Symbiotic mutants of USDA191, a fast-growing Rhizobium that nodulates soybeans

Summary Symbiotic and auxotrophic mutants of Rhizobium japonicum strain USDA191 were isolated using Tn5 mutagenesis and techniques that cause plasmid deletions and plasmid curing. Characterization of several mutants that are unable to nodulate (Nod^-) or unable to fix nitrogen (Fix^_) showed that nod and nif genes are located within one regions of a 200 MD plasmid (pSym191). Blot hybridization analysis of plasmids in other fast-growing R. japonicum strains showed that nod as well as nif sequences are located on plasmids in eight strains but are apparently carried in the chromosome in two strains.     

Nodule formation in soybeans by exopolysaccharide mutants of Rhizobium fredii USDA 191

Production of exopolysaccharides by Rhizobium has been linked with efficient invasion and nodulation of leguminous plant roots by the bacteria. Exopolysaccharide-deficient (exo) mutants of Rhizobium fredii USDA 191 were isolated following Tn5-insertion mutagenesis. Five phenotypically unique exo mutants were investigated for exopolysaccharide synthesis and their ability to nodulate soybeans. The exopolysaccharides produced by these mutants were analysed for polysaccharide composition by column chromatography and thin-layer chromatography. Two mutants designed exo-3 and exo-5 were deficient in both neutral glucan and exopolysaccharide synthesis, but each induced some functional nodules on Glycine max (Peking). The remaining three mutants (exo-1, exo-2 and exo-4) synthesized neutral glucans at levels higher or lower than those in wild-type and exhibited partial exopolysaccharide deficiencies. The data imply that neither exopolysaccharides nor neutral glucans are essential for the induction of determinate nodules by R. fredii.     

Symbiotic Effectiveness and Host-Strain Interactions of Rhizobium fredii USDA 191 on Different Soybean Cultivars

Nodulation, acetylene reduction activity, dry matter accumulation, and total nitrogen accumulation by nodulated plants growing in a nitrogen-free culture system were used to compare the symbiotic effectiveness of the fast-growing Rhizobium fredii USDA 191 with that of the slow-growing Bradyrhizobium japonicum USDA 110 in symbiosis with five soybean (Glycine max (L.) Merr.) cultivars. Measurement of the amount of nitrogen accumulated during a 20-day period of vegetative growth (28 to 48 days after transplanting) showed that USDA 110 fixed 3.7, 39.1, 4.6, and 57.3 times more N₂ than did USDA 191 with cultivars Pickett 71, Harosoy 63, Lee, and Ransom as host plants, respectively. With the unimproved Peking cultivar as the host plant, USDA 191 fixed 3.3 times more N₂ than did the USDA 110 during the 20-day period. The superior N₂ fixation capability of USDA 110 with the four North American cultivars as hosts resulted primarily from higher nitrogenase activity per unit nodule mass (specific acetylene reduction activity) and higher nodule mass per plant. The higher N₂-fixation capability of USDA 191 with the Peking cultivar as host resulted primarily from higher nodule mass per plant, which was associated with higher nodule numbers. There was significant variation in the N₂-fixation capabilities of the four North American cultivar-USDA 191 symbioses. Pickett 71 and Lee cultivars fixed significantly more N₂ in symbiosis with USDA 191 than did the Harosoy 63 and Ransom cultivars. This quantitative variation in N₂-fixation capability suggests that the total incompatibility (effectiveness of nodulation and efficiency of N₂ fixation) of host soybean plants and R. fredii strains is regulated by more than one host plant gene. These results indicate that it would not be prudent to introduce R. fredii strains into North American agricultural systems until more efficient N₂-fixing symbioses between North American cultivars and these fast-growing strains can be developed. When inoculum containing equal numbers of USDA 191 and of strain USDA 110 was applied to the unimproved Peking cultivar in Perlite pot culture, 85% of the 160 nodules tested were occupied by USDA 191. With Lee and Ransom cultivars, 99 and 85% of 140 and 96 nodules tested, respectively, were occupied by USDA 110.     

Transfer of a host range plasmid from Rhizobium leguminosaum to fast-growing bacteria that nodulate soybeans

The Rhizobium leguminosarum host range plasmid pJB5JI was transferred to three fast-growing bacterial strains able to nodulate soybeans. These strains, isolated in China, contained plasmids and were able to transfer pJB5JI back to R. leguminosarum . Soybean strains carrying pJB5JI elicited early stages of nodule development on peas.     

Studies of symbiotic plasmids in Rhizobium trifolii and fast-growing bacteria that nodulate soybeans

The Rhizobium trifolii symbiotic plasmid pRt5a was transferred to the fast-growing soybean strain USDA 194. Transconjugants carrying pRt5a were not able to nodulate clovers and one of the transconjugants had lost its smallest resident plasmid and did not fix nitrogen in soybean. Transconjugants of USDA 194 carrying pRt5a were able to transfer pRt5a back to a non-nodulating R. trifolii which inherited the symbiotic properties of the R. trifolii strain from which the plasmid was derived.     

Symbiotic mutants of Rhizobium meliloti that uncouple plant from bacterial differentiation

Spontaneous mutants at a new symbiotic locus in Rhizobium meliloti SU47 are resistant to several phages and are conditionally insensitive to a monoclonal antibody to the bacterial surface, apparently because they are deficient in a wild-type exopolysaccharide. On alfalfa, the mutants do not curl root hairs, but penetrate the epidermis directly, forming nodules that contain no visible infection threads or "bacteroids," have a few bacteria in superficial intercellular spaces only and not within the nodule cells, and fail to fix nitrogen (Fix-). Evidently, infection threads are not essential for cell proliferation and nodule formation, which are here induced by a bacterial signal at a distance and uncoupled from the bacterial differentiation that normally goes on as well.     

The formation of R-prime deletion mutants and the identification of the symbiotic genes in Rhizobium fredii strain USDA191

Summary R-prime plasmids were formed between the plasmid of Rhizobium fredii strain USDA191 containing nodulation and nitrogen-fixation genes, pRjaUSDA191c, and pRL180, and RP1 derivative. R. fredii USDA191 contains four HindIII fragments that hybridize with an 8.7 kb EcoRI fragment that contains nodulation genes from R. meliloti. These four fragments are on pRjaUSDA191c and are 15.5 kb, 12.5 kb, 6.8 kb, and 5.2 kb in size. A series of R-primes generated in E. coli of pRjaUSDA191c were transferred into a Nod^- Nif^- derivative of strain USDA191 to determine which nodulation region is necessary for nodule formation. Transconjugants containing the 12.5 kb and the 6.8 kb HindIII fragments on segments of pRjaUSDA191c produced nodules on soybean plants. However, transconjugants containing the 12.5 kb HindIII fragment alone were unable to form nodules, suggesting that the 6.8 kb HindIII fragment or the 6.8 kb and the 12.5 kb HindIII fragments together were needed for nodule formation. The 6.8 kb HindIII fragment was subcloned into the vector pVK102 and transferred into transconjugants containing no sequences homologous to R. meliloti nodulation DNA or to transconjugants containing only the 12.5 kb HindIII fragment. Nodules were formed on soybeans only when both the 12.5 kb and the 6.8 kb HindIII fragments were present in R. frediistrain USDA191.     

Symbiotic properties of antibiotic-resistant mutants of Rhizobium galegae

Mutagenesis provoked by exposure to increased concentration of antibiotics of five indigenous Rhizobium galegae strains resulted in the generation of several antibiotic-resistant mutants. The mutants differed from the wild type and one from another in respect to the nodulation capacity, the nitrogenase activity, the nodule ultrastructure, and the plant growth response. Galega plants inoculated with mutants resistant to streptomycin and rifampicin formed nodules with higher nitrogenase activity and accumulated more shoot dry biomass than plants inoculated with the parent strains. Resistance to kanamycin and nalidixic acid was associated with significant decrease of nitrogenase activity. A correlation between nitrogen-fixing efficiency and nodule infected cell ultrastructure was found. When the bacteroids occupied about 10 times higher area in infected cells of nodule than peribacteroid spaces and host cytosol had electron dense and homogenous structure, the nitrogenase activity was the highest. This revised version was published online in August 2006 with corrections to the Cover Date.     

Symbiotic pseudorevertants of Rhizobium meliloti ndv mutants.

Nodule development (ndv) mutants of Rhizobium meliloti cannot invade alfalfa to establish a nitrogen-fixing symbiosis and instead induce the formation of small, white, unoccupied nodules on alfalfa roots. Such mutants also fail to produce the unusual cyclic oligosaccharide beta-(1----2)-glucan and show defects in several aspects of vegetative growth and function. Here we show that ndv mutants are severely reduced, although not totally deficient, in the ability to attach to and initiate infection threads on alfalfa seedlings, and we demonstrate that the symbiotic deficiency can be separated from the rest of the mutant phenotype by isolating second-site pseudorevertants. Pseudorevertants selected for restoration of motility, a vegetative property, regained a substantial amount of attachment capability but only slight infection thread initiation and symbiotic ability. Such strains also regained partial tolerance to growth at low osmolarity, even though they did not recover the ability to synthesize periplasmic beta-(1----2)-glucan. Pseudorevertants selected on alfalfa for restoration of symbiosis were unrestored for beta-(1----2)-glucan production or any other vegetative property and regained little or no attachment or infection thread initiation capability. We take these data to indicate that wild-type R. meliloti normally has considerable excess capability for both attachment and infection thread initiation and that the symbiotic block in ndv mutants lies further along the developmental pathway than either of these processes, probably at the level of infection thread extension. Further, the fact that neither type of pseudorevertant recovered the ability to produce periplasmic beta-(1----2)-glucan raises the possibility that this oligosaccharide is not directly required for nodule development.     

Expression of symbiotic genes of Rhizobium japonicum USDA 191 in other rhizobia.

A 200-megadalton plasmid was mobilized from Rhizobium japonicum USDA 191 to other Rhizobium strains either that cannot nodulate soybeans or that form Fix- nodules on certain cultivars. The symbiotic properties of the transconjugants indicate that both soybean specificity for nodulation and cultivar specificity for nitrogen fixation are plasmid encoded.     

Symbiotic phenotypes of auxotrophic mutants of Rhizobium meliloti 104A14

Auxotrophic mutants of Rhizobium meliloti 104A14 were isolated using nitrous acid mutagenesis followed by penicillin enrichment. Mutants in ornithine transcarbamylase, argininosuccinate synthetase or serine-glycine biosynthesis formed nitrogen-fixing (Fix-nodules on the roost of alfalfa (Medicago sativa). Mutants with defects in ornithine, pyrimidine, purine, asparagine, leucine, methionine or tyrosine biosynthesis, in one-carbon metabolism or in carbamoylphosphate synthetase formed nodules but these nodules were unable to fix nitrogen. Prototrophic revertants were always Fix?Plasmids that would complement many of these auxotrophs were isolated by transduction with a P2 cosmid gene bank of R. meliloti 104A14. These plasmids were then introduced into mutants of the same and different classes and the growth and symbiotic phenotypes of the new strains were determined. In all cases, complementation of the nutritional defect restored symbiotic nitrogen fixation.     

Symbiotic and competitive properties of motility mutants of Rhizobium trifolii TA1

Non-motile mutants of Rhizobium trifolii defective in either flagellar synthesis or function were isolated by transposon Tn5 mutagenesis. they were indistinguishable from motile control strains in growth in both laboratory media and in the rhizosphere of clover roots. When each non-motile mutant was grown together with a motile strain in continuous culture, the numbers of motile and non-motile organisms remained in constant proportion, implying that their growth rates were essentially identical. When inoculated separately onto clover roots, the mutants and wildtype did not differ significantly in the number of nodules produced or in nitrogen fixing activity. However, when mixtures of equal numbers of mutant and wild-type cells were inoculated onto clover roots, the motile strain formed approximately five times more nodules than the flagellate or non-flagellate, non-motile mutants, suggesting that motility is a factor in competition for nodule formation.     

Transfer of the Rhizobium leguminosarum biovar trifolii symbiotic plasmid pRtr5a to a strain of Rhizobium sp. that nodulates on Hedysarum coronarium

The Rhizobium leguminosarum biovar trifolii symbiotic plasmid pRtr5a was transferred to the Rhizobium sp. (Hedysarum coronarium) strain RB16. Transconjugants carrying pRtr5a ineffectively nodulated Trifolium repens, T. pratense and T. alexandrinum and were unable to nodulate H. coronarium plants. Agarose gel electrophoresis of transconjugants showed that all had lost an indigenous plasmid (230 Md). These results suggest that this plasmid harbours the symbiotic determinants for nodulation on H. coronarium.     

Symbiotic properties of C4-dicarboxylic acid transport mutants of Rhizobium leguminosarum.

The transport of succinate was studied in bacteroids of an effective, streptomycin-resistant strain (GF160) of Rhizobium leguminosarum. High levels of succinate transport occurred, and the kinetics, specificity, and sensitivity to metabolic inhibitors were similar to those previously described for free-living cells. The symbiotic properties of two transposon (Tn5)-mediated C4-dicarboxylate transport mutants (strains GF31 and GF252) were determined. Strain GF31 formed ineffective nodules, and bacteroids from these nodules showed no succinate transport activity. Strain GF252 formed partially effective nodules, and bacteroids from these nodules showed about 50% of the succinate transport activity of the parent bacteroids. Another dicarboxylic acid transport mutant (Dct-), strain GFS5, isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, formed ineffective nodules. The ability to form ineffective nodules in strains GF31 and GFS5 was shown to correlate with the Dct- phenotype. The data indicate that the presence of a functional C4-dicarboxylic acid transport system is essential for N2 fixation to occur in pea nodules.     

Symbiotic properties of adenine requiring mutants of Rhizobium meliloti strain L5-30

From the effective and prototrophic Rhizobium meliloti strain L5-30 two auxotrophic mutants were isolated: RM4 and RM221. These two mutants required adenine and adenine with thiamine for their growth, respectively. Both mutants nodulated lucerne plants ineffectively. Electron microscopic observations of the nodule tissue showed that its cells were not occupied by bacteria. Prototrophic revertants and transductants of these mutants showed high symbiotic effectiveness. It is assumed that adenine or adenine and thiamine requirements made impossible release of bacteria from the infection thread.     

Genetic analysis of nitrogen fixation in a tropical fast-growing Rhizobium

The Rhizobium strain ORS571, which is associated with the tropical legume Sesbania rostrata, has the property of growing in the free-living state at the expense of ammonia or N₂ as sole nitrogen source. Five mutants, isolated as unable to form colonies on plates under conditions of nitrogen fixation, were studied. All of them, which appear as Fix^- in planta, are nif mutants. With mutant 5740, nitrogenase activity of the crude extract was restored by addition of pure Mo-Fe protein of Klebsiella pneumoniae. A 13-kb BamHI DNA fragment from the wild-type strain, which hybridized with a probe carrying the nifHDK genes of K. pneumoniae, was cloned in vector pRK290 to yield plasmid pRS1. The extent of homology between the probe and the BamHI fragment was estimated at 4 kb and hybridization with K. pneumoniae nifH, nifK, and possibly nifD was detected. The pRS1 plasmid was introduced into the sesbania rhizobium nif mutants. Genetic complementation was observed with strain 5740(pRS1) both in the free-living state and in planta. It thus appears that biochemistry and genetics of nitrogen fixation in this particular Rhizobium strain can be performed with bacteria grown under non-symbiotic conditions.     

Rhizobium etli USDA9032 Engineered To Produce a Phenazine Antibiotic Inhibits the Growth of Fungal Pathogens but Is Impaired in Symbiotic Performance

Phenazine production was engineered in Rhizobium etli USDA9032 by the introduction of the phz locus of Pseudomonas chlororaphis O6. Phenazine-producing R. etli was able to inhibit the growth of Botrytis cinerea and Fusarium oxysporum in vitro. Black bean inoculated with phenazine-producing R. etli produced brownish Fix⁻ nodules.