Plasmid pSym1-32 from Rhizobium leguminosarum bv. viceae, controlling nitrogen-fixing activity, effectiveness of symbiosis, competitiveness, and acid tolerance

The symbiotic plasmid (pSym1-32) of the highly effective Rhizobium leguminosarum bv. viceae 1-32 strain was identified after the conjugal transfer of replicons carrying Tn5-mob into the plasmidless Agrobacterium tumefaciens Gm1-9023 strain. Plasmid pSym1-32 was transferred into R. leguminosarum bv. viceae strains Y14 (showing low effectiveness of symbiosis with Vicia villosa) and Y57 (unable to fix nitrogen). Transconjugants formed Fix+ nodules on roots of V. villosa and had a highly enhanced nitrogen fixing ability, increased plant weight, and increased nitrogen accumulation compared to the recipient strains. Variation of transconjugants in symbiotic properties (accompanied by alterations in plasmid composition in some of the conjugants) was detected. Moreover, the donor strain R. leguminosarum bv. viceae 1-32 was shown to be more efficient in the competitiveness and acid tolerance than the recipient Y14 strain. Both these properties were transmitted upon transfer of pSym1-32 into the recipient. Thus, plasmid pSym1-32 was shown to carry genes involved in the control of the nitrogen fixing ability, symbiotic effectiveness, competitiveness, and acid tolerance in R. leguminosarum bv. viceae.     

Genetic diversity of indigenous Rhizobium leguminosarum bv. viciae isolates nodulating two different host plants during soil restoration with alfalfa

A total of 360 Rhizobium leguminosarum bv. viciae strains was isolated from three brown-coal mining restoration fields of different age and plant cover (without and in the first and second year of alfalfa, Medicago sativa, cultivation) using two host species (Vicia hirsuta and Pisum sativum) as capture plants. The strains were genetically typed by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-generated 16S-23S ribosomal DNA intergenic spacer regions (IGS-RFLP) and characterized by plasmid profiles and RFLP analysis of amplified nodABC genes. The R. leguminosarum bv. viciae population was dominated by the same group of strains (irrespective of the trap plant used). According to type richness, the genetic diversity of indigenous R. leguminosarum in the second year of restoration was lower than in the first year and it resembled that of the fallow field, except for plasmid types, in which it was higher than that of the fallow field. Some of the less frequent nodABC genotypes were associated with distinct chromosomal IGS genotypes and symbiotic plasmids (pSyms) of different sizes, indicating that horizontal transfer and rearrangements of pSym can occur in natural environments. However, the dominant pSym and chromosomal genotypes were strictly correlated suggesting a genetically stable persistence of the prevailing R. leguminosarum bv. viciae genotypes in the absence of its host plant.     

Diversity and stability of plasmid transfer in isolates from a single field population of Rhizobium leguminosarum bv. viciae

Abstract Isolates of R. leguminosarum bv. viciae from pea and lentil nodules taken at one field site in France were tested in the laboratory for their ability to donate and receive plasmids by conjugation. Five isolates of 20 tested as donors were found to be capable of donating a plasmid which restored the ability to nodulate V. sativa to an isolate which had spontaneously lost this ability. Of 16 isolates tested as recipients all were found to be competent to receive one or more Tn5-labelled test plasmids at a frequency that varied widely (10⁻⁹− 10⁻³ per recipient) dependent upon both the recipient and the plasmid transferred. Three distinct plasmids carrying genes essential for symbiotic functions (pSym) were consistently shown to be transferred at a lower frequency than a cryptic plasmid. Collectively, these results indicate a significant potential for plasmid transfer within the natural soil population. During this work, several independent derivatives were obtained which contained two bv. viciae pSym. These plasmids usually appeared to be compatible together in cells ex planta, but the one acquired in matings was apparently frequently lost (10⁻² per cell) in nodules of V. sativa . Hybrid derivatives containing bv. viciae and bv. phaseoli pSym, apparently retained both plasmids in nodules when P. vulgaris was the host plant but lost the bv. phaseoli pSym at high frequency (4 × 10⁻¹ per cell) in nodules of V. sativa . Structural rearrangements among the plasmids of these transconjugants were also detected in cells recovered from nodules.     

N-acyl-homoserine lactone inhibition of rhizobial growth is mediated by two quorum-sensing genes that regulate plasmid transfer

The growth of some strains of Rhizobium leguminosarum bv. viciae is inhibited by N-(3-hydroxy-7-cis tetradecenoyl)-L-homoserine lactone (3OH-C(14:1)-HSL), which was previously known as the small bacteriocin before its characterization as an N-acyl homoserine lactone (AHL). Tn5-induced mutants of R. leguminosarum bv. viciae resistant to 3OH-C(14:1)-HSL were isolated, and mutations in two genes were identified. These genes, bisR and triR, which both encode LuxR-type regulators required for plasmid transfer, were found downstream of an operon containing trb genes involved in the transfer of the symbiotic plasmid pRL1JI. The first gene in this operon is traI, which encodes an AHL synthase, and the trbBCDEJKLFGHI genes were found between traI and bisR. Mutations in bisR, triR, traI, or trbL blocked plasmid transfer. Using gene fusions, it was demonstrated that bisR regulates triR in response to the presence of 3OH-C(14:1)-HSL. In turn, triR is then required for the induction of the traI-trb operon required for plasmid transfer. bisR also represses expression of cinI, which is chromosomally located and determines the level of production of 3OH-C(14:1)-HSL. The cloned bisR and triR genes conferred 3OH-C(14:1)-HSL sensitivity to strains of R. leguminosarum bv. viciae normally resistant to this AHL. Furthermore, bisR and triR made Agrobacterium tumefaciens sensitive to R. leguminosarum bv. viciae strains producing 3OH-C(14:1)-HSL. Analysis of patterns of growth inhibition using mutant strains and synthetic AHLs revealed that maximal growth inhibition required, in addition to 3OH-C(14:1)-HSL, the presence of other AHLs such as N-octanoyl-L-homoserine lactone and/or N-(3-oxo-octanoyl)-L-homoserine lactone. In an attempt to identify the causes of growth inhibition, a strain of R. leguminosarum bv. viciae carrying cloned bisR and triR was treated with an AHL extract containing 3OH-C(14:1)-HSL. N-terminal sequencing of induced proteins revealed one with significant similarity to the protein translation factor Ef-Ts.     

Transposon mediation allows a symbiotic plasmid of Rhizobium leguminosarum bv. trifolii to become a symbiosis island in Agrobacterium and Rhizobium

The symbiotic plasmid (pSym) of Rhizobium leguminosarum bv. trifolii 4S5, which carries Tn5-mob, was successfully transferred into Agrobacterium tumefaciens A136 by using a conjugation method. The resulting transconjugants induced the development of ineffective nitrogen-fixing nodules on the roots of white clover seedlings. Depending on the manner in which the pSym was retained, the transconjugants were divided into two groups of strains, Afp and Afcs. pSym was retained as a plasmid in the Afp strains but was integrated into the int gene encoding a phage-related integrase on the linear chromosome of A. tumefaciens A136 in strain Afcs1 (one of the Afcs strains) to form a symbiosis island. Conjugation was performed between strain Afcs1 and R. leguminosarum bv. trifolii H1 (a pSym-cured derivative of wild-type strain 4S), and the Rhizobium H1tr strains were screened as transconjugants. Eighteen of the H1tr strains induced effective nitrogen-fixing nodules on the roots of the host plants. pSym was transferred into all of the transconjugants, except for strain H1tr1, at the same size as pSym of strain 4S5. In strain H1tr1, pSym was integrated into the chromosome as a symbiosis island. These data suggest that pSym can exist among Rhizobium and Agrobacterium strains both as a plasmid and as a symbiosis island with transposon mediation.     

Rhizobium leguminosarum exoB mutants are deficient in the synthesis of UDP-glucose 4'-epimerase

Rhizobium leguminosarum bv. viciae Exo- mutant strains RBL5523,exo7::Tn5,RBL5523,exo8::Tn5 and RBL5523,exo52::Tn5 are affected in nodulation and in the syntheses of lipopolysaccharide, capsular polysaccharide, and exocellular polysaccharide. These mutants were complemented for nodulation and for the syntheses of these polysaccharides by plasmid pMP2603. The gene in which these mutants are defective is functionally homologous to the exoB gene of Rhizobium meliloti. The repeating unit of the residual amounts of EPS still made by the exoB mutants of R. leguminosarum bv. viciae lacks galactose and the substituents attached to it. The R. leguminosarum bv. viciae and R. meliloti exoB mutants fail to synthesize active UDP-glucose 4'-epimerase.     

Structural complexity of the symbiotic plasmid of Rhizobium leguminosarum bv. phaseoli.

The complete physical map of the symbiotic plasmid of Rhizobium leguminosarum bv. phaseoli strain CFN42 was established. The data support the concept that Rhizobium symbiotic genes are part of a complex genomic structure which contains a large amount of reiterated DNA sequences. This plasmid is a circular structure of 390 kb with approximately 10 families of internally reiterated DNA sequences of two to three elements each. One family includes two directly oriented nitrogenase operons situated 120 kb apart. We also found several stretches of pSym that are reiterated in other replicons of the cell. Localization of symbiotic gene sequences by heterologous hybridization revealed that nodABC sequences are separated in two regions, each of which contains a nod boxlike element, and it also suggested the presence of two copies of the nifA and nodD gene sequences. We propose that the complex structure of the symbiotic plasmid allows interactions between repeated DNA sequences which, in turn, might result in frequent rearrangements.     

The dnaJ (hsp40) locus in Rhizobium leguminosarum bv. phaseoli is required for the establishment of an effective symbiosis with Phaseolus vulgaris

P121R25 is a Tn5-induced mutant of the effective Rhizobium leguminosarum bv. phaseoli strain P121R that is unable to use glutamate as the sole carbon and nitrogen source and is defective in symbiotic nitrogen fixation. Enzymatic analysis showed that three enzymes implicated in glutamate metabolism (glutamate dehydrogenase, 2-oxoglutarate dehydrogenase, and glutamate synthase) were affected by this mutation. Sequencing of the chromosomal locus bordering the Tn5 in P121R25 indicated the presence of the dnaK and dnaJ genes in an arrangement similar to that described in R. leguminosarum bv. viciae (GenBank accession number Y14649). The mutation was located in the dnaJ (hsp40) gene.     

The construction of recA–deficient Rhizobium meliloti and R. leguminosarum strains marked with gusA or luc cassettes for use in risk–assessment studies

A vector system was developed employing the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar. viciae as target sequences for the stable genomic integration of foreign DNA. The plasmid vectors can be used either as integration vectors (single cross–over), or as gene replacement vectors (double cross–over). Gene replacement results in the antibiotic–marker–free integration of cloned DNA into the recA genes of R. meliloti and R. leguminosarum bv. viciae. Consequently, the recombinant strains become recombination deficient (RecA^-). The expression of integrated genes is under the control of the neomycin phosphotransferase II (nptll) promoter of transposon Tn5. The system was used to construct recA mutant strains of R. meliloti and R. leguminosarum by. viciae, carrying the Escherichia coli gusA gene encoding β–glucuronidase as well as the firefly (Photinus pyralis) luc gene encoding luciferase as marker genes. The GUS activity in the constructed strains was found to be absolutely stable over more than 100 generations of non–selective growth in liquid culture. The stability was also confirmed in root–nodule passages. In addition, the potential use of the luc gene as a stable genetic marker in the unequivocal identification of tagged strains among indigenous microbes in non–sterile soil was demonstrated. It is proposed to use bioluminescent recA mutants as model organisms in risk assessment studies with genetically engineered Rhizobium strains.     

Survival and dispersion of genetically modified rhizobia in the field and genetic interactions with native strains

Abstract A genetically modified strain of the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum biovar viciae was used to inoculate a typical host, pea, and a control non-host cereal crop in the field. The inoculant was monitored for survival and spread from the site of application, and for genetic interactions with the native population. It could be identified by chromosomally located antibiotic resistance markers and additional markers conferred by the transposon Tn 5 inserted on its conjugative symbiotic plasmid. These markers facilitated enumeration of the strain on selective agar, enabling survival and spread to be monitored over a six year period. Although culturable cell numbers dropped two to three orders of magnitude after the first year, subsequently they remained around 10² viable cells per g soil, even in subplots where only the non-host cereals had been grown. However, peas did give the inoculant a small survival advantage compared with non-hosts. Soil cultivation appeared to play a major role in inoculant dissemination from the site of application. Transfer of the Tn 5 marker to other rhizobia could be monitored by screening for isolates with Tn 5 -encoded antibiotic resistance in the absence of the inoculant chromosomal markers. Over three years, more than 4000 pea root nodules were screened for indigenous rhizobia that had acquired the Tn 5 -marked symbiotic plasmid from the inoculant. None were detected, although overall about 2% of nodules contained the inoculant strain, and transfer of the Tn 5 -marked symbiotic plasmid to three out of four R. leguminosarum biovar viciae isolates from the field site could be demonstrated under laboratory conditions.     

Distribution of O-acetyl groups in the exopolysaccharide synthesized by Rhizobium leguminosarum strains is not determined by the Sym plasmid

The patterns of O-acetylation of the exopolysaccharide (EPS) from the Sym plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii strain LPR5, R. leguminosarum bv. trifolii strain ANU843 and R. leguminosarum bv. viciae strain 248 were determined by 1H and 13C NMR spectroscopy. Beside a site indicative of the chromosomal background, these strains have one site of O-acetylation in common, namely residue b of the repeating unit. The O-acetyl esterification pattern of EPS of the Sym plasmid-cured derivatives of strains LPR5, ANU843, and 248 was not altered by the introduction of a R. leguminosarum bv. viciae Sym plasmid or a R. leguminosarum bv. trifolii Sym plasmid. The induction of nod gene expression by growth of the bacteria in the presence of Vicia sativa plants or by the presence of the flavonoid naringenin, produced no significant changes in either amount or sites of O-acetyl substitution. Furthermore, no such changes were found in the EPS from a Rhizobium strain in which the nod genes are constitutively expressed. The substitution pattern of the exopolysaccharide from R. leguminosarum is, therefore, determined by the bacterial genome and is not influenced by genes present on the Sym plasmid. This conclusion is inconsistent with the suggestion of Philip-Hollingsworth et al. (Philip-Hollingsworth, S., Hollingsworth, R. I., Dazzo, F. B., Djordjevic, M. A., and Rolfe, B. G. (1989) J. Biol. Chem. 264, 5710-5714) that nod genes of R. leguminosarum bv. trifolii, by influencing the acetylation pattern of EPS, determine the host specificity of nodulation.     

Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction

Monitoring genetically modified (GM) bacterial inoculants after field release using conventional culture methods can be difficult. An alternative is the detection of marker genes in DNA extracted directly from soil, using specific oligonucleotide primers with the polymerase chain reaction (PCR). The PCR was used to monitor survival of two GM Rhizobium leguminosarum bv. viciae inoculants after release in the field at Rothamsted. One strain, RSM2004, is marked by insertion of transposon Tn 5 ; the second strain, CT0370, released at the same site, is modified by chromosomal integration of a single copy of the gene from E. coli conferring GUS activity. Both GM strains provide a realistic case study for the development of PCR-based detection techniques. Specific primers were developed to amplify regions of the Tn 5 and GUS genetic markers using PCR and conditions optimized for each primer set to routinely detect a signal from 10 fg of purified template DNA, the equivalent of one cell per reaction. Procedures to improve the sensitivity of detection are described, to detect fewer than 50 cells g⁻¹ soil in soil-extracted DNA.     

Rhizobium leguminosarum bv. viciae contains a second fnr/fixK-like gene and an unusual fixL homologue

Genes of Rhizobium leguminosarum bv. viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed. The fixK–fixL region is located upstream of the fixNOQP operon on the non-nodulation plasmid pRleVF39c. The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N-terminal domain of response regulators) fused to its transmitter domain. An oxygen-sensing haem-binding domain, found in other FixL proteins, is conserved in R. leguminosarum bv. viciae FixL. R. leguminosarum bv. viciae possesses a second fnr -like gene, designated fixK , whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK. Individual R. leguminosarum bv. viciae fixK and fixL insertion mutants displayed a Fix⁺ phenotype. A reduced nitrogen-fixation activity was found for a R. leguminosarum bv. viciae fnrN -deletion mutant, whereas no nitrogen-fixation activity was detectable for a fixK / fnrN double mutant. The R. leguminosarum bv. viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis. Another orf was identified downstream of fixK–fixL and encodes a product which has homology to pseudoazurins from different species. Mutation of this azu gene showed that it is dispensable for nitrogen fixation.     

Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids

Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. leguminosarum bv. viciae were made which lack polyhydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wildtype-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutant, or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.     

Symbiotic plasmid rearrangement in Rhizobium leguminosarum bv. viciae VF39SM

A rearrangement between the symbiotic plasmid (pRleVF39d) and a nonsymbiotic plasmid (pRleVF39b) in Rhizobium leguminosarum bv. viciae VF39 was observed. The rearranged derivative showed the same plasmid profile as its parent strain, but hybridization to nod, fix, and nif genes indicated that most of the symbiotic genes were now present on a plasmid corresponding in size to pRleVF39b instead of pRleVF39d. On the other hand, some DNA fragments originating from pRleVF39b now hybridized to the plasmid band at the position of pRleVF39d. These results suggest that a reciprocal but unequal DNA exchange between the two plasmids had occurred.     

Plasmid pSym1-32 of Rhizobium leguminosarumbv. viceae Controlling Nitrogen Fixation Activity,Effectiveness of Symbiosis,Competitiveness, and Acid Tolerance

The symbiotic plasmid (pSym1-32) of the highly effective Rhizobium leguminosarumbv. viceae1-32 strain was identified after the conjugal transfer of replicons carrying Tn5-mobinto the plasmidless Agrobacterium tumefaciensGm1-9023 strain. Plasmid pSym1-32 was transferred intoR. leguminosarumbv. viceaestrains Y14 (showing low effectiveness of symbiosis with Vicia villosa) and Y57 (unable to fix nitrogen). Transconjugants formed Fix⁺nodules on roots of V. villosaand had a highly enhanced nitrogen fixing ability, increased plant weight, and increased nitrogen accumulation compared to the recipient strains. Variation of transconjugants in symbiotic properties (accompanied by alterations in plasmid composition in some of the conjugants) was detected. Moreover, the donor strain R. leguminosarumbv. viceae1-32 was shown to be more efficient in the competitiveness and acid tolerance than the recipient Y14 strain. Both these properties were transmitted upon transfer of pSym1-32 into the recipient. Thus, plasmid pSym1-32 was shown to carry genes involved in the control of the nitrogen fixing ability, symbiotic effectiveness, competitiveness, and acid tolerance in R. leguminosarumbv. viceae.     

A cultivar-specific interaction between Rhizobium leguminosarum bv. trifolii and subterranean clover is controlled by nodM, other bacterial cultivar specificity genes, and a single recessive host gene.

Insertion mutagenesis identified two negatively acting gene loci which restrict the ability of Rhizobium leguminosarum bv. trifolii TA1 to infect the homologous host Trifolium subterraneum cv. Woogenellup. One locus was confirmed by DNA sequence analysis as the nodM gene, while the other locus, designated csn-1 (cultivar-specific nodulation), is not located on the symbiosis plasmid. The presence of these cultivar specificity loci could be suppressed by the introduction of the nodT gene from ANU843, a related R. leguminosarum bv. trifolii strain. Other nod genes, present in R. leguminosarum bv. viciae (including nodX) and R. meliloti, were capable of complementing R. leguminosarum bv. trifolii TA1 for nodulation on cultivar Woogenellup. Nodulation studies conducted with F2 seedlings from a cross between cultivar Geraldton and cultivar Woogenellup indicated that a single recessive gene, designated rwt1, is responsible for the Nod- association between strain TA1 and cultivar Woogenellup. Parallels can be drawn between this association and gene-for-gene systems common in interactions between plants and biotrophic pathogens.     

Comparison of geographically distant populations of Rhizobium isolated from root nodules of Phaseolus vulgaris

Seventy-two rhizobial strains were isolated from the root nodules of french beans ( Phaseolus vulgaris ). They were sampled from two geographically distant field populations and 18 additional different sites in France. They were characterized by a) plasmid profiles, (b) RFLP analysis of total cellular DNA using various chromosomal and symbiotic gene probes (including nif H from Rhizobium etli bv. phaseoli ) and c) their ability to nodulate a potential alternative host, L. leucocephala. Over half of the isolates were ascribed to Rhizobium leguminosarum bv. phaseoli on the basis of the hybridization analysis, the possession of multiple copies of nif H and their inability to nodulate L. leucocephala. The remaining isolates belonged to 2 groups which were shown to be genomically distinct from R. leguminosarum bv. phaseoli, R. etli bv. phaseoli and R. tropici. Most members of these two groups shared with R. tropici the ability to nodulate L. leucocephala and, for isolates of only one of these groups, the presence of one copy of nif H. Members of each of the 3 taxa were widely distributed in France and circumstantial evidence of pSym transfer between them was shown. R. leguminosarum bv. phaseoli and one of the two novel groups co-occurred within the two geographically distant populations. Individual genotypes were conserved between them. The finding of a third taxon at various other locations indicated additional diversity among rhizobia nodulating beans.     

The sym plasmid pRP2JI and at least two other loci of Rhizobium leguminosarum biovar phaseoli can confer resistance to infection by the virulent bacteriophage RL38

Abstract The virulent Rhizobium bacteriophage RL38 did not form plaques on R.leguminosarum by phaseoli but did so at high efficiency on a derivative of that strain lacking its symbiotic plasmid pRP2JI. Other strains with large deletions in pRP2JI which removed many nod and nif genes retained resistance to RL38, showing that the gene which confers phage resistance lies elsewhere on the plasmid. Although the wild-type strain of R. leguminosarum bv. phaseoli failed to plate RL38, it was possible to transduce chromosomal markers into this strain, indicating that the 'block' was not at an early stage in the infection process. Two different recombinant plasmids obtained from a clone bank of genomic DNA of R. leguminosarum bv. phaseoli , which appeared to have no DNA in common, both conferred resistance to RL38. Surprisingly, the DNA cloned in each of these plasmids did not originate from pRP2JI. Therefore, several different loci both on the Sym plasmid and elsewhere on the bacterial genome can be involved in conferring resistance to this bacteriophage.     

Isolation of unique nucleic acid sequences from rhizobia by genomic subtraction: Applications in microbial ecology and symbiotic gene analysis

A subtraction hybridization and PCR amplification procedure was used to isolate two Rhizobium DNA probes which exhibited high degrees of specificity at different levels of taxonomic organization and which could be used as tools for detection of rhizobia in ecological studies. First, a probe was isolated from Rhizobium leguminosarum bv. trifolii strain P3 by removing those Sau3A restriction fragments from a P3 DNA digest which cross hybridized with pooled DNA from seven other strains of the same biovar. The remaining restriction fragments hybridized to DNA from strain P3 but not to DNA from any of the seven other strains. In a similar experiment another DNA probe, specific for the Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici group, was generated by removing sequences from R. leguminosarum bv phaseoli strain Kim 5s with pooled subtracter DNA from eight other Rhizobium, Bradyrhizobium and Agrobacterium species. The same subtraction hybridization technique was also used to isolate symbiotic genes from a Rhizobium species. Results from a 1:1 subtractive DNA hybridization of the broad host range Rhizobium sp NGR234 against highly homologous S. fredii USDA257, combined with those from competitive RNA hybridizations to cosmid digests of the NGR234 symbiotic plasmid, allowed the identification of several NGR234 loci which were flavonoid-inducible and not present in S. fredii USDA257. One of these, ORF-1, was highly homologous to the leucine responsive regulatory protein of E. coli.