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.     

Localization and symbiotic function of a region on the Rhizobium leguminosarum Sym plasmid pRL1JI responsible for a secreted, flavonoid-inducible 50-kilodalton protein.

A previously described (R. A. de Maagd, C. A. Wijffelman, E. Pees, and B. J. J. Lugtenberg, J. Bacteriol. 170:4424-4427, 1988) Sym plasmid-dependent, naringenin-inducible 50-kilodalton protein of Rhizobium leguminosarum biovar viciae is further characterized in this paper. The protein was overproduced by constructing a strain containing multiple copies of the R. meliloti nodD gene, which facilitated its purification. An antiserum was used to screen Tn5 insertion mutants located in the pRL1JI region found to be responsible for the production of the 50-kilodalton protein. These inserts define a new nod locus left of the nod genes identified previously. Mutations in this region affect the nodulation ability in a way which is dependent on the bacterial background as well as on the host plant. The mutants nodulate normally in a strain RBL1532 (R. leguminosarum biovar viciae strain 248, cured of its Sym plasmid) background on all three tested host plant species. In contrast, in a strain RBL5045 (R. leguminosarum biovar trifolii strain RCR5, cured of its Sym plasmid) background, nodulation on Vicia sativa is severely impaired, whereas nodulation on Vicia hirsuta and Trifolium subterraneum is apparently unaltered.     

Evidence for genetic exchange and recombination of Rhizobium symbiotic plasmids in a soil population

A soil population of 16 Rhizobium leguminosarum bv. trifolii isolates was characterized by using three Sym (for symbiotic) plasmid-specific DNA hybridization probes: (i) an R. leguminosarum bv. trifolii-specific, repeated-sequence probe; (ii) a nifHDK gene probe, and (iii) a nod gene probe. A predominant Sym plasmid family was identified among the isolates. Three other unrelated Sym plasmid families were also identified. The isolates were also classified either by using a chromosomal DNA hybridization probe or by serological relatedness to 25 different R. leguminosarum bv. trifolii antisera. With either method, it was possible to group the 16 soil isolates into identical or related families. However, the correlation between the two techniques was not high. Irrespective of the means used to classify the bacterial host strain, it was possible to identify the same Sym plasmids in unrelated strains, as well as unrelated Sym plasmids in identical host strains. These data indicate that, within this soil population, there has been genetic exchange of Sym plasmids, and in one instance the hybridization pattern indicates that in vivo recombination of two different Sym plasmids may have occurred. Symbiotic effectiveness tests on red, strawberry, and subterranean clovers clearly differentiated the isolates. In general, the pattern of response was similar within groupings on the basis of Sym plasmid and chromosomal profiles but different between such groups.     

Chemotaxis of Rhizobium meliloti to the plant flavone luteolin requires functional nodulation genes.

Luteolin is a phenolic compound from plants that acts as a potent and specific inducer of nodABC gene expression in Rhizobium meliloti. We have found that R. meliloti RCR2011 exhibits positive chemotaxis towards luteolin. A maximum chemotactic response was observed at 10(-8) M. Two closely related flavonoids, naringenin and apigenin, were not chemoattractants. The presence of naringenin but not apigenin abolished chemotaxis of R. meliloti towards luteolin. A large deletion in the nif-nod region of the symbiotic megaplasmid eliminated all chemotactic response to luteolin but did not affect general chemotaxis, as indicated by swarm size on semisoft agar plates and chemotaxis towards proline in capillary tubes. Transposon Tn5 mutations in nodD, nodA, or nodC selectively abolished the chemotactic response of R. meliloti to luteolin. Agrobacterium tumefaciens GMI9050, a derivative of the C58 wild type lacking a Ti plasmid, responded chemotactically to 10(-8) M luteolin. The introduction of a 290-kilobase nif-nod-containing sequence of DNA from R. meliloti into A. tumefaciens GMI9050 enabled the recipient to respond to luteolin at concentrations peaking at 10(-6) M as well as at concentrations peaking at 10(-8) M. The response of A. tumefaciens GMI9050 to luteolin was also abolished by the presence of naringenin.     

Sym plasmid genes of Rhizobium trifolii expressed in Lignobacter and Pseudomonas strains.

A 14-kilobase (kb) fragment of Rhizobium trifolii Sym plasmid containing nodulation (nod) genes or the pSym plasmid of R. trifolii cointegrated with a broad-host-range vector R68.45 (pPN1) were transferred to Lignobacter strain K17 and Pseudomonas aeruginosa strain PAO5 by conjugation. Lignobacter transconjugants carrying Sym plasmid pPN1 formed nodules on white, red, and subterranean clover plants. Lignobacter transconjugants containing a 14-kb fragment of nod genes cloned into a multicopy plasmid nodulated only white and subterranean clover plants, whereas transconjugants carrying the same fragment cloned into a low-copy plasmid vector nodulated only white clover plants. All nodules formed by Lignobacter transconjugants showed bacterial release from the infection threads into the host cytoplasm. Pseudomonas transconjugants with plasmid pPN1 formed nodule-like structures on white clover plants. These structures were not invaded by bacteria; however, a few bacteria were found within the intercellular spaces of the outermost cells of the structures. Pseudomonas transconjugants carrying the 14-kb fragment of R. trifolii nod genes did not form nodules on tested clover plants. All clover plants inoculated with either Pseudomonas or Lignobacter transconjugants containing a 14-kb fragment of nod genes (but not entire Sym plasmid) showed the "thick-and-short-root" response when compared to the control plants inoculated with the R. trifolii wild-type strain.     

Distribution of Symbiotic Genotypes in Rhizobium leguminosarum biovar viciae Populations Isolated Directly from Soils

The distribution of symbiotic (Sym) plasmid types across background genotypes was investigated in two field populations of Rhizobium leguminosarum biovar viciae isolated directly from soils. PCR-based methods were used to characterize the background genotypes and the Sym gene types. Identical Sym gene types were associated with a variable range of background genotypes, while the same background genotype could harbor distinct Sym gene types. Random distributions of Sym gene types in the background genotypes were observed in the two soil populations. These results suggest that Sym plasmid transfer is less restricted than previously thought on the basis of the analysis of strains isolated from legume nodules.     

Jasmonic acid stimulates the expression of nod genes in Rhizobium

Jasmonates and salicylic acid are considered to be signal molecules that induce a variety of plant genes involved in wound or defence response, as well as affecting nos promoter activity. In this paper we examined whether these chemicals could also affect nod genes from isogenic rhizobia strains. Isogenic strains contain the Rhizobium leguminosarum nodA promoter fused to the lacZ gene of Escherichia coli and differ only in the source of the regulatory nodD gene. Naringenin, jasmonic acid and methyl jasmonate induced expression of nod genes in strain RBL1284 and salicylic acid showed no activity alone or when used in combination with other compounds; addition of naringenin + jasmonic acid produced a synergistic effect. Results obtained with strain RBL5284 were similar to those for RBL1284 albeit the combination of naringenin with the other compounds markedly inhibited nod gene expression. Whereas RBL5283 responded to naringenin with a strong induction, jasmonic acid, methyl jasmonate or salicylic acid showed no significant responses. The inhibitory effect of salicylic acid on nod gene expression indicates that the induction mechanism of jasmonic acid, methyl jasmonate, N-propyldihydrojasmonate and naringenin is probably different from that of salicylic acid.     

Detection and subcellular localization of two Sym plasmid-dependent proteins of Rhizobium leguminosarum biovar viciae.

The previously described Sym plasmid-dependent 24-kilodalton rhi protein of Rhizobium leguminosarum biovar viciae was localized in the cytosol fraction. Another Sym plasmid-dependent protein of 50 kilodaltons is secreted into the growth medium, and its expression is dependent on both the nodD gene and a nod gene inducer.     

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.     

A Structural Comparison of the Acidic Extracellular Polysaccharides from Rhizobium trifolii Mutants Affected in Root Hair Infection

The structures of the acidic extracellular polysaccharides (EPSs) from several R. trifolii mutants were compared by examining their compositions and their sugar linkages as determined by methylation analysis. These mutant strains were derived from the wild-type R. trifolii ANU843 and were unable to induce normal root hair curling (Hac- phenotype) or nodulation response (Nod- phenotype) in clover plants. These strains included several transposon Tn5-induced Nod-mutants, strain ANU871, which possesses a 40 to 50 kilobase deletion of the resident Sym plasmid, and strain ANU845 which is missing the Sym plasmid (pSym-). Strains ANU845(pSym-) containing either plasmid pRt150 or pBR1AN were also used. The recombinant plasmid pRt150 restores only root hair curling capacity to ANU845 while plasmid pBR1AN (an R. trifolii pSym) restores both root hair curling and nodulation capacity to this strain. Our composition and methylation results show that the EPSs from all these strains have the same glycosyl and pyruvyl linkages. Thus we suggest that neither the nod genes involved in root hair curling nor the entire pSym encodes for the arrangement of glycosyl or pyruvyl residues in these EPSs. Whether or not the nod genes dictate the location of acetyl or β-hydroxybutyrate substituent groups remains to be determined.     

Regulation of Syrm and Nodd3 in Rhizobium Meliloti

The early steps of symbiotic nodule formation by Rhizobium on plants require coordinate expression of several nod gene operons, which is accomplished by the activating protein NodD. Three different NodD proteins are encoded by Sym plasmid genes in Rhizobium meliloti, the alfalfa symbiont. NodD1 and NodD2 activate nod operons when Rhizobium is exposed to host plant inducers. The third, NodD3, is an inducer-independent activator of nod operons. We previously observed that nodD3 carried on a multicopy plasmid required another closely linked gene, syrM, for constitutive nod operon expression. Here, we show that syrM activates expression of the nodD3 gene, and that nodD3 activates expression of syrM. The two genes constitute a self-amplifying positive regulatory circuit in both cultured Rhizobium and cells within the symbiotic nodule. We find little effect of plant inducers on the circuit or on expression of nodD3 carried on pSyma. This regulatory circuit may be important for regulation of nod genes within the developing nodule.     

Specific flavonoids induced nod gene expression and pre-activated nod genes of Rhizobium leguminosarum increased pea (Pisum sativum L.) and lentil (Lens culinaris L.) nodulation in controlled growth chamber environments.

The gram-negative soil bacteria Rhizobium spp. infect and establish a nitrogen-fixing symbiosis with legume crops which involves the mutual exchange of diffusable signal molecules. In this study, Rhizobium leguminosarum containing a nod-lacZ gene fusion was used to screen the most effective plant-to-bacteria signal molecules for pea and lentil and the induction conditions. Out of a number of signal compounds including apigenin, daidzein, genistein, hesperetin, kaempferol, luteolin, naringenin, and rutin, hesperetin and naringenin were found to be the most effective plant-to-bacteria signal molecules. The induction of nod genes was temperature-dependent, where nod gene induction was decreased with dropping incubation temperature. The combination of hesperetin at 7 microM and naringenin at 3 microM resulted in better induction of nod gene activities compared to either hesperetin or naringenin alone. Nodulation and plant dry matter accumulation of pea and lentil plants receiving preinduced R. leguminosarum were higher than those of plants receiving uninduced R. leguminosarum cells in controlled environment growth chamber conditions. Preinduced Rhizobium with hesperetin at a concentration of 10 microM increased nodule number on average by 60.5% and dry matter accumulation by 14% in field pea at 17 degrees C, while it was 32% and 9% at 24 degrees C, respectively. Similarly, averaged over two rhizobial strains, a 59% and 6% increase in nodule number and biomass production at 17 degrees C, and a 39% and 27% at 24 degrees C, were obtained from lentil inoculated with hesperetin-induced R. leguminosarum, respectively.     

Melanin production encoded by a cryptic plasmid in a Rhizobium leguminosarum strain

Rhizobium leguminosarum strain VF39, isolated from nodules of field-grown faba beans in the Federal Republic of Germany, was shown to contain six plasmids ranging in molecular weight from 90 to 400 Md. Hybridisation to nif gene probes, plasmid curing, and mobilisation to other strains of Rhizobium and to Agrobacterium showed that the third largest plasmid, pRleVF39d (220 Md), carried genes for nodulation and nitrogen fixation. This plasmid was incompatible with pRL10JI, the Sym plasmid of R. leguminosarum strain JB300. Of the other plasmids, the two smallest (pRleVF39a and pRleVF39b, 90 and 160 Md respectively) were shown to be self-transmissible at a low frequency. Although melanin production is as yet unreported in strains of R. leguminosarum biovar viceae, strain VF39 produced a dark pigment, which, since it was not produced on minimal media and its production was greatly enhanced by the presence of tyrosine in the media, is probably melanin-like. Derivatives of VF39 cured of pRleVF39a no longer produced this pigment, but regained the ability to produce it when this plasmid was transferred into them. Strains of Agrobacterium tumefaciens, R. meliloti, and some strains of R. leguminosarum carrying pRleVF39a did not produce this pigment, indicating perhaps that some genes elsewhere on the VF39 genome are also involved in pigment production. Plasmid pRleVF39a appeared to be incompatible with the cryptic Rhizobium plasmids pRle336b and pRL8JI (both ca. 100 Md), but was compatible with the R. leguminosarum biovar phaseoli Sym plasmids pRP1JI, pRP2JI and pRph51a, all of which also code for melanin production. The absence of pRleVF39a in cured derivatives of VF39 had no effect on the symbiotic performance or competitive ability of this strain.     

Expression by Soil Bacteria of Nodulation Genes from Rhizobium leguminosarum biovar trifolii

Gram-negative, rod-shaped bacteria from the soil of white clover-ryegrass pastures were screened for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia and for DNA homology with genomic DNA from Rhizobium leguminosarum biovar trifolii ICMP2668 (NZP582). Of these strains, 3.2% were able to hybridize with strain ICMP2668 and nodulate white clover and approximately 19% hybridized but were unable to nodulate. Strains which nodulated but did not hybridize with strain ICMP2668 were not detected. DNA from R. leguminosarum biovar trifolii (strain PN165) cured of its symbiotic (Sym) plasmid and a specific nod probe were used to show that the relationship observed was usually due to chromosomal homology. Plasmid pPN1, a cointegrate of the broad-host-range plasmid R68.45 and a symbiotic plasmid pRtr514a, was transferred by conjugation to representative strains of nonnodulating, gram-negative, rod-shaped soil bacteria. Transconjugants which formed nodules were obtained from 6 of 18 (33%) strains whose DNA hybridized with that of PN165 and 1 of 9 (11%) strains containing DNA which did not hybridize with that of PN165. The presence and location of R68.45 and nod genes was confirmed in transconjugants from three of the strains which formed nodules. Similarly, a pLAFR1 cosmid containing nod genes from a derivative of R. leguminosarum biovar trifolii NZP514 formed nodules when transferred to soil bacteria.     

转座子Tn5-Mob对菜豆根瘤菌共生基因的诱变、转移和初步定位

转座子Tn5-Mob在质粒RP4-4配合下能诱动(Mobilization)菜豆根瘤菌RCR3622内源质粒的诱动转移。在种间根瘤菌杂交过程中,二个巨型质粒的转移频率均大于10~(-3);分子量约为285kb的psym3622是带有结瘤(nod)和产黑素(mel)基因的共生质粒(Symbiotic plasmid);这二个基因的最大距离不超过70kb左右。另一个分子量约为135kb的质粒在试验中为不具结瘤功能的隐蔽质粒。将psym3622共生质粒导入不结瘤(Nod-)的豌豆根瘤菌菌株B151,能够使后者在菜豆植物上表达结瘤的特性,形成无效根瘤。将psym3622共生质粒导入不结瘤的菜豆根瘤菌菌株JI8400,能够在菜豆植物上形成正常发育的有效根瘤。     

Identification and characterization of the nodD gene in Rhizobium leguminosarum strain 1001

A gene library of the symbiotic 240-kb plasmid of Rhizobium leguminosarum strain 1001 was constructed in pUC18. The clones showing homology with a 6.6-kb fragment containing nodEFDABC from the Sym plasmid pRLlJI were detected by colony hybridization. Additional probes from the symbiotic region of pRLlJI were used to localize the corresponding genes on the map of pRle1001a. The relative positions of nod and nif gene clusters are different than those of pRLlJI. A comparison of the amino acid sequence for NodD from pRle1001a with NodD proteins from other Rhizobium species showed a high degree of sequence conservation at the amino terminus of the protein.     

Analysis of three nodD genes in Rhizobium leguminosarum biovar phaseoli; nodD 1 is preceded by nolE, a gene whose product is secreted from the cytoplasm

In a strain of Rhizobium leguminosarum biovar phaseoli, three copies of the regulatory nodulation gene nodD were identified on the Sym plasmid and sequenced. Two were closely linked to each other and the third was near, but not adjacent, to the nodABC genes. Each of these nodD genes could correct the Nod- defect of a nodD mutant strain of R. leguminosarum biovar viciae on peas. A truncated form of nodD2 could also correct this mutant, indicating that the C-terminus of NodD2 is not needed for inducing activity. Upstream of nodD1 and in the same operon is a newly described gene, noIE, whose product appears to be exported into the periplasm. Close to nodD2 is another gene, noIP, with no known counterpart in other rhizobia. Both noIP and noIE-nodD1 are preceded by 'nod-box' sequences and, in the former case, there appear to be two tandemly repeated nod-box sequences. Mutations in each of the nodD genes and in the noIE and noIP genes did not abolish nodulation or nitrogen fixation on beans.