APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1990, p. 98-103 Vol. 56, No. 1 0099-2240/90/010098-06$02.00/O Copyright © 1990, American Society for Microbiology Construction of a Symbiotically Effective Strain of Rhizobium leguminosarum bv. trifolii with Increased Nodulation Competitiveness ERIC W. TRIPLETT* Department ofAgronomy and Center for the Study of Nitrogen Fixation, 1575 Linden Drive, University of Wisconsin-Madison, Madison, Wisconsin 53706 Received 22 June 1989/Accepted 4 October 1989 Genes involved in nodulation competitiveness (tfx) were inserted by marker exchange into the genome of the effective strain Rhizobium leguminosarum bv. trifolii TAl. Isogenic strains of TAl were constructed which differed only in their ability to produce trifolitoxin, an antirhizobial peptide. Trifolitoxin production by the ineffective strain R. leguminosarum bv. trifolii T24 limited nodulation of clover roots by trifolitoxin-sensitive strains of R. leguminosarum bv. trifolii. The trifolitoxin-producing exconjugant TA1::10-15 was very competitive for nodulation on clover roots when coinoculated with a trifolitoxin-sensitive reference strain. The nonproducing exconjugant TA1::12-10 was not competitive for nodule occupancy when coinoculated with the reference strain. Tetracycline sensitivity and Southern analysis confirmed the loss of vector DNA in the exconjugants. Trifolitoxin production by TA1::10-15 was stable in the absence of selection pressure. Transfer of tfx to TAl did not affect nodule number or nitrogenase activity. These experiments represent the first stable genetic transfer of genes involved in nodulation competitiveness to a symbiotically effective Rhizobium strain. Inoculation of legume plants with superior strains of Barry (2, 3), who has developed transposon Tn7 vectors for Rhizobium often fails to improve legume productivity. This this purpose. Another technique is marker exchange, in failure is caused by the ability of ineffective or inefficient which the DNA on a vector is homologous with a region on native strains of Rhizobium to prevent nodulation by the the chromosome. An incompatible plasmid and the appro- inoculum strains (9, 11, 17, 24, 25). Several laboratories are priate selection pressure are then used to force the DNA on working on various strategies to address this problem. In this the vector to exchange with the homologous region on the laboratory, we are characterizing the production of a very chromosome. Williams et al. (26) and O'Gara et al. (16) have potent antirhizobial compound, referred to as trifolitoxin, developed integration vectors for this purpose which include which is produced by Rhizobium leguminosarum bv. trifolii a gene from Rhizobium meliloti. The R. meliloti gene is used T24 (20-23). as a source of homology for the marker exchange event. Trifolitoxin production by T24 limits nodulation of clover Genes are chosen for this purpose which do not affect the roots by other strains of R. leguminosarum bv. trifolii (19, symbiosis. A multiple cloning site is included within the R. 21). Strain T24 is not useful as a solution to the Rhizobium meliloti gene. Insert DNA in that cloning site can then be competition problem since it induces ineffective nodules on integrated into the R. meliloti gene. clover. The trifolitoxin genes have been cloned and trans- In this article, the stable insertion and expression of the ferred to effective strains of Rhizobium by conjugation of a trifolitoxin genes into an effective strain of Rhizobium by a recombinant plasmid, pTFX1, which possesses the genes marker exchange event that utilizes homologous DNA necessary for trifolitoxin production and resistance (20). present in pTFX1 are described. However, recombinant plasmids based on pLAFR1 or pLAFR3, such as pTFX1, are commonly unstable in the MATERIALS AND METHODS absence of selection pressure (13, 15). Thus, Rhizobium transconjugants with pTFX1 are not likely to be able to limit Bacteria and plasmids. The bacterial strains and plasmids nodulation by trifolitoxin-sensitive indigenous strains of used in this work are listed in Table 1. Rhizobium under agricultural conditions where tetracycline Bacterial growth conditions. Rhizobium strains were cul- application is impractical. Although pTFX1 has been trans- tured at 28°C on Bergersen synthetic medium (BSM) as ferred to effective strains of Rhizobium (20), the nodulation described by Bergersen (4). Strains of Escherichia coli were competitiveness of the Rhizobium pTFX1-carrying transcon- cultured at 37°C on Luria-Bertani (LB) medium. Antibiotics jugants has not been tested because of the suspected insta- were added as needed at the following final concentrations: bility of pTFX1 following inoculation on legume roots. kanamycin, 50 ,ug/ml; tetracycline, 12.5 ,ug/ml; spectinomy- Experiments to test the nodulation competitiveness of trifo- cin, 50 ,ug/ml; streptomycin, 50 Fig/ml; gentamicin, 25 ,ug/ml; litoxin-producing, effective rhizobia have been delayed until nalidixic acid, 10 ,ug/ml; and neomycin, 75 ,ug/ml. the trifolitoxin production and resistance gene (tfx) has been Bacterial conjugations. Conjugation of the pTFX1::TnS inserted stably into the Rhizobium genome. mutants into Rhizobium strains was performed as described Two methods are now available for the insertion of foreign previously (21) with some modifications. The donor, recipi- genes into the chromosome of rhizobia and other gram- ent, and helper strains were mixed in a 1:1:1 ratio in water, negative bacteria. One such method is that described by each at a cell density of approximately 5 x 107 per ml. After being vortexed, a 5-pl suspension of this mixture was placed on a YM/KB (21) plate with 3% agar. After incubation for 2 * Electronic mail address: triplett@wiscmac3. days at 28°C, each mating mix was suspended in 0.1 ml of 98 VOL. 56, 1990 EFFECTIVE, TRIFOLITOXIN-PRODUCING RHIZOBIUM STRAIN 99 TABLE 1. Bacteria and plasmids Strain or plasmid Genotype or relevant characteristicsa Source or reference R. leguminosarum bv. trifolii T24 Tfx+ Tfxr Fix- Cmp+ 19 2046 Tfxs Fix' USDAb Beltsville Rhizobium collection TAl Tfxs Fix+ A. Gibson, CSIROC TA1::10-15 Tfx+ Tfxr Fix' Cmp+ TnS, Kmr Smr Tcs This work TA1::12-10 Tfx- Tfxr Fix+ Cmp- Tn5, Kmr Smr Tcs This work TA1(pTFX1) Tfx+ Tfxr Tcr TnS, Kmr Smr 20 TA1(pTFX1::10-15) Tfx+ Tfxr Tcr TnS, Kmr Smr This work TA1(pTFX1::12-10) Tfx- Tfxr Tcr Tn5, Kmr Smr This work E. coli DH5a Bethesda Research Laboratories Plasmids pTFX1 pLAFR3 derivative with tfx genes, Tcr 20 pTFX1::10-15 pTFX1 derivative with a Tn5 insertion adjacent to tfx 23 pTFX1::12-10 pTFX1 derivative with a Tn5 insertion inside tfx This work pRK2013 KmrTraT Mob' ColEl replicon 10 pRK2073 pRK2073 Spr::Tn7 14 pPHlJI Tra+ Mob' Gmr Spr Smr Cmr IncP replicon 5 a Tfx+, Trifolitoxin producing; Tfx-, non-trifolitoxin producing; Tfxr, trifolitoxin resistant; Tfxs, trifolitoxin sensitive; Fix', effective symbiotic nitrogen fixation; Fix-, incapable of symbiotic nitrogen fixation (ineffective); Nod, nodule induction; Tra, plasmid transfer function; Mob, plasmid mobilization function; Cmp, competitive for nodulation; Km, kanamycin; Tc, tetracycline; Sp, spectinomycin; Sm, streptomycin; Gm, gentamicin; Nal, nalidixic acid; Nm, neomycin. b USDA, U.S. Department of Agriculture. C CSIRO, Commonwealth Scientific and Industrial Research Organisation. water and spread-plated on a BSM plate prepared with and 5 ,ul of that suspension was spotted in the center of a Noble agar and supplemented with tetracycline and strepto- BSM plate for the assay of trifolitoxin production. A single mycin. The use of Noble agar in the interruption medium colony from the initial plate was used to inoculate a second decreased the background of the parental strains on the plate. After 2 days, confluent growth on the second plate was plates. After 5 days, transconjugants were observed. used to assay trifolitoxin. The assays continued for 10 such Conjugations involving the transfer of plasmid DNA be- purifications to single colonies or until trifolitoxin production tween strains of E. coli were conducted as described above was no longer observed. except that 5 ,ul of the mixture of donor, recipient, and Southern analysis. Total DNA of the TAl exconjugants helper strains was placed on an LB plate and incubated at was digested to completion with EcoRI. The fragments were 37°C overnight. Interruptions were done as described above separated by gel electrophoresis and blotted onto a nylon with the appropriate selective medium on solid LB medium. membrane (Nytran; Schleicher & Schuell, Keene, N.H.). In the transfer of plasmid DNA from E. coli to Rhizobium The blot was then probed with a digoxigenin-labeled strains, E. coli DH5a(pRK2013) was used as the helper pLAFR3 probe as described by the manufacturer (Genius strain. Kit; Boehringer Mannheim Corp., Indianapolis, Ind.). DNA isolation. Large-scale plasmid preparations were Plant culture and inoculation. Clover plants were cultured purified by the boiling method described by Holmes and and inoculated as described previously (21). The following Quigley (12). For restriction analysis of small amounts of plasmid DNA, plasmids were purified by the alkaline lysis method described by Ausubel et al. (1). Total genomic DNA 2046 TAl from strains of Rhizobium was isolated as described by Ausubel et al. (1). The plasmid pTFX1 was mapped by TnS mutagenesis and restriction enzyme analysis as described previously (23). Marker exchange. The method of Ditta (8) was used for the marker exchange of the trifolitoxin genes into the genome of R. leguminosarum bv. trifolii TAl (Fig. 1). The incompatible plasmid pPHlJI was conjugated into two TAl transconju- gants with pTFX1::TnS. The conjugation was interrupted on BSM prepared in Noble agar and supplemented with genta- micin, kanamycin, and spectinomycin. The resulting excon- jugants were replica-plated on BSM with tetracycline. The tetracycline-resistant colonies were discarded. Determination of the stability of trifolitoxin production in the absence of selection pressure. Strains T24, TA1(pTFX1), and trifolitoxin-producing TA1(pTFX1: :TnS) transconju- gants and TAl::TFX-TnS exconjugants were streaked to single colonies on BSM medium in the absence of selective FIG.