Rhizobium Spp Research Articles

Functional expression of Escherichia coli fhuA gene in Rhizobium spp. of Cajanus cajan provides growth advantage in presence of Fe3+: ferrichrome as iron source

Cajanus cajan rhizobial isolates were found to be unable to utilize iron bound to ferrichrome, desferrioxamine B or rhodotorulic acid, all being hydroxamate type siderophores. A broad host range expression vector containing the Escherichia coli fhuA gene, encoding the outer membrane receptor for Fe-ferrichrome, was constructed. The plasmid construct (pGR1), designed to express fhuA under the lac promoter of E. coli, complemented E. coli MB97 DeltafhuA mutant for ferri-ferrichrome utilization and also allowed Rhizobium spp. ST1 and Rhizobium spp. IC3123 to grow using iron bound to ferrichrome. Sensitivity to the antibiotic albomycin, transported via the FhuA receptor, was found in case of MB97 as well as rhizobial transformants harboring pGR1. The rhizobial transformants expressing fhuA showed growth stimulation when co-inoculated with Ustilago maydis, a fungal species known to produce ferrichrome under iron starved conditions. Growth stimulation was also observed in the presence of externally supplied ferrichrome. The significance of these findings in terms of the potential for improving the survivability of rhizobial bioinoculant strains in natural soils is discussed.

Isolation and characterisation of sulfur-oxidising bacteria, including strains of Rhizobium, from calcareous sandy soils and their effects on nutrient uptake and growth of maize (Zea mays L.)

Four sulfur-oxidising bacteria were selected among 427 bacterial isolates from calcareous sandy soils in the United Arab Emirates (UAE). These isolates were selected based on their strong ability to oxidise elemental sulfur (S°) in vitro and were identified as Paracoccus versutus CBS 114155, Paracoccus pantotrophus CBS 114154, and 2 strains as Rhizobium spp. NCCB 100053 and NCCB 100054. This is the first published report of a Rhizobium species capable of S° oxidation and also the first record of sulfur-oxidising bacteria from UAE soils. These isolates were tested in a greenhouse in the presence and absence of S° to study their effects on maize growth. Best growth was observed in the treatment with P. versutus application combined with S°, which significantly reduced soil pH, increased soil SO4 level and the uptake of N, S, Fe, Mn, and Zn in maize roots and shoots. The P and Cu uptake in the shoots of maize plants was not significant compared with the treatment that received the application of S° alone. There was no response in plant growth to treatments that included the application of S° combined with P. pantotrophus or Rhizobium strain NCCB 100053 compared with the treatment that received the application of S° alone. There was significant growth inhibition of maize plants in the treatment receiving Rhizobium strain NCCB 100054 with or without the application of S° compared with the treatment that included the application of S° alone. This growth inhibition was associated with a significant decrease in the levels of N, P, S, Fe, Mn, Zn, and Cu in roots and shoots in the absence of S°. Rhizobium strain NCCB 100054 applied with S° significantly decreased the levels of N, S, and Fe in the roots and the levels of N, P, S, Fe, Mn, and Cu in the shoots of maize, with no significant differences in the levels of P and Mn in the roots and in the levels of Zn in the shoots, compared with the treatment with S° alone. These results indicate that the treatment P. versutus combined with S° can be effective as a soil conditioner for horticultural production in calcareous sandy soils.

Production of amino acids by Rhizobium, Mesorhizobium and Sinorhizobium strains in chemically defined media.

Five strains of Rhizobium spp, one strain of Mesorhizobium loti and two strains of Sinorhizobium meliloti were tested for their ability to grow in chemically-defined medium lacking growth factor. Qualitative and quantitative production of aspartic acid, serine, glutamic acid, glycine, histidine, threonine, arginine, alanine, proline, cysteine, tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine was determined by the use of mannitol as sole carbon source. Strains of Rhizobium spp. and Sinorhizobium sp. produced all the amino acids analysed with the exception of cysteine and high biological levels of serine, glycine and alanine were detected after 2 days of culture in mineral medium. Strain U226 of M. loti only produced small amounts of amino acids and glutamic acid, histidine, arginine, cysteine, methionine, lysine and phenylalanine was not liberated into the media.

Polyphasic taxonomy of symbiotic rhizobia from wild leguminous plants growing in Egypt.

About 20 strains of rhizobia from wild legumes were characterized based on numerical analysis of phenotypic characteristics, nodulating ability, fatty acid methyl esters (FAME) and SDS-PAGE profiles of whole cell proteins. FAME analysis revealed that palmitic (16:0), stearic (18:0) and arachidonic (20:0) were detected in most of wild-legume rhizobia, the latter being uncommon in fatty acid profiles of Rhizobium and Sinorhizobium. Numerical analysis of FAME classified strains of wild-legume rhizobia into 9 clusters and one heterogeneous group. There was both agreement and disagreement with the clustering data based on phenotypic analysis and FAME analysis. Four strains were grouped together in the same cluster based on both methods. However, 4 another strains, which were placed in one cluster of phenotypic analysis, were distributed in several clusters after FAME analysis. SDS-PAGE of whole-cell proteins revealed that the rhizobial strains exhibited protein profiles with peptide bands ranging from 5-19 band per profile and showed molar mass of 110-183 kDa. As in the case of FAME analysis, numerical analysis of protein bands was compared with clustering of phenotypic analysis. Agreement of the two methods was obvious when clustering some strains but conflicted in the classification of some other strains. However, integration of the three methods could be the basis of a polyphasic taxonomy. The twenty strains of wild-legume rhizobia were finally classified as follows: 12 strains related to Rhizobium leguminosarum, 5 strains related to Sinorhizobium meliloti and 3 strains to Rhizobium spp. Rhizobia nodulating wild herb legumes are among indigenous strains nodulating crop legumes in cultivated as well as noncultivated lands.

Characterization of the nodulation plasmid encoded chemoreceptor gene mcpG from Rhizobium leguminosarum

BackgroundIn general, chemotaxis in Rhizobium has not been well characterized. Methyl accepting chemotaxis proteins are sensory proteins important in chemotaxis of numerous bacteria, but their involvement in Rhizobium chemotaxis is unclear and merits further investigation.ResultsA putative methyl accepting chemotaxis protein gene (mcpG) of Rhizobium leguminosarum VF39SM was isolated and characterized. The gene was found to reside on the nodulation plasmid, pRleVF39d. The predicted mcpG ORF displayed motifs common to known methyl-accepting chemotaxis proteins, such as two transmembrane domains and high homology to the conserved methylation and signaling domains of well-characterized MCPs. Phenotypic analysis of mcpG mutants using swarm plates did not identify ligands for this putative receptor. Additionally, gene knockouts of mcpG did not affect a mutant strain's ability to compete for nodulation with the wild type. Notably, mcpG was found to be plasmid-encoded in all strains of R. leguminosarum and R. etli examined, though it was found on the nodulation plasmid only in a minority of strains.ConclusionsBased on sequence homology R. leguminosarum mcpG gene codes for a methyl accepting chemotaxis protein. The gene is plasmid localized in numerous Rhizobium spp. Although localized to the sym plasmid of VF39SM mcpG does not appear to participate in early nodulation events. A ligand for McpG remains to be found. Apparent McpG orthologs appear in a diverse range of proteobacteria. Identification and characterization of mcpG adds to the family of mcp genes already identified in this organism.

Novel plant-microbe rhizosphere interaction involving Streptomyces lydicus WYEC108 and the pea plant (Pisum sativum).

A previously undescribed plant-microbe interaction between a root-colonizing Streptomyces species, S. lydicus WYEC108, and the legume Pisum sativum is described. The interaction is potentially of great importance to the health and growth in nature of this nodulating legume. The root-colonizing soil actinomycete S. lydicus WYEC108 influences pea root nodulation by increasing root nodulation frequency, possibly at the level of infection by Rhizobium spp. S. lydicus also colonizes and then sporulates within the surface cell layers of the nodules. Colonization leads to an increase in the average size of the nodules that form and improves the vigor of bacteroids within the nodules by enhancing nodular assimilation of iron and possibly other soil nutrients. Bacteroid accumulation of the carbon storage polymer, poly-beta-hydroxybutyrate, is reduced in colonized nodules. Root nodules of peas taken from agricultural fields in the Palouse hills of northern Idaho were also found to be colonized by actinomycete hyphae. We hypothesize that root and nodule colonization is one of several mechanisms by which Streptomyces acts as a naturally occurring plant growth-promoting bacterium in pea and possibly other leguminous plants.

Nematode communities under stress: the long-term effects of heavy metals in soil treated with sewage sludge

The long-term effects of Ni, Zn+Ni, Cu, Zn+Cu and Zn on the nematode communities in soil treated with sewage sludge were studied in an agroecosystem. The soil was a sandy loam with 9% clay, 1.6–4.0% organic matter (OM) and pH 5.8–7.2. Sewage sludge, artificially enriched with four concentrations of each heavy metal or combinations was applied in 1982 and again in 1986 to some of the plots. Ni and Zn+Ni treatments did not have significant effects on the soil nematode assemblages. Some of the Cu, Zn+Cu and Zn treatments had adverse effects, which correlated with the following changes in their structure, decrease in genus/family richness, decrease in maturity indices (MI and MI 2–5) of the free-living nematode assemblages, reduction or elimination of the persisters ( K-strategists) in favor of colonizers ( r-strategists), decrease or elimination of omnivorous nematodes (Order Dorylaimida, Suborder Dorylaimina) and predatory nematodes (Order Dorylaimida, Suborder Mononchina). The increase in plant-feeding nematodes in the Zn treated plots may be due to the negative effects of the heavy metals on some of their antagonists (e.g., mycorrhizal fungi, root-nodule bacteria from Rhizobium spp .) and the elimination of predacious nematodes in these treatments. Some bacterial-feeding nematodes were found to increase, which was related to the indirect long-term effects of the heavy metals (e.g., lower competition and reduced predation pressure). The most common responses of the nematode communities to soil heavy metal pollution were found to be in agreement with the trends expected in stressed ecosystems and communities, predicted by the hypothesis that stress forces ecosystems into an early stage of development.

Nitrate reductase activity, distribution, and response to nitrate in two contrasting Phaseolus species inoculated with Rhizobium spp.

The nitrate reductase activity distribution and response of two nodulated species of Phaseolus ( Phaseolus vulgaris-common bean, and Phaseolus lunatus-lima bean) to different exogenous nitrate levels were studied during the vegetative period. These Phaseolus species showed to be very contrasting in respect to the pattern of nitrate reductase (NR) activity distribution thought the plant. The highest level of NR activity in P. vulgaris was clearly shown to occur in leaves in contrast with the lowest one detected in roots and nodules as widely seen for other tropical species of the Phaseoleae tribe. Conversely, P. lunatus had higher NR activity in the nodules, whereas its leaves exhibited a steadily decrease during the plant development. Indeed, at 32 days after emergence (pre-flowering stage), the nodulated P. vulgaris had approximately 95% of the total NR activity localized in its leaves, whereas in P. lunatus it was equally distributed in the nodules and in the leaves. Under long-term exposure to increasing exogenous level of nitrate, the leaf-NR activity of nodulated P. vulgaris presented a positive response, whereas the enzyme activity was very low and unresponsive in P. lunatus. In contrast, the nodule-NR activity showed a reverse response to the increasing NO 3 − level. The nodule-NR activity of P. lunatus significantly increased whereas in the P. vulgaris nodules it was very low and unresponsive. This present study suggests that P. lunatus inoculated with Rhizobium tropici presents a singular pattern of nitrate reduction distribution among leaves and nodules during the vegetative development. It is speculated that the nodulated Phaseolus lunatus may have different NR isoforms in their leaves (at least a constitutive type) and an inducible form in their nodules, responsive to long-term exposure to nitrate.

Clogging of a limestone fracture by stimulating groundwater microbes

Biological clogging is promoted in aquifers either to contain or to remediate groundwater. In this study, an apparatus able to detect small changes in hydraulic conductivity (K) was developed to measure the clogging of a single fracture in limestone, following microbial stimulation. The fracture had a 2.5mm2 section and was 50cm long. Prior to the inoculation of the limestone, the sequencing of representative clones from 16S rRNA genes isolated from groundwater, showed significant affiliation with Cytophaga spp., Arcobacter spp. and Rhizobium spp. These bacteria are known to secrete extracellular polymeric substances and form biofilms. When nutrients were added to the inoculated limestone, a decrease in K occurred after 8 days, reaching 0.8% of its initial value after 22 days (Kfi=340cmmin−1). This study showed that a stimulation of indigenous microbes from groundwater effectively clogged a macrofracture in limestone, suggesting the potential application of biobarriers in fractured rock aquifers.

Interaction of pea ( Pisum sativum L.) lectins with rhizobial strains

Lectins from two varieties (PG-3 and LFP-48) of pea have been purified by affinity chromatography on Sephadex G-50. The specific activity increased by 23 and 25 folds, respectively. These lectins from both the varieties were found to be specific for mannose. The purified fluorescein isothiocyanate (FITC)-labelled lectins showed binding reaction with homologous as well as heterologous strains of Rhizobium spp. The results revealed that pea lectins are not highly specific to their respective rhizobia. Moreover, these lectins showed a greater stimulatory effect on homologous Rhizobium leguminosarum strains.

Dual inoculation of a woody legume (Centrolobium tomentosum) with rhizobia and mycorrhizal fungi in south-eastern Brazil

The integration of N2 fixing trees into stable agroforestry systems in the tropics is being tested due to their ability to produce high biomass N and P yields, when symbiotically associated with rhizobia and mycorrhizal fungi. The growth of Centrolobium tomentosum Guill. ex Benth, a native leguminous tree from the Brazilian Atlantic Forest, was assessed with dual inoculation of Rhizobium spp and mycorrhizal fungi under field conditions. Complete fertilization was compared to treatments of inoculation with selected rhizobia strains BHICB-Ab1 or BHICB-Ab3, associated or not to arbuscular mycorrhizal (AM) fungi. The dual inoculation increased the height and growth in relation to the plants treated with rhizobia alone. Plants inoculated with strain BHICB-Ab1 and arbuscular mycorrhizal fungi (AMF) exhibited an increase of 56% dry matter over uninoculated control and nitrogen accumulation was greater than with BHICB-Ab3 inoculated plants. Strain BHICB-Ab1 presented a synergetic relationship with mycorrhizal fungi since the combined inoculation with BHICB-Ab1 enhanced plant height and dry weight more than single inoculation while the growth of BHICB-Ab3 plants was not modified by AMF inoculation. Arbuscular mycorrhizal fungi enhanced plants survival and seemed to favour the nodule occupation by rhizobia strains as compared to the non-mycorrhizal plants. Inoculation with selected rhizobia and AMF improved the growth of C. tomentosum under field conditions.

Rhizobia can induce nodules in white clover by "hijacking" mature cortical cells activated during lateral root development.

We examined a range of responses of root cortical cells to Rhizobium sp. inoculation to investigate why rhizobia preferentially nodulate legume roots in the zone of emerging root hairs, but generally fail to nodulate the mature root. We tested whether the inability to form nodules in the mature root is due to a lack of plant flavonoids to induce the bacterial genes required for nodulation or a failure of mature cortical cells to respond to Rhizobium spp. When rhizobia were inoculated in the zone of emerging root hairs, changes in beta-glucuronidase (GUS) expression from an auxin-responsive promoter (GH3), expression from three chalcone synthase promoters, and the accumulation of specific flavonoid compounds occurred in cortical cells prior to nodule formation. Rhizobia failed to induce these responses when inoculated in the mature root, even when co-inoculated with nod gene-inducing flavonoids. However, mature root hairs remained responsive to rhizobia and could support infection thread formation. This suggests that a deficiency in signal transduction is the reason for nodulation failure in the mature root. However, nodules could be initiated in the mature root at sites of lateral root emergence. A comparison between lateral root and nodule formation showed that similar patterns of GH3:gusA expression, chalcone synthase gene expression, and accumulation of a particular flavonoid compound occurred in the cortical cells involved in both processes. The results suggest that rhizobia can "hijack" cortical cells next to lateral root emergence sites because some of the early responses required for nodule formation have already been activated by the plant in those cells.

The replicator of the nopaline-type Ti plasmid pTiC58 is a member of the repABC family and is influenced by the TraR-dependent quorum-sensing regulatory system.

The replicator (rep) of the nopaline-type Ti plasmid pTiC58 is located adjacent to the trb operon of this conjugal element. Previous genetic studies of this region (D. R. Gallie, M. Hagiya, and C. I. Kado, J. Bacteriol. 161:1034-1041, 1985) identified functions involved in partitioning, origin of replication and incompatibility, and copy number control. In this study, we determined the nucleotide sequence of a 6,146-bp segment that encompasses the rep locus of pTiC58. The region contained four full open reading frames (ORFs) and one partial ORF. The first three ORFs, oriented divergently from the traI-trb operon, are closely related to the repA, repB, and repC genes of the octopine-type Ti plasmid pTiB6S3 as well as to other repA, -B, and -C genes from the Ri plasmid pRiA4b and three large plasmids from Rhizobium spp. The fourth ORF and the partial ORF are similar to y4CG and y4CF, respectively, of the Sym plasmid pNGR234a. The 363-bp intergenic region between traI and repA contained two copies of the tra box which is the cis promoter recognition site for TraR, the quorum-sensing activator of Ti plasmid conjugal transfer. Expression of the traI-trb operon from the tra box II-associated promoter mediated by TraR and its acyl-homoserine lactone ligand, AAI, was negatively influenced by an intact tra box III. On the other hand, the region containing the two tra boxes was required for maximal expression of repA, and this expression was enhanced slightly by TraR and AAI. Copy number of a minimal rep plasmid increased five- to sevenfold in strains expressing traR but only when AAI also was provided. Consistent with this effect, constitutive expression of the quorum-sensing system resulted in an apparent increase in Ti plasmid copy number. We conclude that Ti plasmid copy number is influenced by the quorum-sensing system, suggesting a connection between conjugal transfer and vegetative replication of these virulence elements.

Effects of nitrogen source and defoliation on growth and biological dinitrogen fixation of Gliricidia sepium seedlings.

Effects of four N sources and two defoliation treatments on growth and nitrogenase activity of Gliricidia sepium (Jacq.) Walp seedlings were studied in a greenhouse. All nutrients were supplied in irrigation water to the sterile growing medium. The N sources were: (1) 100 mg l(-1) of N supplied as NO(3) (-) (high-NO(3) (-)), (2) 50 mg l(-1) of N supplied as NO(3) (-) and inoculation with Rhizobium spp. medium-NO(3) (-)), (3)100 mg l(-1) of N supplied as NH(4)NO(3), and (4) inoculation with Rhizobium spp without mineral N (N(2)). At 35 weeks after sowing, mean total biomass was 130.5, 50.5, 22.9 and 17.4 g seedling(-1) in the NH(4)NO(3), N(2), medium-NO(3) (-) and high-NO(3) (-) treatments, respectively. The root/shoot ratio was high in all of the N treatments (1.73-2.77) because the seedlings had big taproots. The medium-NO(3) (-) treatment completely inhibited nodulation, whereas seedlings in the N(2) treatment were profusely nodulated. At 32 weeks after sowing, groups of seedlings in the N(2) and high-NO(3) (-) treatments were subjected to 50 or 100% defoliation. Closed-chamber acetylene reduction assays of intact root systems were conducted to compare nitrogenase activity at 7, 14 and 28 days after defoliation (DAD). At 7 and 14 DAD, nitrogenase activity of completely and partially defoliated seedlings was about 10 and 60%, respectively, of that of undefoliated controls. At 28 DAD, nitrogenase activity of completely defoliated seedlings was twice the predefoliation value, whereas nitrogenase activity of partially defoliated seedlings was only 87% of the predefoliation value. Recovery of nitrogenase activity was strongly correlated with foliage regrowth in the completely defoliated seedlings, but not in the partially defoliated seedlings. Abundant belowground C and N reserves in the large taproot probably contributed to the rapid recovery from defoliation. Accumulation of belowground biomass may also improve defoliation tolerance of mature trees.

The apoplast‐habitat of endophytic dinitrogen‐fixing bacteria and their significance for the nitrogen nutrition of nonleguminous plants

AbstractRecent attention has been drawn to plant‐growth‐promoting bacteria which colonize not only the rhizosphere and the surface of roots but also the interior of roots and shoots of higher plants. The bacteria are found in the apoplast i.e. intercellular spaces and xylem vessels. These so‐called endophytic bacteria are considered to be more effective in providing the host plant with nitrogen than associative rhizosphere bacteria on the root surface and appear to play an important role in biological nitrogen fixation (BNF) in nonleguminous plants. This is discussed in detail. Bacteria which have been found in the apoplast of nonleguminous plants include Azospirillum spp., Pantoea agglomerans, Acetobacter diazotrophicus, Herbaspirillum spp., Azoarcus spp. and Rhizobium spp. The significance of the apoplastic colonization of these bacteria for the nitrogen nutrition of tropical non‐legumes is discussed.

Type III protein secretion systems in bacterial pathogens of animals and plants.

Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli, and Chlamydia spp. and the plant pathogens Pseudomonas syringae, Erwinia spp., Ralstonia solanacearum, Xanthomonas campestris, and Rhizobium spp.

Biodegradation of phosphonomycin by Rhizobium huakuii PMY1.

The biodegradation by Rhizobium huakuii PMY1 of up to 10 mM phosphonomycin as a carbon, energy, and phosphorus source with accompanying P(i) release is described. This biodegradation represents a further mechanism of resistance to this antibiotic and a novel, phosphate-deregulated route for organophosphonate metabolism by Rhizobium spp.

Structural characterization of the K antigens from Rhizobium fredii USDA257: evidence for a common structural motif, with strain-specific variation, in the capsular polysaccharides of Rhizobium spp.

Rhizobium fredii participates in a nitrogen-fixing symbiosis with soybeans, in a strain-cultivar-specific interaction, and past studies have shown that the cell surface and extracellular polysaccharides of rhizobia function in the infection process that leads to symbiosis. The structural analysis of the capsular polysaccharides (K antigens) from strain USDA257 was performed in this study. The K antigens were extracted from cultured cells with hot phenol-water and purified by size exclusion chromatography. We isolated two structurally distinct K antigens, both containing a high proportion of 3-deoxy-D-manno-2-octulosonic acid (Kdo). The polysaccharides were characterized by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry, nuclear magnetic resonance spectrometry, and gas chromatography-mass spectrometry analyses. The primary polysaccharide, which constituted about 60% of the K-antigen preparation, consisted of repeating units of mannose (Man) and Kdo, [-->)3-beta-D-Manp-(1-->5)-beta-D-Kdop-(2-->], and a second polysaccharide consisted of 2-O-MeMan and Kdo, [-->)3-beta-D-2-O-MeManp-(1-->5)-beta-D-Kdop-(2-->]. These structures are similar to yet distinct from those of other strains of R. fredii and R. meliloti, and this finding provides further evidence that the K antigens of rhizobia are strain-specific antigens which are produced within a conserved motif.

Novel and complex chromosomal arrangement of Rhizobium loti nodulation genes.

A mutational and structural analysis of Rhizobium loti nodulation genes in strains NZP2037 and NZP2213 was carried out. Unlike the case with other Rhizobium strains examined to date, nodB was found on an operon separate from nodACIJ. Sequence analysis of the nodACIJ and nodB operon regions confirm that R. loti common nod genes have a gene organization different from that of other Rhizobium spp. At least 4 copies of nodD-like sequences were identified in R. loti. The complete nucleotide sequence of one of these, nodD3, was determined. A new host-specific nod gene, nolL, was identified adjacent to nodD3. NolL shares homology with NodX and other O-acetyl transferases. Mutational analysis of the nod regions of strains NZP2037 and NZP2213 showed that nodD3, nodI, nodJ, and nolL were all essential for R. loti strains to effectively nodulate the extended host Lotus pedunculatus, but were not necessary for effective nodulation of the less restrictive host, Lotus corniculatus. Both nodD3 and nolL were essential for R. loti strains to nodulate Leucaena leucocephala.

Differentiation of Rhizobium strains using the polymerase chain reaction with random and directed primers

Total genomic DNA isolated from Rhizobium spp was analysed by the polymerase chain reaction (PCR) in conjunction with either arbitrary oligonucleotide primers or a 20-base oligonucleotide primer corresponding to a conserved nif gene promoter region. The amplified fragment length polymorphisms (amplification profiles) generated by the 2 arbitrary primers (RPO4 and RPO5), effectively differentiated a diverse collection of Rhizobium meliloti, R. leguminosarum bv. trifolii and R. l. bv. viciae strains. The nif-directed primer (RPO1) was also highly discriminatory on Rhizobium DNA and generated unique amplification profiles for each strain. For selected strains, reproducible amplification profiles were obtained using a variety of DNA sources, and these were achievable over a 125-fold range in template concentration. Amplification profiles generated by the primers RPO5 and RPO1 were shown to be temperature dependant, with the RPO1 primer capable of generating amplification profiles at annealing temperatures up to 65°C. Reproducible amplification profiles were generated from either purified total genomic DNA or from template DNA present in freeze-thaw bacterial cell lysates. Moreover, the RPO1 primer was used to positively identify specific R. l. bv. trifolii strains directly in crude extracts prepared from squashed clover-root nodules. The significance of these results is discussed in relation to the development of strategies for using PCR-based methodologies in Rhizobium ecological studies.