Alleviation of aluminum toxicity to Rhizobium leguminosarum bv. viciae by the hydroxamate siderophore vicibactin

Acid rain solubilises aluminum which can exert toxic effects on soil bacteria. The root nodule bacterium Rhizobium leguminosarum biovar viciae synthesises the hydroxamate siderophore vicibactin in response to iron limitation. We report the effect of vicibactin on the toxicity of aluminum(III) to R. leguminosarum and kinetic studies on the reaction of vicibactin with Al(III) and Fe(III). Aluminum (added as the nitrate) completely inhibited bacterial growth at 25 M final concentration, whereas the preformed Al-vicibactin complex had no effect. When aluminum and vicibactin solutions were added separately to growing cultures, growth was partly inhibited at 25 M final concentration of each, but fully inhibited at 50 M final concentration of each. Growth was not inhibited at 50 M Al and 100 M vicibactin, probably reflecting the slow reaction between Al and vicibactin; this results in some aluminum remaining uncomplexed long enough to exert toxic effects on growth, partly at 25 M Al and vicibactin and fully at 50 M Al and vicibactin. At 100 M vicibactin and 50 M Al, Al was complexed more effectively and there was no toxic effect. It was anticipated that vicibactin might enhance the toxicity of Al by transporting it into the cell, but the Al-vicibactin complex was not toxic. Several explanations are possible: the Al-vicibactin complex is not taken up by the cell; the complex is taken up but Al is not released from vicibactin; Al is released in the cell but is precipitated immediately. However, vicibactin reduces the toxicity of Al by complexing it outside the cell.     

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain TA1

Rhizobium leguminosarum bv. trifolii strain TA1 is an aerobic, motile, Gram-negative, non-spore-forming rod that is an effective nitrogen fixing microsymbiont on the perennial clovers originating from Europe and the Mediterranean basin. TA1 however is ineffective with many annual and perennial clovers originating from Africa and America. Here we describe the features of R. leguminosarum bv. trifolii strain TA1, together with genome sequence information and annotation. The 8,618,824 bp high-quality-draft genome is arranged in a 6 scaffold of 32 contigs, contains 8,493 protein-coding genes and 83 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.     

Symbiotic effects of deltamatB Rhizobium leguminosarum bv. trifolii mutant on clovers

The role of malonate in symbiotic nitrogen metabolism has long been controversial, although it is known to occur in legume roots, especially in the nodules. Here we report that malonate metabolism plays a key role in the differentiation of bacteroids Rhizobium leguminosarum bv. trifolii in clover nodules. An operon, mat, that consists of three consecutive genes (matABC) has been discovered. Mat encodes enzymes that catalyze the uptake and conversion of malonate to acetyl-CoA through malonyl-CoA. A mutant bacteria, which replaced matB that encodes malonyl-CoA synthetase with a kanamycin resistant gene, was generated and infected with white clover. Clover growth was considerably reduced, even though nodules were formed. However, the nodules were filled with vacuoles, but not with bacteroids. This indicates that malonate metabolism is an important requirement for the formation of mature nodules that are filled with bacteroids.     

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain SRDI565.

Rhizobium leguminosarum bv. trifolii SRDI565 (syn. N8-J) is an aerobic, motile, Gram-negative, non-spore-forming rod. SRDI565 was isolated from a nodule recovered from the roots of the annual clover Trifolium subterraneum subsp. subterraneum grown in the greenhouse and inoculated with soil collected from New South Wales, Australia. SRDI565 has a broad host range for nodulation within the clover genus, however N₂-fixation is sub-optimal with some Trifolium species and ineffective with others. Here we describe the features of R. leguminosarum bv. trifolii strain SRDI565, together with genome sequence information and annotation. The 6,905,599 bp high-quality-draft genome is arranged into 7 scaffolds of 7 contigs, contains 6,750 protein-coding genes and 86 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.     

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain SRDI943.

Rhizobium leguminosarum bv. trifolii SRDI943 (strain syn. V2-2) is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Trifolium michelianum Savi cv. Paradana that had been grown in soil collected from a mixed pasture in Victoria, Australia. This isolate was found to have a broad clover host range but was sub-optimal for nitrogen fixation with T. subterraneum (fixing 20-54% of reference inoculant strain WSM1325) and was found to be totally ineffective with the clover species T. polymorphum and T. pratense. Here we describe the features of R. leguminosarum bv. trifolii strain SRDI943, together with genome sequence information and annotation. The 7,412,387 bp high-quality-draft genome is arranged into 5 scaffolds of 5 contigs, contains 7,317 protein-coding genes and 89 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.     

Cloning and characterization of ce/8A gene from Rhizobium leguminosarum bv. trifolii 1536

AIMS: To isolate the cellulase gene from Rhizobium leguminosarum bv. trifolii 1536. METHODS AND RESULTS: By the shot-gun method a clone (cel8A) harbouring 3.1 kb genomic DNA fragment from R. leguminosarum bv. trifolii 1536 was obtained. The cel8A gene coded 348 amino acids and it belongs to the glycosyl hydrolase family 8. The molecular mass of Cel8A protein induced from Escherichia coli DH5alpha, appeared to be 35 kDa. The optimum pH and optimum temperature was 7.0, and about 30 degrees C for its enzymatic activity respectively. CONCLUSIONS: R. leguminosarum bv. trifolii 1536 had cel8A gene having an open reading frame of 1047 bp coded for the activity of hydrolyzation of carboxymethyl cellulose. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of celluloytic enzyme by R. leguminosarum bv. trifolii was confirmed, which would play specific roles in rhizobia. Future study should focus on its role in the infection and nodulation phenomena.     

A hierarchical analysis of population genetic structure in Rhizobium leguminosarum bv. trifolii

Little is known about the population processes that shape the genetic diversity in natural populations of rhizobia. A sample of 912 Rhizobium leguminosarum biovar trifolii isolates were collected from naturalized red clover populations ( Trifolium pratense ) and analyzed for 15 allozyme loci to determine the levels and distribution of genetic diversity. Hierarchical analyses compared different sampling levels, geographical separation, and temporal separation. Total genetic diversity across all isolates was H = 0.426, with 57.6% of the total diversity found among isolates obtained from individual red clover plants. Relatively low genetic differentiation among populations and high differentiation among plants within populations was observed; this suggests that gene flow and founder effect act differently at geographical and local scales. Significant differences were observed in (i) allele frequencies among populations and among plants within populations, and (ii) the frequency distribution of the most widespread and the most abundant strains. When multilocus linkage disequilibrium was calculated, significant levels of disequilibrium were observed in the total sample and in three of the eight populations.     

Genetic and Metabolic Divergence within a Rhizobium leguminosarum bv. trifolii Population Recovered from Clover Nodules

Rhizobia are able to establish symbiosis with leguminous plants and usually occupy highly complex soil habitats. The large size and complexity of their genomes are considered advantageous, possibly enhancing their metabolic and adaptive potential and, in consequence, their competitiveness. A population of Rhizobium leguminosarum bv. trifolii organisms recovered from nodules of several clover plants growing in each other''s vicinity in the soil was examined regarding possible relationships between their metabolic-physiological properties and their prevalence in such a local population. Genetic and metabolic variability within the R. leguminosarum bv. trifolii strains occupying nodules of several plants was of special interest, and both types were found to be considerable. Moreover, a prevalence of metabolically versatile strains, i.e., those not specializing in utilization of any group of substrates, was observed by combining statistical analyses of Biolog test results with the frequency of occurrence of genetically distinct strains. Metabolic versatility with regard to nutritional requirements was not directly advantageous for effectiveness in the symbiotic interaction with clover: rhizobia with specialized metabolism were more effective in symbiosis but rarely occurred in the population. The significance of genetic and, especially, metabolic complexity of bacteria constituting a nodule population is discussed in the context of strategies employed by bacteria in competition.The soil bacterium Rhizobium leguminosarum bv. trifolii is capable of symbiotic interaction with the host plant Trifolium spp. (clover). The symbiotic process involves an exchange of chemical signals between both organisms, resulting in the expression of specific bacterial and plant genes. In response to flavonoid signals from legumes, bacterial lipochitooligosaccharides (Nod factors) are synthesized and in turn trigger the expression of plant genes and root nodule formation (9). Rhizobia invade the root nodules and differentiate into bacteroids that fix nitrogen (14, 16, 21, 36, 37). Atmospheric dinitrogen converted into ammonia is further transported and assimilated by the plant, which, reciprocally, provides photosynthates (42, 43, 50). The range of plant benefits varies and depends on the effectiveness of the bacterial strains as well as the legume plant genotype (8).A common feature of rhizobial genomes is the complexity and diversity of genomic organization, with a single chromosome and large plasmids ranging in size from ca. 100 kb up to 2 Mb (34). The genes encoding symbiotic functions usually constitute independent replicons known as symbiotic plasmids (pSym), or symbiotic islands when incorporated into the chromosome (25). The plasmids constitute a pool of accessory genetic information (18, 53) and contribute to the plasticity and dynamic state of the genome commonly observed among members of the Rhizobiaceae family (4, 25, 28, 34). Rhizobia occupy highly complex soil habitats, and their large and multipartite genomes, which encode many potentially useful metabolic traits, might be advantageous, enhancing their adaptive potential (33). Local populations of rhizobia may differ significantly on both the genetic and physiological levels. The diverse metabolic capacities of different strains and species of rhizobia might be important in their adaptation and survival in the rhizospheres of host plants. Plant root exudates contain a great number of chemical compounds, comprising sugars, amino acids, amines, aliphatic and aromatic acids, phenols, and others (2, 3, 15, 38, 49), thus potentially influencing the structure of the bacterial community in the rhizosphere. It was demonstrated that more metabolically versatile strains of R. leguminosarum were better competitors (51). Several studies showed that the nutritional diversity of soil habitats and the rhizosphere influences the number of rhizobia and that competition for root nodule colonization can take place even inside the infection threads, occupied, in some cases, by more than one strain (32, 38, 47). Up-to-date research on the diversity and competition of rhizobia focused on strains colonizing the soil or particular species of legume plants (8, 12, 24, 31, 35). Comprehensive analyses of the genetic and, especially, metabolic variability in rhizobia that occupy a spatially restricted area, for instance, all the nodules of a legume plant root system coexisting in one place, are still lacking.In this work, we investigated the degree of genetic and metabolic variability within the R. leguminosarum bv. trifolii strains occupying a spatially restricted area—the nodules of several clover plants—focusing on estimation of possible interconnections between the metabolic-physiological properties of strains and their frequency of occurrence.     

Selection of Portuguese Rhizobium leguminosarum bv. trifolii strains for production of legume inoculants

From several native clover species, growing in six different soil types, 170 Rhizobium leguminosarum biovar trifolii strains were isolated, covering the central and southern regions of Portugal. The effectiveness of the strains varied from ineffective to highly effective on T. subterraneum cv. Clare and on T. fragiferum cv. Palestine, with a predominance of medium and high effectiveness on both host plants. The effectiveness was not influenced by provenence (soil or plant), except for the strains from the rankers soils and for the strains isolated from T. pratense, that were ineffective or medium effective on T. subterraneum.Selected strains were evaluated for effectiveness on T. subterraneum cv. Clare, using the commercial strain TA1 as reference. Several of the isolated strains were more effective than TA1, indicating that local strains may be used to produce better inoculants.     

Structure and role in symbiosis of the exoB gene of Rhizobium leguminosarum bv trifolii

The Rhizobium leguminosarum bv trifolii exoB gene has been isolated by heterologous complementation of an exoB mutant of R. meliloti. We have cloned a chromosomal DNA fragment from the R. leguminosarum bv trifolii genome that contains an open reading frame of 981 bp showing 80% identity at the amino acid level to the UDP-glucose 4-epimerase of R. meliloti. This enzyme produces UDP-galactose, the donor of galactosyl residues for the lipid-linked oligosaccharide repeat units of various heteropolysaccharides of rhizobia. An R. leguminosarum bv trifoliiexoB disruption mutant differed from the wild type in the structure of both the acidic exopolysaccharide and the lipopolysaccharide. The acidic exopolysaccharide made by our wild-type strain is similar to the Type 2 exopolysaccharide made by other R. leguminosarum bv trifolii wild types. The exopolysaccharide made by the exoB mutant lacked the galactose residue and the substitutions attached to it. The exoB mutant induced the development of abnormal root nodules and was almost completely unable to invade plant cells. Our results stress the importance of exoB in the Rhizobium-plant interaction.     

Rhizobium leguminosarum bv. trifolii PssP protein is required for exopolysaccharide biosynthesis and polymerization

Rhizobium leguminosarum bv. trifolii produces an acidic exopolysaccharide (EPS) that is important for the induction of nitrogen-fixing nodules on clover. Recently, three genes, pssN, pssO, and pssP, possibly involved in EPS biosynthesis and polymerization were identified. The predicted protein product of the pssP gene shows a significant sequence similarity to other proteins belonging to the PCP2a family that are involved in the synthesis of high-molecular-weight EPS. An R. leguminosarum bv. trifolii TA1 mutant with the entire coding region of pssP deleted did not produce the EPS. A pssP mutant with the 5' end of the gene disrupted produced exclusively low-molecular-weight EPS. A mutant that synthesized a functional N-terminal periplasmic domain but lacked the C-terminal part of PssP produced significantly reduced amounts of EPS with a slightly changed low to high molecular form ratio. Mutants affected in the PssP protein carrying a stable plasmid with a constitutively expressed gusA gene induced nodules on red clover that were not fully occupied by bacteria. A mutant with the entire pssP gene deleted infected only a few plant cells in the nodule. The pssP promoter-gusA reporter fusion was active in bacteroids during nodule development.     

Population level processes in Rhizobium leguminosarum bv. trifolii: the role of founder effects

The importance of genotype-specific selection between host and symbiont, founder effect, and clonal reproduction in Rhizobia leguminosarum biovar trifolii populations is relatively unknown. A field experiment was conducted to sample 1268 isolates of R. l. bv. trifolii from four genotypically distinct Trifolium pratense plants for allozyme variation at nine loci. Genetic and genotypic variation, population genetic substructure, and linkage disequilibrium were estimated. Of the 1268 isolates 188 genotypically distinct strains (electrophoretic types or ETs) were identified with an average of 11.04 different ETs per plant. Total genetic diversity in the plot was 0.346 and most of the variation was found within plants (= 80%). Our data suggests that genotype-specific selection between the rhizobia and the four host-plant genotypes tested does not influence local population structure, but evidence of founder effect was present. Significant linkage disequilibrium was observed and is most likely due to the clonal reproduction of R. l. bv. trifolii.     

Genome sequence of the Trifolium rueppellianum -nodulating Rhizobium leguminosarum bv. trifolii strain WSM2012.

Rhizobium leguminosarum bv. trifolii WSM2012 (syn. MAR1468) is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an ineffective root nodule recovered from the roots of the annual clover Trifolium rueppellianum Fresen growing in Ethiopia. WSM2012 has a narrow, specialized host range for N₂-fixation. Here we describe the features of R. leguminosarum bv. trifolii strain WSM2012, together with genome sequence information and annotation. The 7,180,565 bp high-quality-draft genome is arranged into 6 scaffolds of 68 contigs, contains 7,080 protein-coding genes and 86 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.     

The response of the Rhizobium leguminosarum bv. trifolii wild-type and exopolysaccharide-deficient mutants to oxidative stress

Aims

The aim of this study was investigation of the response of R. leguminosarum bv. trifolii wild-type and its two rosR and pssA mutant strains impaired in exopolysaccharide (EPS) synthesis to oxidative stress conditions caused by two prooxidants: a superoxide anion generator- menadione (MQ) and hydrogen peroxide (H₂O₂).

Methods

The levels of enzymatic (catalase, superoxide dismutase, pectinase and β-glucosidase) and non-enzymatic (superoxide anion generator, formaldehyde, phenolic compounds) biomarkers were monitored using biochemical methods in both the supernatants and rhizobial cells after treatment with 0.3?mM MQ and 1.5?mM H₂O₂. The viability of bacterial cells was estimated using fluorescent dyes and confocal laser scanning microscopy. In addition, the effect of prooxidants on symbiosis of the R. leguminosarum bv. trifolii strains with clover was established.

Results

The tested stress factors significantly changed enzymatic patterns of the rhizobial strains, and the wild-type strain proved to be more resistant to these prooxidants than both pssA and rosR mutants. Significantly higher activities of both catalase and superoxide dismutase have been detected in these mutants in comparison to the wild-type strain. H₂O₂ and MQ also increased the levels of pectinase and β-glucosidase activities in the tested strains. Moreover, pre-incubation of R. leguminosarum bv. trifolii strains with the prooxidants negatively affected the viability of bacterial cells and the number of nodules elicited on clover plants.

Conclusions

EPS produced in large amounts by R. leguminosarum bv. trifolii plays a significant protective role as a barrier against oxidative stress factors and during symbiotic interactions with clover plants.     

Isolation and partial characterization of phenolate siderophore from Rhizobium leguminosarum IARI 102

Abstract Rhizobium leguminosarum IARI 102 produced 2,3-dihydroxy benzoic acid, a type of phenolate siderophore, under iron-starved conditions. Hydroxamic acids were not detected. Maximum production of siderophore was found at 26 h of growth in a chemically defined medium at 28°C with shaking. Threonine was detected as the amino acid conjugate of the siderophore. Addition of Fe³⁺ to the culture medium increased the growth yield significantly, but depressed the production of the iron chelating compound.