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Despite the importance of mutualism as a key ecological process, its persistence in nature is difficult to explain since the existence of exploitative, "cheating" partners that could erode the interaction is common. By analogy with the proposed policing strategy stabilizing intraspecific cooperation, host sanctions against non-N(2) fixing, cheating symbionts have been proposed as a force stabilizing mutualism in legume-Rhizobium symbiosis. Following this proposal, penalizations would include decreased nodular rhizobial viability and/or early nodule senescence in nodules occupied by cheating rhizobia. In this work, we analyse the stability of Rhizobium-legume symbiosis when non-fixing, cheating strains are present, using an experimental and modelling approach. We used split-root experiments with soybean plants inoculated with two rhizobial strains, a cooperative, normal N(2) fixing strain and an isogenic non-fixing, "perfect" cheating mutant derivative that lacks nitrogenase activity but has the same nodulation abilities inoculated to split-root plants. We found no experimental evidence of functioning plant host sanctions to cheater rhizobia based on nodular rhizobia viability and nodule senescence and maturity molecular markers. Based on these experiments, we developed a population dynamic model with and without the inclusion of plant host sanctions. We show that plant populations persist in spite of the presence of cheating rhizobia without the need of incorporating any sanction against the cheater populations in the model, under the realistic assumption that plants can at least get some amount of fixed N(2) from the effectively mutualistic rhizobia occupying some nodules. Inclusion of plant sanctions leads to the unrealistic effect of ultimate extinction of cheater strains in soil. Our simulation results are in agreement with increasing experimental evidence and theoretical work showing that mutualisms can persist in presence of cheating partners.

Plant-interacting micro-organisms can establish either mutualistic or pathogenic associations. Although the outcome is completely different, common molecular mechanisms that mediate communication between the interacting partners seem to be involved. Specifically, nitrogen-fixing bacterial symbionts of legume plants, collectively termed rhizobia, and phytopathogenic bacteria have adopted similar strategies and genetic traits to colonize, invade and establish a chronic infection in the plant host. Quorum-sensing signals and identical two-component regulatory systems are used by these bacteria to coordinate, in a cell density-dependent manner or in response to changing environmental conditions, the expression of important factors for host colonization and infection. The success of invasion and survival within the host also requires that rhizobia and pathogens suppress and/or overcome plant defence responses triggered after microbial recognition, a process in which surface polysaccharides, antioxidant systems, ethylene biosynthesis inhibitors and virulence genes are involved.

Rhizobium etli type strain CFN42 contains six plasmids. We analyzed the distribution of genetic markers from some of these plasmids in bean-nodulating strains belonging to different species (Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum, and Sinorhizobium fredii). Our results indicate that independent of geographic origin, R. etli strains usually share not only the pSym plasmid but also other plasmids containing symbiosis-related genes, with a similar organization. In contrast, strains belonging to other bean-nodulating species seem to have acquired only the pSym plasmid from R. etli.

Characterization of nine transposon-induced mutants of Rhizobium tropici with decreased salt tolerance (DST) allowed the identification of eight gene loci required for adaptation to high external NaCl. Most of the genes also were involved in adaptation to hyperosmotic media and were required to overcome the toxicity of LiCl. According to their possible functions, genes identified could be classified into three groups. The first group included two genes involved in regulation of gene expression, such as ntrY, the sensor element of the bacterial ntrY/ntrX two-component regulatory system involved in regulation of nitrogen metabolism, and greA, which encodes a transcription elongation factor. The second group included genes related to synthesis, assembly, or maturation of proteins, such as alaS coding for alanine-tRNA synthetase, dnaJ, which encodes a molecular chaperone, and a nifS homolog probably encoding a cysteine desulfurase involved in the maturation of Fe-S proteins. Genes related with cellular build-up and maintenance were in the third group, such as a noeJ-homolog, encoding a mannose-1-phosphate guanylyltransferase likely involved in lipopolysaccharide biosynthesis, and kup, specifying an inner-membrane protein involved in potassium uptake. Another gene was identified that had no homology to known genes but that could be conserved in other rhizobia. When inoculated on Phaseolus vulgaris growing under nonsaline conditions, all DST mutants displayed severe symbiotic defects: ntrY and noeJ mutants were impaired in nodulation, and the remaining mutants formed symbiosis with very reduced nitrogenase activity. The results suggest that bacterial ability to adapt to hyperosmotic and salt stress is important for the bacteroid nitrogen-fixing function inside the legume nodule and provide genetic evidence supporting the suggestion that rhizobia face severe environmental changes after their release into plant cells.

Two transposon Tn5-induced mutants of wild-type broad-host-range Rhizobium sp. GRH2 were isolated and found to harbour different alterations in surface polysaccharides. These mutants, designated GRH2-14 and GRH2-50, induced a few, empty nodules on Acacia and lost the ability to nodulate most host herbaceous legumes. Whereas mutant GRH2-14 produces an acidic exopolysaccharide (EPS) similar to the wild-type, the acidic EPS of mutant GRH2-50 lacks galactose and the pyruvyl and 3-hydroxybutyryl substituents attached to this sugar moiety. In addition, both mutants GRH2-50 and GRH2-14 were altered in smooth lipopolysaccharides (LPS). DNA sequence analyses of the corresponding Tn5 insertions revealed that strain GRH2-50 was mutated in a DNA locus homologous to galE, and in vitro enzyme assays indicated that the UDPglucose 4-epimerase (GalE) activity was missing in this mutant strain. DNA hybridization studies showed that the GRH2-50 mutant DNA has homologous sequences within the different biovars of Rhizobium leguminosarum. However, no DNA homology to GRH2-14 altered DNA was found in those rhizobial strains, indicating that it represents a new chromosomal lps locus in Rhizobium sp. (Acacia) involved in symbiotic development.

The nfe genes located on the large plasmid pRmeGR4b are involved in the nodulation efficiency and competitiveness of Rhizobium meliloti GR4 on alfalfa roots. One hundred twenty-eight base-pairs downstream of nfe2 gene we found an open reading frame designated ORFC, 970 bp long and potentially coding for a 320 amino acid long protein. The amino acid sequence of the putatively encoded ORFC product shows similarity with ornithine cyclodeaminase (OCD) of Agrobacterium tumefaciens an unusual enzyme that converts ornithine into proline. The gene product of ORFC was identified as a 37-kDa protein by in vitro-coupled transcription-translation and in vivo by the T7 RNA polymerase/promoter system. DNA hybridization studies showed that strain GR4 carries a single copy of the ocd-like gene. No homologous sequences to GR4 ORFC DNA were found in other R. meliloti strains or Rhizobium spp. assayed. Furthermore, a GR4 derivative mutant obtained by plasmid disruption of ORFC showed an impaired nodulation efficiency as compared to that of the wild-type strain GR4. Thus, the former locus should be considered a novel nfe gene. We propose to rename the nfe genes, nfe1, 2 and ORFC as nfeA, B, and D, respectively.

The large plasmid, pRmeGR4a, of Rhizobium meliloti GR4 is a nonsymbiotic, self-transmissible replicon that shows a high degree of stability. Plasmid replication and stabilization functions are located on a 4.8-kb PstI fragment. Analysis of the nucleotide sequence of this DNA region revealed the presence of six open reading frames (ORFs) with coding capabilities; all are transcribed in the same direction. Only one of the ORFs (ORF3) seems to be essential for replication. Its predicted protein sequence shows homology with RepC, a protein that has been suggested to be involved in the replication of Agrobacterium Ri and Ti plasmids.

Rhizobium sp. wild-type strain GRH2 was originally isolated from root nodules of the leguminous tree Acacia cyanophylla and has a broad host range which includes herbaceous legumes, e.g., Trifolium spp. We examined the extracellular exopolysaccharides (EPSs) produced by strain GRH2 and found three independent glycosidic structures: a high-molecular-weight acidic heteropolysaccharide which is very similar to the acidic EPS produced by Rhizobium leguminosarum biovar trifolii ANU843, a low-molecular-weight native heterooligosaccharide resembling a dimer of the repeat unit of the high-molecular-weight EPS, and low-molecular-weight neutral beta (1,2)-glucans. A Tn5 insertion mutant derivative of GRH2 (exo-57) that fails to form acidic heteropolysaccharides was obtained. This Exo- mutant formed nitrogen-fixing nodules on Acacia plants but infected a smaller proportion of cells in the central zone of the nodules than did wild-type GRH2. In addition, the exo-57 mutant failed to nodulate several herbaceous legume hosts that are nodulated by wild-type strain GRH2.

We have previously demonstrated that the Rhizobium meliloti large plasmid pRmeGR4b carries the gene locus nodule formation efficiency (nfe) which is responsible for nodulation efficiency and competitive ability of strain GR4 on alfalfa roots. In this study we report that expression of nfe-lacZ fusions in Escherichia coli is activated in the presence of the cloned nifA gene of R. meliloti. This activation was found to be oxygen sensitive and to require the E. coli ntrA gene product. In contrast to the R. meliloti nifA, the cloned nifA gene of Klebsiella pneumoniae was able to activate expression of nfe in aerobically grown cells of both E. coli and R. meliloti. Hybridization experiments did not show homology to nfe in four R. meliloti wild-type strains tested. These strains were uncompetitive when coinoculated with a GR4 derivative carrying plasmid pRmeGR4b, but were competitive when coinoculated with a GR4 derivative carrying a single transposon mutation into the nfe region. When nfe DNA was introduced into the four wild-type strains, a significant increase in the competitive ability of two of them was observed, as deduced from their respective percentages of alfalfa root nodule occupancy in two-strains coinoculation experiments.

A resident plasmid of wild-type strains of Rhizobium meliloti of 59.6 megadaltons has been shown to be transferred at a high frequency to "cured" strains of this bacterial species. This plasmid, named pEZ1, that confers phage-sensitivity to cells carrying it is also transmissible to Escherichia coli and from it to "cured" R. meliloti strains.

Nitrite production by nodules and roots of pea plants (Pisum sativum L., cultivar Alaska) inoculated with Rhizobium leguminosarum strain 3855 has been studied. Nitrate reductase (NR) activity and nitrite reductase (NiR) activity of the bacteroidal and cytosolic fractions of the nodules were also determined, as well as the nitrite content of the nodules cytosol. Nitrite production by nodules and roots from plants treated with 5 mM KNO3 was higher than that of nodules and roots from plants not treated with nitrate, and regardless of the nitrate treatment, nitrite production increased with the incubation period. The presence of nitrate, propanol or both compounds in the incubation mixtures significantly increased the nitrite production by nodules and roots. Nitrite reductase activity was detected in fresh by isolated bacteroids of R. leguminosarum strain 3855, although the presence of nitrate reductase activity could not be detected both in bacteroids of nodules isolated from plants treated or not with 5 mM KNO3. After isolation, when bacteroids were incubated in a mixture with nitrate, nitrate reductase activity developed after incubation for 12 h. Consequently, there was an increase in nitrite reductase activity, which resulted in the disappearance of the nitrite previously accumulated in the incubation medium. Nitrate utilization by bacteroids was not detected until 5 h from the beginning of the incubation period. Since the presence of chloramphenicol or rifampicin in the incubation medium prevented the development of the nitrate reductase activity, such activity was induced in bacteroids. Nitrite content and nitrate reductase and nitrite reductase activities of the cytosol from nodules of pea plants treated or not with 5 mM KNO3 varied with the buffer used for nodules homogenization. However, no nitrite was found when nodules were homogenized with ethanol, what indicates that nitrite accumulation in the cytosol occurs during the homogenization process of the nodules.

A derivative of the IncP1 plasmid RP4, carrying the thermoinducible prophage Mucts62, was obtained in Escherichia coli K12 J53 (RP4). It was impossible to maintain the hybrid plasmid RP4::Mucts62 in Rhizobium meliloti GR4. Thus, it was used as a vehicle for introducing the ampicillin-resistant transposon Tn1 into the R. meliloti genome. Transposition of Tn1 did not generate auxotrophic strains, suggesting that the insertion of Tn1 into the R. meliloti genome was relatively specific. Two chromosomal hot spots for Tn1 insertion were identified by cotransductional analysis, after general transduction by phage DF2. Plasmid-curing experiments, carried out by heat treatment, revealed that symbiotic plasmid(s) also contain at least one site for Tn1 insertion.

A mutant of Rhizobium meliloti that elicited the formation of inactive nodules in alfalfa was found not to form beta-(1----2) glucan in vivo or in vitro. It was nonmotile because it lacks flagella. The 235-kilodalton protein which acts as an intermediate in beta-(1----2) glucan synthesis was undetectable in the mutant. These properties of the mutant are common to those of chvB mutants of Agrobacterium tumefaciens. Exopolysaccharide formation by the R. meliloti mutant was about double that by the wild type.

The nitrogenase activity, nitrate reductase activity and oxygen uptake as well as the hydrogen incorporation and ATP content were examined in the root nodules and bacteroids, respectively, formed by Rhizobium leguminosarum strains 128C53 (hydrogenase positive) and 300 (hydrogenase negative) in symbiosis with Pisum sativum plants grown in the presence of 2 mM KNO3. The strain 128C53 showed the greatest values for all parameters analyzed, except for the nitrate reductase activity, which was higher for the strain 300. Similarly, nodule nitrate reductase activity in strain 300 was greater than that in strain 128C53 when plants grew in the absence of combined nitrogen. In general, the highest values were obtained when determinations were made after 7 hours of plant illumination. However, the hydrogenase activity of strain 128C53 and the nitrate reductase activities of both strains increased with the light period, reaching a maximum after 14 hours of illumination. These results suggest that the benefits derived from the superior symbiotic properties and from the presence of hydrogenase activity in strain 128C53 could be counteracted by the higher rates of the nodule nitrate reductase activity in strain 300.

The ability of extracellular polysaccharides of different strains of Rhizobium meliloti to induce the production of polygalacturonase by roots of Medicago sativa seedlings has been studied. Some strains showed a high inducing activity while those derived from them, after treatment with acridine orange and in which extrachromosomal DNA was absent, did not show this character. A comparative study of polysaccharide production and preliminary studies on the chemical composition of the active fractions obtained after Sephadex G-25 filtration indicated that the monomers which form the active fractions are qualitatively and quantitatively different according to their origin.

We examined the contribution of a cryptic plasmid, pRmeGR4b, to the nodulation of Medicago sativa by strain GR4 of Rhizobium meliloti. A 905-base-pair PstI DNA fragment in pRmeGR4b was found to hybridize DNA of the R. meliloti fixA promoter region as a probe. Sequence analysis of the PstI fragment showed a 206-base-pair region displaying high homology with the DNA upstream of the RNA start points of the P1 and P2 symbiotic promoters. Putative nif promoter consensus sequences were conserved in this DNA segment. Expression of DNA downstream of the nif promoterlike sequence, monitored by beta-galactosidase activity of different lacZ fusions, was demonstrated to depend on a functional nifA gene, both in microaerobically free-living cells and in nodules. Individual transposon Tn3-HoHo1 insertions in this DNA region caused a reduced nodulation competitiveness. This new symbiotic region, occupying approximately 5 kilobases of pRmeGR4b DNA, was called nfe (nodule formation efficiency).

A simiplified version of bacteriophage DF2 transduction, using the thermosensitive mutant DF2tsC, is described. The transduction frequencies of Rhizobium meliloti markers by DF1tsC are of about 10(-6) or higher. The development of transductants at 34 degrees C does not require the addition of antiserum for phage neutralization.

The ability of Rhizobium meliloti to induce polygalacturonase production in legume roots decreased during culture under laboratory conditions but was inducible with mitomycin C. This character was irreversibly lost after treatment with acridine orange. Extrachromosomal DNA of molecular weight 5.9 x 10(6) daltons was detectable in neutral sucrose gradient but was absent from cells 'cured' with acridine orange. Therefore, the ability to induce the enzyme production may be controlled by a plasmid.

A circular linkage map of the Rhizobium meliloti chromosome, obtained from R68.45-mediated crosses, has been revised by cotransductional analysis, after general transduction by DF2 phage. Three short chromosomal regions have been mapped by cotransduction. Comparison between conjugal and cotransductional data suggests that R68.45-mediated linkage measures are indeed rough. Cotransduction seems to be a useful tool for improving the linkage map of R. meliloti.

Nitrate reductase activity (NRA) was measured in leaves, stems, roots and nodules of four cultivars of Pisum sativum L. supplied with 2 mM KNO3 and nodulated by selected Rhizobium leguminosarum strains. As a control, non-nodulated (uninoculated) plants were grown in the same nutrient medium. NRA was determined by an in vitro assay; it varied widely among strains and cultivars. Although differences in tissue NRA in nodulated plants were mainly related to cultivar genotype, nodulation much affected the distribution of NRA within the plant. There was also some cultivar × bacteria strain effect. Non-nodulated plants had higher leaf and root NRA than nodulated cultivars, but nodulated plants had twice as high stem NRA as non-nodulated plants. These differences correlated with nitrate concentrations which were depressed in leaves and roots, and increased in stems, of nodulated plants. Nitrate accumulation was increased in leaves, stems and roots of nodulated plants in comparison with non-nodulated ones. There were no differences in the root to shoot ratio between nodulated and non-nodulated plants. The relationships between tissue nitrate reduction and nodulation are discussed. [ABSTRACT FROM AUTHOR]