Genetic Analysis of Rhizobium leguminosarum bv. Phaseoli Mutants Defective in Nodulation and Nodulation Suppression

Nodulation-defective rhizobia and their nodule-forming derivatives containing cloned DNA from the wild type were used to study nodulation suppression in Phaseolus vulgaris L. Non-nitrogen-fixing derivatives which formed rhizobia-containing white nodules induced partial suppression. Comparison of this with the complete suppression by Fix⁺ derivatives and a Fix^- mutant which formed rhizobia-containing pink nodules suggests that the extent of suppression may be related to successive stages of nodule development.     

Rhizobium meliloti nodD genes mediate host-specific activation of nodABC.

To differentiate among the roles of the three nodD genes of Rhizobium meliloti 1021, we studied the activation of a nodC-lacZ fusion by each of the three nodD genes in response to root exudates from several R. meliloti host plants and in response to the flavone luteolin. We found (i) that the nodD1 and nodD2 products (NodD1 and NodD2) responded differently to root exudates from a variety of hosts, (ii) that NodD1 but not NodD2 responded to luteolin, (iii) that NodD2 functioned synergistically with NodD1 or NodD3, (iv) that NodD2 interfered with NodD1-mediated activation of nodC-lacZ in response to luteolin, and (v) that a region adjacent to and upstream of nodD2 was required for NodD2-mediated activation of nodC-lacZ. We also studied the ability of each of the three R. meliloti nodD genes to complement nodD mutations in R. trifolii and Rhizobium sp. strain NGR234. We found (i) that nodD1 complemented an R. trifolii nodD mutation but not a Rhizobium sp. strain NGR234 nodD1 mutation and (ii) that R. meliloti nodD2 or nodD3 plus R. meliloti syrM complemented the nodD mutations in both R. trifolii and Rhizobium sp. strain NGR234. Finally, we determined the nucleotide sequence of the R. meliloti nodD2 gene and found that R. meliloti NodD1 and NodD2 are highly homologous except in the C-terminal region. Our results support the hypothesis that R. meliloti utilizes the three copies of nodD to optimize the interaction with each of its legume hosts.     

Nod factor signaling genes and their function in the early stages of Rhizobium infection

A lipochitosaccharide-based signal molecule that is secreted by Rhizobium, named Nod factor (NF), induces root nodule formation in legumes. This molecule is also essential for the establishment of bacterial infection. Genetic analyses in the legume species Lotus japonicus and Medicago truncatula have led to the identification of many components of the NF signaling cascade. At least three of these genes do not function exclusively in the Rhizobium symbiosis but are also essential for the formation of mycorrhiza, an endosymbiosis found in many higher plant species. Recent studies have advanced our understanding of the functions of NF signaling genes in the Rhizobium infection process and the extent to which these genes are unique to legumes.     

The expression of the nodD and nodABC genes of Rhizobium leguminosarum is not regulated in response to combined nitrogen

Abstract Using a Rhizobium leguminosarum bv. viciae strain harboring nodD :: lacZ or nodC :: lacZ translational fusions, grown in minimal media containing different concentrations of nitrate and/or ammonium salts, lacZ expression was monitored. Based on these experiments it is shown that the induction of Rhizobium leguminosarum bv. viciae nodD and nodABC operons by the flavanone naringenin is not regulated in response to nitrate and/or ammonium salts.     

Regulation of tryptophan genes in Rhizobium leguminosarum.

Twelve tryptophan auxotrophs of Rhizobium leguminosarum were characterized biochemically. They were grown in complex and minimal media with several carbon sources, in both limiting and excess tryptophan. Missing enzyme activities allowed assignment of all mutant to the trpE, trpD, trpB, or trpA gene, confirming earlier results with the same mutants (Johnston et al., Mol. Gen. Genet. 165:323-330, 1978). In regulatory experiments, only the first enzyme of the pathway, anthranilate synthase, responded (about 15-fold) to tryptophan excess or limitation.     

Characterization and functional analysis of seven flagellin genes in Rhizobium leguminosarum bv. viciae. Characterization of R. leguminosarum flagellins

Background

Rhizobium leguminosarum bv. viciae establishes symbiotic nitrogen fixing partnerships with plant species belonging to the Tribe Vicieae, which includes the genera Vicia, Lathyrus, Pisum and Lens. Motility and chemotaxis are important in the ecology of R. leguminosarum to provide a competitive advantage during the early steps of nodulation, but the mechanisms of motility and flagellar assembly remain poorly studied. This paper addresses the role of the seven flagellin genes in producing a functional flagellum.

Results

R. leguminosarum strains 3841 and VF39SM have seven flagellin genes (flaA, flaB, flaC, flaD, flaE, flaH, and flaG), which are transcribed separately. The predicted flagellins of 3841 are highly similar or identical to the corresponding flagellins in VF39SM. flaA, flaB, flaC, and flaD are in tandem array and are located in the main flagellar gene cluster. flaH and flaG are located outside of the flagellar/motility region while flaE is plasmid-borne. Five flagellin subunits (FlaA, FlaB, FlaC, FlaE, and FlaG) are highly similar to each other, whereas FlaD and FlaH are more distantly related. All flagellins exhibit conserved amino acid residues at the N- and C-terminal ends and are variable in the central regions. Strain 3841 has 1-3 plain subpolar flagella while strain VF39SM exhibits 4-7 plain peritrichous flagella. Three flagellins (FlaA/B/C) and five flagellins (FlaA/B/C/E/G) were detected by mass spectrometry in the flagellar filaments of strains 3841 and VF39SM, respectively. Mutation of flaA resulted in non-motile VF39SM and extremely reduced motility in 3841. Individual mutations of flaB and flaC resulted in shorter flagellar filaments and consequently reduced swimming and swarming motility for both strains. Mutant VF39SM strains carrying individual mutations in flaD, flaE, flaH, and flaG were not significantly affected in motility and filament morphology. The flagellar filament and the motility of 3841 strains with mutations in flaD and flaG were not significantly affected while flaE and flaH mutants exhibited shortened filaments and reduced swimming motility.

Conclusion

The results obtained from this study demonstrate that FlaA, FlaB, and FlaC are major components of the flagellar filament while FlaD and FlaG are minor components for R. leguminosarum strains 3841 and VF39SM. We also observed differences between the two strains, wherein FlaE and FlaH appear to be minor components of the flagellar filaments in VF39SM but these flagellin subunits may play more important roles in 3841. This paper also demonstrates that the flagellins of 3841 and VF39SM are possibly glycosylated.     

Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum.

A gene library of genomic DNA from the hydrogen uptake (Hup)-positive strain 128C53 of Rhizobium leguminosarum was constructed by using the broad-host-range mobilizable cosmid vector pLAFR1. The resulting recombinant cosmids contained insert DNA averaging 21 kilobase pairs (kb) in length. Two clones from the above gene library were identified by colony hybridization with DNA sequences from plasmid pHU1 containing hup genes of Bradyhizobium japonicum. The corresponding recombinant cosmids, pAL618 and pAL704, were isolated, and a region of about 28 kb containing the sequences homologous to B. japonicum hup-specific DNA was physically mapped. Further hybridization analysis with three fragments from pHU1 (5.9-kb HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) showed that the overall arrangement of the R. leguminosarum hup-specific region closely parallels that of B. japonicum. The presence of functional hup genes within the isolated cosmid DNA was demonstrated by site-directed Tn5 mutagenesis of the 128C53 genome and analysis of the Hup phenotype of the Tn5 insertion strains in symbiosis with peas. Transposon Tn5 insertions at six different sites spanning 11 kb of pAL618 completely suppressed the hydrogenase activity of the pea bacteroids.     

Physiological Studies on Nodule Formation. The Characteristics and Inheritance of Abnormal Nodulation of Trifolium pratense by Rhizobium leguminosarum

Five to 7 per cent of plants of Trifolium repens L. and T.pratenseL. and 100 per cent of plants of T. subterraneum L. were nodulatedby Rhizobium leguminosarum but none of T. hybridum L., T. glomeratumL. or T parvifirum Ehrh. The frequency of nodulation of T. pratenseby R. leguminosarum was much increased by breeding from susceptibleplants. Such plants were not nodulated by bacteria isolatedfrom any other cross-inoculation group, but remained fully susceptibleto R. trifolii. The nodules formed by R. leguminosarum are generallyassociated with lateral roots and are ineffective.     

Competition Among Rhizobium leguminosarum Strains for Nodulation of Lentils (Lens esculenta)

Thirty-one cultures of Rhizobium leguminosarum were screened for effectiveness (C₂H₂ reduction) on lentils (Lens esculenta). Fluorescent antibodies prepared against three of the most effective strains (Hawaii 5-0, Nitragin 92A3, and Nitragin 128A12) exhibited a high degree of strain specificity; the antibodies reacted strongly with their homologous rhizobia in culture and with bacteroids in nodules. They did not cross-react with one another, and only weakly with 5 of the 47 other R. leguminosarum cultures tested. In competition studies in the growth chamber, whenever strain Nitragin 92A3 was included in the inoculum mixture, it consistently (but not always significantly, P = 0.05) occupied the majority of nodules on all four cultivars used. However, some degree of strain X cultivar interaction was apparent: Hawaii 5-0 was of equal competitiveness (P = 0.05) with Nitragin 92A3 on three of the varieties (Commercial, Tekoa, and Benewah), but inferior (P = 0.01) on the Chilean variety; Nitragin 92A3 completely dominated (P = 0.01) Nitragin 128A12 on all cultivars; and Hawaii 5-0 was of equal competitiveness (P = 0.05) to Nitragin 128A12 on the Chilean variety and more competitive (P = 0.01) on the commercial variety and less so on the other two varieties. In field experiments, Hawaii 5-0 proved of equal competitiveness (P = 0.01) with Nitragin 92A3 in one soil (an Inceptisol) and superior (P ≤ 0.05) to it in another (an Oxisol). Incidence of double-strain occupancy of nodules varied from 0 to 36% in vermiculite, depending on the strains in the mixture and the host variety, and from 0 to 38% in the field, depending on the strains in the mixture and the soil type. The results suggest a close relationship between the competitiveness of a strain and its occurrence in doubly infected nodules.     

Nodulation of Trifolium repens at low pH by single and paired strains of Rhizobium leguminosarum biovar trifolii

Detailed individual nodulation profiles were obtained for five strains of Rhizobium leguminosarum biovar trifolii inoculated onto roots of Trifolium repens seedlings growing on an agar medium of pH 4.5. The time of appearance and the location of every nodule were noted for a period of 10 days after inoculation. Using these nodulation frequency profiles, pairings of strains were identified and six mixed-strain inoculation (1:1 ratio) experiments were subsequently performed at pH 4.5. Results from the mixed-inoculum experiments showed that the performance of a Rhizobium strain in single culture could not be reliably used to predict the outcome of a paired-inoculation study and that some seedlings were exclusively nodulated by rhizobia that performed poorly at low pH in single-culture inoculations. Received: 26 November 1996 / Accepted: 18 April 1997     

Root Exudates of Various Host Plants of Rhizobium leguminosarum Contain Different Sets of Inducers of Rhizobium Nodulation Genes

Rhizobium promoters involved in the formation of root nodules on leguminous plants are activated by flavonoids in plant root exudate. A series of Rhizobium strains which all contain the inducible Rhizobium leguminosarum nodA promoter fused to the Escherichia coli lacZ gene, and which differ only in the source of the regulatory nodD gene, were recently used to show that the regulatory nodD gene determines which flavonoids are able to activate the nodA promoter (HP Spaink, CA Wijffelman, E Pees, RJH Okker, BJJ Lugtenberg 1987 Nature 328: 337-340). Since these strains therefore are able to discriminate between various flavonoids, they were used to determine whether or not plants that are nodulated by R. leguminosarum produce different inducers. After chromatographic separation of root exudate constituents from Vicia sativa L. subsp. nigra (L.), V. hirsuta (L.) S.F. Gray, Pisum sativum L. cv Rondo, and Trifolium subterraneum L., the fractions were tested with a set of strains containing a nodD gene of R. leguminosarum, R. trifolii, or Rhizobium meliloti, respectively. It appeared that the source of nodD determined whether, and to what extent, the R. leguminosarum nodA promoter was induced. Lack of induction could not be attributed to the presence of inhibitors. Most of the inducers were able to activate the nodA promoter in the presence of one particular nodD gene only. The inducers that were active in the presence of the R. leguminosarum nodD gene were different in each root exudate.     

High-efficiency transformation of Rhizobium leguminosarum by electroporation.

Electrotransformation of Rhizobium leguminosarum was successfully carried out with a 15.1-kb plasmid, pMP154 (Cmr), containing a nodABC-lacZ fusion by electroporation. The maximum transformation efficiency, 10(8) transformants/microg of DNA, was achieved at a field strength of 14 kV/cm with a pulse of 7.3 ms (186 Omega). The number of transformants was found to increase with increasing cell density, with no sign of saturation. In relation to DNA dosage, the maximum transformation efficiency (5.8 x 10(8) transformants/microg of DNA) was obtained with 0.5 microg of DNA/ml of cell suspension, and a further increase in the DNA concentration resulted in a decline in transformation efficiency.     

Nodulation protein NodL of Rhizobium leguminosarum O-acetylates lipo-oligosaccharides, chitin fragments and N-acetylglucosamine in vitro

Upon induction of their nodulation genes, the root nodule-inducing Rhizobium bacteria produce lipo-oligosaccharide signal molecules. All lipo-oligosaccharides identified from Rhizobium leguminosarum bv. viciae carry an O-acetyl group at the C-6 position of the non-reducing terminal sugar, the presence of which is important for biological activity and host specificity. Previously we showed that a functional nodL gene product is required for the presence of this O-acetyl moiety. The production of polyclonal antibodies against isolated NodL protein, using a NodL- overproducing Escherichia coli strain is described. These antibodies were used (i) to elucidate the subcellular localization of the NodL protein, which appeared to be present in the cytosol, and (ii) for the purification of native NodL protein from E. coli. Here we provide biochemical proof that purified NodL protein has transacetylating activity in vitro with acetyl-CoA as the acetyl donor. NodL protein appeared to be able to acetylate various substrates, such as lipo-oligosaccharides, chitin fragments and N-acetylglucosamine. For chitinpentaose as the substrate we have shown, using mass spectrometry and NMR spectroscopy, that NodL protein substitutes one O-acetyl group at the C-6 position of the non-reducing terminal sugar.     

Cloning of Rhizobium leguminosarum genes for competitive nodulation blocking on peas.

One type of competitive interaction among rhizobia is that between nonnodulating and nodulating strains of Rhizobium leguminosarum on primitive pea genotypes. Pisum sativum cv. Afghanistan nodulates effectively with R. leguminosarum TOM, and this can be blocked in mixed inoculations by R. leguminosarum PF2, which does not nodulate this cultivar. We termed this PF2 phenotype Cnb+, for competitive nodulation blocking. Strain PF2 contains three large plasmids including a 250-kilobase-pair symbiotic (Sym) plasmid. Transfer of this plasmid, pSymPF2, to nonblocking rhizobia conferred the Cnb+ phenotype on recipients in mixed inoculations on cultivar Afghanistan with TOM. A library of the PF2 genome constructed in the vector pMMB33 was used to isolate two cosmid clones which hybridize to pSymPF2. These cosmids, pDD50 and pDD58, overlapped to the extent of 23 kilobase pairs and conferred a Cnb+ phenotype on recipient Cnb- rhizobia, as did pSD1, a subclone from the common region.     

Hydrogenase genes are uncommon and highly conserved in Rhizobium leguminosarum bv. viciae

A screening for hydrogen uptake (hup) genes in Rhizobium leguminosarum bv. viciae isolates from different locations within Spain identified no Hup+ strains, confirming the scarcity of the Hup trait in R. leguminosarum. However, five new Hup+ strains were isolated from Ni-rich soils from Italy and Germany. The hup gene variability was studied in these strains and in six available strains isolated from North America. Sequence analysis of three regions within the hup cluster showed an unusually high conservation among strains, with only 0.5-0.6% polymorphic sites, suggesting that R. leguminosarum acquired hup genes de novo in a very recent event.