Knlr Tc r plasmids (R-prime plasmids) harboured symbiotic genes, which were not expressed in an Agrobacterium tumefaciens background, some of these plasmids were transferred back into the pSymRhizobium "hedysari" strain RJ243. It was not possible to transfer the R-prime plasmids from GMI9023 to RJ243 in one step since both strains are rifampicin-resistant. Therefore the plasmids were first transferred from GMI9023 transconjugants to A. tumefaciens C58C1 (Em r, Cm r) and then to RJ243. The R-prime plasmids present in strains RJ118, RJ120 and RJ121, with a molecular mass of about 100, 85 and 70 MDa and named pR1, pR2 and pR3, respectively, were transferred to C58C1, the transconjugants being selected on Lb medium, supplemented with chloramphenicol and kanamycin. One transconjugant of each cross was chosen as a donor strain to transfer the different plasmids to RJ243. These transconjugants were Rift, Km r, Tc ~, 3-ketolactose-negative and did not grow on Lb medium. Electrophoretic studies showed that the transfer of the different R-prime plasmids from GMI9023 to C58C1 and further from C58C1 to RJ243 did not affect the plasmids'
Fig. 1. Plasmid profiles in strains: A, RJ122; B, GMI9023, C, RJ121; D, RJ120; E, RJ77; F, RJll9; G, RJll8; H, GMI9023; I, RJ117; J, RJll6; K, RJ77; L, RJll5. RJll9 (lane F) harbours two plasmids, the R-prime and pJB3JI, suggesting that in this strain the R-prime may release the inserted fragment.
Table 2 Antibiotic markers of nodule isolates induced by RJ243(pR1) and RJ243(pR2) on sulla Strains
Nodules Markers Rifr/Rif s Tcr/TCs Kmr/Kms
1 2 3 4 5 6 7 8 9 10 11 12
10/0 10/0 10/0 10/0 10/0 10/0 10/0 10/0 10/0 10/0 10/0 10/0
10/0 10/0 10/0 9/1" 10/0 10/0 9/1 a 10/0 10/0 10/0 10/0 10/0
7/3 6/4 1/9 6/4 10/0 8/2 6/4 1/9 6/4 6/4 8/2 1/9
2 3 4 5 6 7 8 9 10 11 12
10/0 10/0 10/0 10/0 10/0 10/0 10/0 10/0 10/0 10/0 10/0
10/0 10/0 10/0 10/0 10/0 10/0 10/0 10/0 10/0 10/0 10/0 10/0
2/8 4/6 4/6 10/0 4/6 8/2 6/4 7/3 4/6 4/6 8/2 7/3
Fig. 2. Plasmid profiles in strains: A, RJ120; B, RJ243(pR2); C, RJll8; D, RJ243(pR1); E, GMI9023; F, RJ243; G, RJ243(pR3); H, RJ121.
mobility, so they have presumably maintained the same physical structure even after passing through different recipient backgrounds. Figure 2 shows the electrophoretic profiles of strains GMI9023 and RJ243 harbouring pR1, pR2 and pR3 plasmids. Strains RJ243(pR1) and RJ243(pR2) induced non-nitrogen-fixing nodules on sulla. Bacteria were isolated from the nodules on a non-selective medium, then tested for antibiotic resistance (Table 2) and plasmid profiles. Km r Tc r isolates harboured the two plasmids of the recipient strain RJ243 and plasmids pR1 or pR2, and were Nod ÷ Fix- on sulla. The Km ~ Tc ~ isolates harboured a band corresponding to pJB3JI and Km s Tc s isolates had the same electrophoretic profile as RJ243. All Km s isolates were N o d - on sulla. In order to determine the transfer frequency of pR1 and pR2, both plasmids were transferred from RJ243 to the p S y m - R. "hedysari" strain RJ68. The transfer frequency was about 1 0 - 4 / r e cipient cell. Transconjugants RJ68(pR1) and RJ68(pR2) were Nod + Fix- on sulla. On the other hand, the helper plasmid pJB3JI was transferred to the same recipient strain R J68 at a frequency of about 5 × 10-2/recipient cell. The restoration of the phenotype Nod + on sulla in the Sym plasmid cured strains upon introduction of plasmids pR1 and pR2 indicates that both R-primes harbour all essential nod genes for nodulation on sulla. The culture stability of pR1 and pR2 in the RJ243 background were tested. Results are shown
a The clone Tcs also was Kms. Rhizobia isolated from 12 nodules induced by RJ77 on sulla were Smr, Tcr and Kmr.
in Table 3 and indicate that both plasmids are relatively stable in culture. Banfalvi et al.  found that R-primes are unstable both in culture and after the passage in plant of strains harbouring them. The same au-
Table 3 Culture stability of pR1, pR2, pJB3JI and pRHclJ plasmids Strains RJ243(pR1) RJ243(pR2) RJ77 a
98/2 99/1 100/0
100/0 100/0 100/0
a RJ77 harbours the symbiotic plasmid pRHclJ (Kmr) and pJB3JI (Tcr). The three Kms clones did not harbour either pRl or pR2 but a band corresponding to pJB3JI.
thors suggest that this instability could be due to homologous recombination between the host D N A and that of the R-prime. This is not true in our case, since we transferred to a pSym-cured strain R-primes harbouring a fragment of the pSym; therefore, an instability could be explained by homologous recombination only in case of homology between the pSym and the Rhizobium chromosome. In fact, the R-primes are fairly stable in culture (Table 3). Instability after passage in sulla (Table 2) could be explained by extra copies of nod genes in R-primes, with respect to those present in the pSym. In fact, Knight et al.  have reported that extra copies of nodABC genes in a wild-type strain inhibited the nodulation and that those few nodules formed eventually contained bacteria which had lost the plasmid carrying nodABC. It has been reported previously that strain IS123 is able to induce 'erroneous nodulation' on red clover plants , and it has also been demonstrated that p R H c l J (the tagged symbiotic plasmid of IS123) has the necessary genetic information to nodulate red clover . In order to determine whether the genetic information present in pR1 a n d / o r pR2 was sufficient to induce this nodulation, RJ243(pR1) and RJ243(pR2) were inoculated on red clover, where both strains were N o d - . This would indicate that other genes present in p R H c l J , but not in R-primes, are necessary for 'erroneous' clover nodulation. However, these genes are not necessary for nodulation on sulla. The R-prime plasmids pR1 and pR2 were also transferred to R. leguminosarum biovar trifolii RS169S14 at a frequency of about 10-6/recipient cell and to its pSym- derivative RS169NA4 at a frequency of about 1 0 - a / r e c i p i e n t cell. The presence of pR1 and pR2 in transconjugants was demonstrated by electrophoretic studies. When p R H c l J was transferred to RS164S14, but not to RS169NA4, rearrangements between symbiotic plasmids were observed that indicate incompatibility between the two Sym replicons (data not shown). However, no incompatibility was observed when pR1 or pR2 were transferred to this R. leguminosarum biovar trifolii background, suggesting that the gene(s) responsible for this phe-
nomenon is (are) not in the nod region cloned in R-primes. Transconjugants RS169S14(pR1) and RS169S14(pR2) were Fix-- on red, white and subterranean clover and Nod + Fix- on sulla. Rhizobia isolated from clover nodules were Sm r, Tc r and Km s and they had lost pR1 or pR2 plasmids, harbouring a band corresponding to the helper plasmid pJB3JI. Rhizobia isolated from sulla nodules were always Sm r, Tc r but from a single nodule it was possible to isolate Km r and Km S clones. Km r clones had the same electrophoretic profile that transconjugants had before passing through sulla and were Nod + Fix- on sulla. The Km s clones did not harbour pR1 or pR2 but a band corresponding to pJB3JI and were N o d - on sulla. Transconjugants RS169NA4(pR1) and RS169NA4(pR2) were N o d - on red, white and subterranean clover and also on sulla. The fact that RS169S14 (pR1 or pR2) were able to induce sulla nodulation while, surprisingly, strain NA4 (Sym- derivative of RS169S14) harbouring pR1 or pR2 was not, could indicate that some gene(s) in the R. leguminosarum biovar trifolii Sym plasmid is (are) necessary for sulla nodulation besides the cloned genes present in the R-primes.
Acknowledgement We thank Prof. A. Squartini for critical reading of and comments on the manuscript.
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