Meliloti L5-30 Research Articles

Soil factors exhibit greater influence than bacterial inoculation on alfalfa growth and nitrogen fixation

In order to study the effects of soil factors and bacterial inoculation on alfalfa (Medicago sativa), plants were inoculated with Ensifer meliloti L33 and Azospirillum brasilense Sp7 in pot experiments using two different soils separately as well as in a mixture. One soil was contaminated with chemical waste products; the other was an arable soil. Soil factors, including the availability of macro- and micronutrients as well as carbon and nitrogen contents, were found to exhibit a much greater influence on the growth of alfalfa than any of the inoculations. In contaminated soil, the shoot and root growth of alfalfa was decreased and nodules were diminished and ineffective. Bacterial inoculations did not significantly improve this hostile growth environment. However, in a mixture (44% arable, 22% contaminated soil, 34% vermiculite), growth conditions for alfalfa were improved so that shoot dry weight and nodule numbers increased up to 100- and 20-fold, respectively, compared with the contaminated soil. For the strain L33, its persistence in the rhizosphere was correlated to the presence of its host plant, but its dynamics were influenced by competition with indigenous rhizobia. The strain Sp7, once provided with a suitable soil, was not dependent on the plant's rhizosphere, but it enhanced the performance of L33 and native rhizobia.

Structural, Functional and Calorimetric Investigation of MosA, a Dihydrodipicolinate Synthase from Sinorhizobium meliloti L5–30, does not Support Involvement in Rhizopine Biosynthesis

MosA is an enzyme from Sinorhizobium meliloti L5-30, a beneficial soil bacterium that forms a symbiotic relationship with leguminous plants. MosA was proposed to catalyze the conversion of scyllo-inosamine to 3-O-methyl-scyllo-inosamine (compounds known as rhizopines), despite the MosA sequence showing a strong resemblance to dihydrodipicolinate synthase (DHDPS) sequences rather than to methyltransferases. Our laboratory has already shown that MosA is an efficient catalyst of the DHDPS reaction. Here we report the structure of MosA, solved to 1.95 A resolution, which resembles previously reported DHDPS structures. In this structure Lys161 forms a Schiff base adduct with pyruvate, consistent with the DHDPS mechanism. We have synthesized both known rhizopines and investigated their ability to interact with MosA in the presence and absence of methyl donors. No MosA-catalyzed methyltransferase activity is observed in the presence of scyllo-inosamine and S-adenosylmethionine (SAM). 2-Oxobutyrate can form a Schiff base with MosA, acting as a competitive inhibitor of MosA-catalyzed dihydrodipicolinate synthesis. It can be trapped on the enzyme by reaction with sodium borohydride, but does not act as a methyl donor. The presence of rhizopines does not affect the kinetics of dihydrodipicolinate synthesis. Isothermal titration calorimetry (ITC) shows no apparent interaction of MosA with rhizopines and SAM. Similar experiments with pyruvate as titrant demonstrate that the reversible Schiff base formation is largely entropically driven. This is the first use of ITC to study Schiff base formation between an enzyme and its substrate.

Crystallization, preliminary X-ray diffraction and structure solution of MosA, a dihydrodipicolinate synthase from Sinorhizobium meliloti L5-30.

The structure of MosA, a dihydrodipicolinate synthase and reported methyltransferase from Sinorhizobium meliloti, has been solved using molecular replacement with Escherichia coli dihydrodipicolinate synthase as the model. A crystal grown in the presence of pyruvate diffracted X-rays to 2.3 A resolution using synchrotron radiation and belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 69.14, b = 138.87, c = 124.13 A.

MosA, a Protein Implicated in Rhizopine Biosynthesis in Sinorhizobium meliloti L5-30, is a Dihydrodipicolinate Synthase

MosA is a gene product encoded on a pSym megaplasmid of Sinorhizobium meliloti L5-30. The gene is part of an operon reported to be essential for the synthesis of the rhizopine 3- O-methyl- scyllo-inosamine. MosA has been assigned the function of an O-methyltransferase. However, the reported sequence of this protein is very much like that of dihydrodipicolinate synthase (DHDPS), except for a 40 amino acid residue C-terminal domain. This similarity contradicts accepted ideas regarding structure–function relationships of enzymes. We have cloned and overexpressed the recombinant gene in Escherichia coli, and discovered that the reported sequence contains an error resulting in a frame-shift. The correct sequence contains a new stop codon, truncating the C-terminal 41 amino acid residues of the reported sequence. The expressed protein, bearing an N-terminal polyhistidine tag, catalyzes the condensation of pyruvate and aspartate β-semialdehyde efficiently, suggesting that this activity is not a side-reaction, but an activity for which this enzyme has evolved. Electro-spray mass spectrometry experiments and inhibition by l-lysine are consistent with the enzyme being a DHDPS. E. coli AT997, a mutant host normally requiring exogenous diaminopimelate for growth, could be complemented by transformation with a plasmid bearing the gene encoding MosA. A role for this enzyme in rhizopine synthesis cannot be ruled out, but is called into question.

Resilience of a soil-established, genetically modified Sinorhizobium meliloti inoculant to soil management practices

Microcosm studies were conducted in the greenhouse to evaluate management strategies for eliminating a genetically modified bacterial inoculant from soil. Sinorhizobium meliloti L33, genetically tagged by the chromosomal insertion of a luciferase ( luc) gene, was inoculated into soil seeded with its leguminous host plant, alfalfa ( Medicago sativa). Within 10 weeks the titer decreased from 10 6 cfu g −1 soil to approximately 10 4 cfu g −1 soil, a level which was maintained without significant deviation until the end of the experiment after 83 weeks. At the point of equilibration, six different treatments were initiated: crop rotation to clover and rye as non-host plants, inoculation with two competitor strains—the non-modified parental strain S. meliloti 2011 or with S. meliloti F16, a strain previously isolated from the experimental soil—and the application of two pesticides, intending to eliminate either the host plant by Roundup ® or the rhizobia by exploiting their heavy metal sensitivity towards copper. The efficiency of these treatments was studied and compared by monitoring the titer of S. meliloti L33 in bulk soil, rhizospheres and in alfalfa root nodules. In bulk soil, only the application of Roundup ® resulted in a significant reduction of S. meliloti L33 to 4.5×10 1 cfu g −1 soil while all other treatments had no significant effect. However, in rhizospheres of clover and rye the concentrations of S. meliloti were approximately 300-fold lower than those found in the rhizosphere of alfalfa after 57 weeks of growth. Additionally, analysis of the alfalfa nodule occupancy revealed a partial replacement of S. meliloti L33 by the strain F16, indicating that cell numbers of the inoculants were not decisive but their competitiveness was. Thus, crop rotation and inoculation with a superior competitor affected S. meliloti L33 on different levels, but only the removal of the host plant by application of a herbicide was an efficient means to reduce the strain in soil.

Effect of field inoculation with Sinorhizobium meliloti L33 on the composition of bacterial communities in rhizospheres of a target plant (Medicago sativa) and a non-target plant (Chenopodium album)-linking of 16S rRNA gene-based single-strand conformation polymorphism community profiles to the diversity of cultivated bacteria.

Fourteen weeks after field release of luciferase gene-tagged Sinorhizobium meliloti L33 in field plots seeded with Medicago sativa, we found that the inoculant also occurred in bulk soil from noninoculated control plots. In rhizospheres of M. sativa plants, S. meliloti L33 could be detected in noninoculated plots 12 weeks after inoculation, indicating that growth in the rhizosphere preceded spread into bulk soil. To determine whether inoculation affected bacterial diversity, 1,119 bacteria were isolated from the rhizospheres of M. sativa and Chenopodium album, which was the dominant weed in the field plots. Amplified ribosomal DNA restriction analysis (ARDRA) revealed plant-specific fragment size frequencies. Dominant ARDRA groups were identified by 16S rRNA gene nucleotide sequencing. Database comparisons indicated that the rhizospheres contained members of the Proteobacteria (alpha, beta, and gamma subgroups), members of the Cytophaga-Flavobacterium group, and gram-positive bacteria with high G+C DNA contents. The levels of many groups were affected by the plant species and, in the case of M. sativa, by inoculation. The most abundant isolates were related to Variovorax sp., Arthrobacter ramosus, and Acinetobacter calcoaceticus. In the rhizosphere of M. sativa, inoculation reduced the numbers of cells of A. calcoaceticus and members of the genus Pseudomonas and increased the number of rhizobia. Cultivation-independent PCR-single-strand conformation polymorphism (SSCP) profiles of a 16S rRNA gene region confirmed the existence of plant-specific rhizosphere communities and the effect of the inoculant. All dominant ARDRA groups except Variovorax species could be detected. On the other hand, the SSCP profiles revealed products which could not be assigned to the dominant cultured isolates, indicating that the bacterial diversity was greater than the diversity suggested by cultivation.

Variation of Microbial Rhizosphere Communities in Response to Crop Species, Soil Origin, and Inoculation with Sinorhizobium meliloti L33.

A greenhouse study with soil-plant microcosms was conducted in order to compare the effect of crop species, soil origin, and a bacterial inoculant on the establishment of microbial communities colonizing plant roots. Two crop species, alfalfa (Medicago sativa) and rye (Secale cereale), were grown separately in two soils collected from agricultural fields at different locations and with differing histories of leguminous crop rotation. A subset of microcosms was inoculated at 10(6) cfu g(-1) soil with the luciferase marker gene-tagged Sinorhizobium meliloti strain L33, a symbiotic partner of M. sativa. Microbial consortia were collected from the rhizospheres of alfalfa after 10 weeks of incubation and from rye after 11 weeks. S. meliloti L33 populations were one to two orders of magnitude higher in the rhizospheres of alfalfa than of rye. In soil with previous alfalfa cultivation, 80% of the alfalfa nodules were colonized by indigenous bacteria, while in the other soil alfalfa was colonized almost exclusively (>90%) with S. meliloti L33. Three community-level targeting approaches were used to characterize the variation of the extracted microbial rhizosphere consortia: (1) Community level physiological profiles (CLPP), (2) fatty acid methyl ester analysis (FAME), and (3) diversity of PCR amplified 16S rRNA target sequences from directly extracted ribosomes, determined by temperature gradient gel electrophoresis (TGGE). All approaches identified the crop species as the major determinant of microbial community characteristics. Consistently, the influence of soil was of minor importance, while a modification of the alfalfa-associated microbial community structure after inoculation with S. meliloti L33 was only consistently observed by using TGGE.

Fate of plasmid-bearing, luciferase marker gene tagged bacteria after feeding to the soil microarthropod Onychiurus fimatus (Collembola)

In order to study the potential impact of the soil microarthropod Onychiurus fimatus (Collembola) on the microbial community, we analysed the fate of luciferase marker gene tagged bacterial strains fed to young adult specimens in petri dish microcosm experiments. In faeces collected from O. fimatus, Escherichia coli S17-1/pRP4luc and Sinorhizobium meliloti L33 were only detectable for 2 days after feeding whereas strain HR2/pRP4luc, a close relative of Stenotrophomonas maltophilia, isolated from another collembolan species, could be detected for 16 days. The amount of shed cells of strain HR2 increased during the frequent releases of the cast-off skins (exuvia). In order to analyse whether gut associated bacteria could serve as recipients for mobile genetic elements, plasmid-bearing E. coli donor strains were incubated with faeces in filter mating-like experiments and, in other experiments, directly fed to O. fimatus specimens. Transconjugants were obtained with both the conjugative self-transferable broad host range plasmid pRP4luc and the mobilisable (Mob +) broad host range plasmid pSUP104luc, the latter, however, only with a mobilising donor strain. No transfer was detected with the narrow host range plasmids pSUP202luc (Mob +), pUC18luc (Mob −), or with the broad host range transposon delivery plasmid pUTluxCDABE (Mob +). Transconjugants of pRP4luc were detected within one day of the beginning of a feeding experiment and then throughout the incubation period of two weeks, with gaps of no detection after 5, 12 and 14 days, probably caused by moulting. The results of this study indicate that feeding activities of collembola can modify the structure of soil-inhabiting microbial communities and enhance the spread of plasmids from non-indigenous to indigenous soil bacteria.

Fate of plasmid-bearing, luciferase marker gene tagged bacteria after feeding to the soil microarthropod Onychiurus fimatus (Collembola).

In order to study the potential impact of the soil microarthropod Onychiurus fimatus (Collembola) on the microbial community, we analysed the fate of luciferase marker gene tagged bacterial strains fed to young adult specimens in petri dish microcosm experiments. In faeces collected from O. fimatus, Escherichia coli S17-1/pRP4luc and Sinorhizobium meliloti L33 were only detectable for 2 days after feeding whereas strain HR2/pRP4luc, a close relative of Stenotrophomonas maltophilia, isolated from another collembolan species, could be detected for 16 days. The amount of shed cells of strain HR2 increased during the frequent releases of the cast-off skins (exuvia). In order to analyse whether gut associated bacteria could serve as recipients for mobile genetic elements, plasmid-bearing E. coli donor strains were incubated with faeces in filter mating-like experiments and, in other experiments, directly fed to O. fimatus specimens. Transconjugants were obtained with both the conjugative self-transferable broad host range plasmid pRP4luc and the mobilisable (Mob(+)) broad host range plasmid pSUP104luc, the latter, however, only with a mobilising donor strain. No transfer was detected with the narrow host range plasmids pSUP202luc (Mob(+)), pUC18luc (Mob(-)), or with the broad host range transposon delivery plasmid pUTluxCDABE (Mob(+)). Transconjugants of pRP4luc were detected within one day of the beginning of a feeding experiment and then throughout the incubation period of two weeks, with gaps of no detection after 5, 12 and 14 days, probably caused by moulting. The results of this study indicate that feeding activities of collembola can modify the structure of soil-inhabiting microbial communities and enhance the spread of plasmids from non-indigenous to indigenous soil bacteria.

The gut of the soil microarthropod Folsomia candida (Collembola) is a frequently changeable but selective habitat and a vector for microorganisms.

Interaction potentials between soil microarthropods and microorganisms were investigated with Folsomia candida (Insecta, Collembola) in microcosm laboratory experiments. Microscopic analysis revealed that the volumes of the simple, rod-shaped guts of adult specimens varied with their feeding activity, from 0.7 to 11.2 nl. A dense layer of bacterial cells, associated with the peritrophic membrane, was detected in the midgut by scanning electron microscopy. Depending on the molting stage, which occurred at intervals of approximately 4 days, numbers of heterotrophic, aerobic gut bacteria changed from 4.9 x 10(2) to 2.3 x 10(6) CFU per specimen. A total of 11 different taxonomic bacterial groups and the filamentous fungus Acremonium charticola were isolated from the guts of five F. candida specimens. The most abundant isolate was related to Erwinia amylovora (96.2% DNA sequence similarity to its 16S rRNA gene). F. candida preferred to feed on Pseudomonas putida and three indigenous gut isolates rather than eight different type culture strains. When luciferase reporter gene-tagged bacterial strains were pulse fed to F. candida, gut isolates were continuously shed for 8 days to several weeks but Escherichia coli HB101 was shed for only 1 day. Ratios of ingested to released bacterial cells demonstrated that populations of nonindigenous gut bacteria like Sinorhizobium meliloti L33 and E. coli HB101 were reduced by more than 4 orders of magnitude but that the population of gut isolate Alcaligenes faecalis HR4 was reduced only 500-fold. This work demonstrates that F. candida represents a frequently changeable but selective habitat for bacteria in terrestrial environments and that microarthropods have to be considered factors that modify soil microbial communities.

Ecological pre-release risk assessment of two genetically engineered, bioluminescent Rhizobium meliloti strains in soil column model systems

In order to identify potential ecological risks associated with the environmental release of two Rhizobium meliloti strains, genetically engineered with the firefly-derived luciferase gene (luc), a pre-release greenhouse investigation was conducted. The upper 4 cm of soil columns (30 cm diameter; 65 cm depth), which were filled according to the horizons of an agricultural field (loamy sand), were inoculated with seeds of Medicago sativa (alfalfa) and R. meliloti cells at approximately 5×106 cells·g–1 soil. Four treatments were tested: inoculation with a non-engineered wild type strain (2011), strain L33 (luc +), strain L1(luc +, recA–) and non-inoculated controls. The fate of the engineered strains was followed by two methods: (1) selective cultivation and subsequent detection of bioluminescent colonies and (2) PCR detection of the luc gene in DNA, directly extracted from soil. Strain R. meliloti L33 declined to 9.0×104 cfu·g–1 soil within 24 weeks and to 2.8×103 cfu·g–1 soil within 85 weeks in the upper 25 cm of the soil columns. Decline rates for R. meliloti L1 were not significantly different. Vertical distribution analysis of the recombinant cells after 37 weeks revealed that in three of four columns tested, the majority of cells (>98%) remained above 10 cm soil depth and no recombinant cells occurred below 20 cm depth. However, in one column all horizons below 20 cm were colonized with 2.2×104 to 6.8×104 cfu g–1 soil. Ecological monitoring parameters included organic substance, total nitrogen, ammonium and nitrate, microbial biomass, culturable bacteria on four different growth media and the immediate utilization of 95 carbon sources (BiologGN) by soil-extracted microbial consortia. None of the parameters was specifically affected by the genetically engineered cells.

An Experimental Test of the Rhizopine Concept in Rhizobium meliloti

In some Rhizobium-legume symbioses, compounds known as rhizopines are synthesized by bacteroids and subsequently catabolized by free-living cells of the producing strain. It has been suggested than rhizopines act as proprietary growth substrates and enhance the competitive ability of the producing strain in its interactions with the diverse microbial community found within the rhizosphere. Wild-type, rhizopine-producing Rhizobium meliloti L5-30 and mutant L5-30 strains deficient for either rhizopine synthesis or catabolism were inoculated onto lucerne host plants in competition experiments. These experiments demonstrated that no apparent advantage resulted from the ability to synthesize a rhizopine, whereas the ability to catabolize rhizopine provided a clear advantage when an organism was in competition with a strain without this ability. The results suggest that when an organism is in competition with a catabolism-deficient mutant, the ability to catabolize rhizopine results in enhanced rates of nodulation. The results of the experiments were not consistent with the hypothesis that the sole role of rhizopines is to act as proprietary growth substrates for the free-living population of the producing strain.

Rhizobium meliloti lacking mosA synthesizes the rhizopine scyllo-inosamine in place of 3-O-methyl-scyllo-inosamine.

The Rhizobium meliloti Rm220-3 mos locus producing the rhizopine scyllo-inosamine (SI) in nodules is shown to consist of three ORFs (ORF1, mosB and mosC) arranged in an operon structure. This differs from the R. meliloti L5-30 mos locus, which produces 3-O-methyl-scyllo-inosamine (3-O-MSI), by the complete absence of mosA. The deletion covers a region of 1235 nt and includes the entire mosA gene as well as the sequence both upstream and downstream. As a result, Rm220-3 mos ORF1 shares a 5' region common with L5-30 ORF1 but includes an additional 10 bp insertion and a section in the L5-30 mosA and mosB intercistronic region. Antibodies against L5-30 Mos proteins detected MosB and MosC proteins in Rm220-3-induced nodules but no translation product for either ORF1 or mosA was detected. A construct prepared from the L5-30 mos locus which has a truncated mosA gene produces SI rather than 3-O-MSI, confirming this gene is involved in a methylation step in the production of 3-O-MSI. Further, changes made to this ORF result in reduced levels of the rhizopine.

Molecular and genetic characterization of the rhizopine catabolism (mocABRC) genes of Rhizobium meliloti L5-30.

Rhizopine (L-3-O-methyl-scyllo-inosamine, 3-O-MSI) is a symbiosis-specific compound, which is synthesized in nitrogen-fixing nodules of Medicago sativa induced by Rhizobium meliloti strain L5-30. 3-O-MSI is thought to function as an unusual growth substrate for R. meliloti L5-30, which carries a locus (mos) responsible for its synthesis closely linked to a locus (moc) responsible for its degradation. Here, the essential moc genes were delimited by Tn5 mutagenesis and shown to be organized into two regions, separated by 3 kb of DNA. The DNA sequence of a 9-kb fragment spanning the two moc regions was determined, and four genes were identified that play an essential role in rhizopine catabolism (mocABC and mocR). The analysis of the DNA sequence and the amino acid sequence of the deduced protein products revealed that MocA resembles NADH-dependent dehydrogenases. MocB exhibits characteristic features of periplasmic-binding proteins that are components of high-affinity transport systems. MocC does not share significant homology with any protein in the database. MocR shows homology with the GntR class of bacterial regulator proteins. These results suggest that the mocABC genes are involved in the uptake and subsequent degradation of rhizopine, whereas mocR is likely to play a regulatory role.

Characterization of genes for synthesis and catabolism of a new rhizopine induced in nodules by Rhizobium meliloti Rm220-3: extension of the rhizopine concept

Rhizopines are selective growth substrates synthesized in nodules only by strains of rhizobia capable of their catabolism. We report the isolation and study of genes for the synthesis and catabolism of a new rhizopine, scyllo-inosamine (sIa), from alfalfa nodules induced by Rhizobium meliloti Rm220-3. This compound is similar in structure to the previously described rhizopine 3-O-methyl-scyllo-inosamine from R. meliloti L5-30 (P.J. Murphy, N. Heycke, Z. Banfalvi, M.E. Tate, F.J. de Bruijn, A. Kondorosi, J. Tempé, and J. Schell, Proc. Natl. Acad. Sci. USA 84:493-497, 1987). The synthesis (mos) and catabolism (moc) genes for the Rm220-3 rhizopine are closely linked and located on the nod-nif Sym plasmid. The mos genes are directly controlled by the NifA/NtrA regulatory system. A comparison of the sequence of the 5' regions of the two mos loci shows very extensive conservation of sequence as well as strong homology to the nifH coding region. Restriction mapping and hybridization to DNA from the four open reading frames (ORFs) of the L5-30 mos locus indicate the absence of mosA and presence of the other three ORFs (ORF1 and mosB and -C) in Rm220-3. We suggest that the L5-30 mosA gene product is involved in the conversion of scyllo-inosamine to 3-O-methyl-scyllo-inosamine. Restriction fragment length polymorphism analysis of the moc regions of both strains shows that they are very similar. Regulation studies indicate that the moc region is not controlled by the common regulatory gene nifA, ntrA, and ntrC. We discuss the striking similarities in gene structure, location, and regulation between these two rhizopine loci in relation to the rhizopine concept.

Induction of C4-dicarboxylate transport genes by external stimuli in Rhizobium meliloti.

We have characterized two mutants of Rhizobium meliloti L5-30 obtained by random mutagenesis using Mu-lacZ that were defective in transport of C4-dicarboxylic acids. These mutants induced ineffective nodules on alfalfa. Mutations in the two strains appeared to be located in a dctA gene. Levels of dctA gene expression were determined under different environmental conditions using dctA-lacZ fusions, and beta-galactosidase activities increased in response to osmotic stress and also when the cells were incubated in medium low in calcium. The transcriptional induction of the dctA gene by environmental signals was decreased by DNA gyrase inhibitors such as novobiocin and coumermycin.

Early recognition in the Rhizobium meliloti-alfalfa symbiosis: root exudate factor stimulates root adsorption of homologous rhizobia

Adsorption of Rhizobium meliloti to alfalfa roots before their infection and nodule formation shows the specificity of the symbiotic association (G. Caetano-Anollés and G. Favelukes, Appl. Environ. Microbiol. 52:377-382, 1986). The time course of specific adsorption of R. meliloti (10(3) to 10(4) cells per ml) to roots shows an initial lag period of 3 h, suggesting that either or both symbionts must become conditioned for the adsorption process. Preincubation of R. meliloti L5-30 for 3 h with dialyzed alfalfa root exudate (RE) markedly increased early adsorption of rhizobia to alfalfa roots. The activity in RE was linked to a nondialyzable, thermolabile, trypsin-sensitive factor(s), very different from the root-exuded flavonoid compounds also involved in early Rhizobium-legume interactions. The lack of activity in the RE from plants grown in 5 mM NO3- suggested its negative regulation by the nitrogen nutritional status of the plant. Preincubation of R. meliloti with heterologous clover RE did not stimulate adsorption of rhizobial cells to roots. A short pretreatment of RE with homologous (but not heterologous) strains eliminated the stimulatory activity from solution. The stimulation of adsorption of R. meliloti to alfalfa roots was strongly dependent on the growth phase of the rhizobia, being greater at the late exponential stage. Nevertheless, the capacity of R. meliloti L5-30 to eliminate from solution the stimulatory activity in RE appeared to be constitutive in the rhizobia. The low concentration of rhizobial cells used in these experiments was critical to detect the stimulation of adsorption. The early interaction of spontaneously released alfalfa root macromolecular factor(s) and free-living R. meliloti, which shows the specificity and regulatory properties characteristic of infection and nodulation, would be an initial recognition event in the rhizosphere which triggers the process of symbiotic association.

Adsorption ofRhizobium meliloti to alfalfa roots: Dependence on divalent cations and pH

Adsorption ofRhizobium meliloti L5-30 in low numbers to alfalfa (Medicago sativa L.) roots was dependent on the presence of divalent cations, and required neutral pH. Adsorption was proportional to Ca and/or Mg concentrations up to 1.5 mM. Ca was not substituted by Sr, Ba or Mn. Adsorption was abolished and viability decreased at pH≤6. When lowering pH, higher Ca concentrations were required to attain similar adsorption levels, indicating a marked interactive effect between Ca and H ions. Pretreatment of the roots with Ca and low pH did not affect subsequent adsorption of the bacteria. However, Ca pretreatment ofR. meliloti sustained further adsorption at low Ca levels and low pH substantially affected their ability to adsorb. Low pH appears to affect the stability of binding causing desorption of the previously bound bacteria. The presence of saturating concentrations of heterologousR. leguminosarum bv.trifolii A118, did not prevent the expression of divalent cations and pH requirements, as well as their interaction. Our results suggest that rhizobial binding to the root surface already shows the Ca and pH dependence of alfalfa nodulation, which was generally associated to some event prior to rhizobial penetration of root hairs.

Role of Motility and Chemotaxis in Efficiency of Nodulation by Rhizobium meliloti

Spontaneous mutants of Rhizobium meliloti L5-30 defective in motility or chemotaxis were isolated and compared against the parent with respect to symbiotic competence. Each of the mutants was able to generate normal nodules on the host plant alfalfa (Medicago sativa), but had slightly delayed nodule formation, diminished nodulation in the initially susceptible region of the host root, and relatively low representation in nodules following co-inoculation with equal numbers of the parent. When inoculated in growth pouches with increasing dosages of the parental strain, the number of nodules formed in the initially susceptible region of the root increased sigmoidally, with an optimum concentration of about 10(5) to 10(6) bacteria/plant. The dose-response behavior of the nonmotile and nonchemotactic mutants was similar, but they required 10- to 30-fold higher concentrations of bacteria to generate the same number of nodules. The distribution frequencies of nodules at different positions along the primary root were very similar for the mutants and parent, indicating that reduced nodulation by the mutants in dose-response experiments probably reflects reduced efficiency of nodule initiation rather than developmentally delayed nodule initiation. The number of bacteria that firmly adsorbed to the host root surface during several hours of incubation was 5- to 20-fold greater for the parent than the mutants. The mutants were also somewhat less effective than their parent as competitors in root adsorption assays. It appears that motility and chemotaxis are quantitatively important traits that facilitate the initial contact and adsorption of symbiotic rhizobia to the host root surface, increase the efficiency of nodule initiation, and increase the rate of infection development.

Transport and catabolism of D-mannose in Rhizobium meliloti.

Rhizobium meliloti L5-30 grows on D-mannose as the sole carbon source. The catabolic pathway of D-mannose was characterized. The following activities were present: mannose transport system, mannokinase, and mannosephosphate isomerase. Several mannose-negative mutants were selected; they were classified into three functional groups: group I, mannokinase and mannosephosphate isomerase defective: group II, mannokinase defective; and group III, mannosephosphate isomerase defective. Mannose uptake was an active process, since it was inhibited by azide, dinitrophenol, and cyanide, but not by fluoride or arsenate. Growth on succinate repressed mannose uptake activity. The mannose transport system was present in all the mutants. Uptake studies showed that mannose-negative mutants did not metabolize this sugar.