Establishment of a root proteome reference map for the model legume Medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting.

We have established a proteome reference map for Medicago truncatula root proteins using two-dimensional gel electrophoresis combined with peptide mass fingerprinting to aid the dissection of nodulation and root developmental pathways by proteome analysis. M. truncatula has been chosen as a model legume for the study of nodulation-related genes and proteins. Over 2,500 root proteins could be displayed reproducibly across an isoelectric focussing range of 4-7. We analysed 485 proteins by peptide mass fingerprinting, and 179 of those were identified by matching against the current M. truncatula expressed sequence tag (EST) database containing DNA sequences of approximately 105,000 ESTs. Matching the EST sequences to available plant DNA sequences by BLAST searches enabled us to predict protein function. The use of the EST database for peptide identification is discussed. The majority of identified proteins were metabolic enzymes and stress response proteins, and 44% of proteins occurred as isoforms, a result that could not have been predicted from sequencing data alone. We identified two nodulins in uninoculated root tissue, supporting evidence for a role of nodulins in normal plant development. This proteome map will be updated continuously (http://semele.anu.edu.au/2d/2d.html) and will be a powerful tool for investigating the molecular mechanisms of root symbioses in legumes.

Characterisation of rice anther proteins expressed at the young microspore stage.

In combination with two-dimensional polyacrylamide gel electrophoresis (2-DE) protein mapping and mass spectrometry analysis, the pattern of gene expression in specific tissues at a specific stage can be displayed and characterised. We used this approach for rice (Oryza sativa L. cultivar Doongara) to display and assign identity to proteins in the anthers at the young microspore stage. Over 4000 anther proteins in the pI range of 4-11 and molecular mass range of 6-122 kDa were reproducibly resolved after silver staining, representing about 10% of the estimated total genomic output of rice. Two hundred and seventy-three protein spots have been extracted either from polyninylidene diffluoride membrane blots or from colloidal Coomassie blue stained 2-DE gels and analysed by N-terminal sequencing, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MS) analysis or tandem MS sequencing. This enabled identification of 53 anther protein spots representing 43 different proteins. Using the publicly available rice expressed sequence tag (EST) database at the National Centre for Biotechnology Information, a further 37 protein spots were matched to ESTs. After BLAST searching with these ESTs, we were able to predict the identity of 22 of these protein spots. Proteome reference maps of rice anthers have been constructed according to the SWISS-2DPAGE standards and are available for public access at http://semele.anu.edu.au/2d/2d.html.

Elevated levels of synthesis of over 20 proteins results after mutation of the Rhizobium leguminosarum exopolysaccharide synthesis gene pssA.

The protein expression profiles of Rhizobium leguminosarum strains in response to specific genetic perturbations in exopolysaccharide (EPS) biosynthesis genes were examined using two-dimensional gel electrophoresis. Lesions in either pssA, pssD, or pssE of R. leguminosarum bv. viciae VF39 or in pssA of R. leguminosarum bv. trifolii ANU794 not only abolished the capacity of these strains to synthesize EPS but also had a pleiotropic effect on protein synthesis levels. A minimum of 22 protein differences were observed for the two pssA mutant strains. The differences identified in the pssD and pssE mutants of strain VF39 were a distinct subset of the same protein synthesis changes that occurred in the pssA mutant. The pssD and pssE mutant strains shared identical alterations in the proteins synthesized, suggesting that they share a common function in the biosynthesis of EPS. In contrast, a pssC mutant that produces 38% of the EPS level of the parental strain showed no differences in its protein synthesis patterns, suggesting that the absence of EPS itself was contributing to the changes in protein synthesis and that there may be a complex interconnection of the EPS biosynthetic pathway with other metabolic pathways. Genetic complementation of pssA can restore wild-type protein synthesis levels, indicating that many of the observed differences in protein synthesis are also a specific response to a dysfunctional PssA. The relevance of these proteins, which are grouped as members of the pssA mutant stimulon, remains unclear, as the majority lacked a homologue in the current sequence databases and therefore possibly represent a novel functional network(s). These findings have illustrated the potential of proteomics to reveal unexpected higher-order processes of protein function and regulation that arise from mutation. In addition, it is evident that enzymatic pathways and regulatory networks are more interconnected and more sensitive to structural changes in the cell than is often appreciated. In these cases, linking the observed phenotype directly to the mutated gene can be misleading, as the phenotype could be attributable to downstream effects of the mutation.

Temporal and spatial order of events during the induction of cortical cell divisions in white clover by Rhizobium leguminosarum bv. trifolii inoculation or localized cytokinin addition.

We examined the timing and location of several early root responses to Rhizobium leguminosarum bv. trifolii infection, compared with a localized addition of cytokinin in white clover, to study the role of cytokinin in early signaling during nodule initiation. Induction of ENOD40 expression by either rhizobia or cytokinin was similar in timing and location and occurred in nodule progenitor cells in the inner cortex. Inoculation of rhizobia in the mature root failed to induce ENOD40 expression and cortical cell divisions (ccd). Nitrate addition at levels repressing nodule formation inhibited ENOD40 induction by rhizobia but not by cytokinin. ENOD40 expression was not induced by auxin, an auxin transport inhibitor, or an ethylene precursor. In contrast to rhizobia, cytokinin addition was not sufficient to induce a modulation of the auxin flow, the induction of specific chalcone synthase genes, and the accumulation of fluorescent compounds associated with nodule initiation. However, cytokinin addition was sufficient for the localized induction of auxin-induced GH3 gene expression and the initiation of ccd. Our results suggest that rhizobia induce cytokinin-mediated events in parallel to changes in auxin-related responses during nodule initiation and support a role of ENOD40 in regulating ccd. We propose a model for the interactions of cytokinin with auxin, ENOD40, flavonoids, and nitrate during nodulation.

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.

Identification of nolR-regulated proteins in Sinorhizobium meliloti using proteome analysis.

Extractable proteins from Sinorhizobium meliloti strains AK631 and EK698 (a Tn5-induced noIR-deficient mutant of AK631), grown in tryptone agar (TA) medium with or without the addition of the plant signal luteolin, were separated by two-dimensional gel electrophoresis and compared. Analysis of silver-stained gels showed that the noIR mutant had 189 proteins that were significantly altered in their levels (101 protein spots up- and 88 downregulated). Coomassie-stained preparative two-dimensional (2-D) gels or polyvinylidene difluoride (PVDF) membranes blotted from preparative gels showed that at least 52 of the altered proteins could be reproducibly detected and isolated from the noIR mutant. These 52 altered protein spots were classified into five groups based on an assessment of protein abundance by computer analysis and the effect of the presence or absence of luteolin addition to the growth medium. N-terminal microsequencing of 38 proteins revealed that the most striking feature of the consequence of the noIR mutation is the number and broad spectrum of cellular functions that are affected by the loss of the NoIR function. These include proteins involved in the tricarboxylic acid (TCA) cycle, heat shock and cold shock proteins, protein synthesis, a translation elongation factor, oxidative stress and cell growth and maintenance functions. We propose that the NoIR repressor is a global regulatory protein which responds to environmental factors to fine-tune intracellular metabolism.

Proteome analysis demonstrates complex replicon and luteolin interactions in pSyma-cured derivatives of Sinorhizobium meliloti strain 2011.

Sinorhizobium meliloti was studied by proteomic analysis to investigate the contribution made by plasmid-encoded functions on the intracellular regulation of this bacterium. Protein profiles of strain 2011 were compared with those from its mutant strains which were either cured of their pRme2011a (also called pSyma) plasmid (strain 818), or contained an extensive deletion of this plasmid (strain SmA146). Plasmid pSyma contains the nodulation and nitrogen fixation genes and is 1.4 Mbp with an estimated coding potential of 1,400 proteins. However, under the growth conditions used we could detect 60 differences between the parent strain and its pSyma-cured derivative, strain 818. While the majority of these differences were due to regulatory changes, such as up- and downregulation, some proteins were totally missing in some strains. These 60 proteins were classified into 21 subgroups, A to U, based on their measured protein levels when the cells were grown in the presence or absence of luteolin. Comparisons were made between the different strains to assess the possible interactions of the different proteins of the subgroups and plasmid pSyma. These results suggest that pSyma has a role in the regulation of the expression of genes from the other replicons (3.5 Mbp chromosome and the 1.7 Mbp pSymB plasmid) present in the S. meliloti cells. Proteome analysis provides a sensitive tool to examine the functional organisation of the S. meliloti genome and the intracellular gene interactions between replicons and will provide a powerful analytical tool to complement the genome sequencing of strain 1021.

Proteome analysis of the model microsymbiont Sinorhizobium meliloti: isolation and characterisation of novel proteins.

Sinorhizobium meliloti is an agriculturally and ecologically important microbe due to its capacity to establish nitrogen-fixing symbiosis with plant legumes. Two-dimensional gel electrophoresis of total cellular protein was used to establish a proteome reference map for the model microsymbiont Sinorhizobium meliloti strain 1021. The extent of changes in the gene expression of cells grown in a defined medium at different growth phases was established. After examination of over 2000 resolved protein spots, a minimum of 52 reproducible changes in protein expression levels were detected when early exponential phase cells were compared to late exponential phase cells. In contrast, induction of nodulation gene expression by the addition of the flavonoid luteolin to cells did not result in detectable changes in protein expression at either early or late exponential phase. N-terminal microsequencing of eighteen unknown constitutive proteins plus four proteins, induced or up-regulated in late exponential phase cells, allowed the identification of proteins not previously described in rhizobia. These included an amide-binding protein, a putative hydrolase of the glyoxalase II protein family, a nucleoside diphosphate kinase, and a 5'-nucleotidase. N-terminal microsequencing was also valuable in revealing N-terminal post-translational processing and assigning a subcellular location to the analysed protein. Proteome analysis will provide a powerful analytical tool to complement the sequencing of the genome of strain 1021.

Comparison of characteristics of the nodX genes from various Rhizobium leguminosarum strains.

We have analyzed the nucleotide sequences of the nodX genes from two strains of Rhizobium leguminosarum bv. viciae able to nodulate Afghan peas (strains A1 and Himalaya) and from two strains of R. leguminosarum bv. trifolii (ANU843 and CSF). The nodX genes of strains A1 and ANU843 were shown to be functional for the induction of nodules on Afghan peas. To analyze the cause of phenotypic differences of strain A1 and strain TOM we have studied the composition of the lipochitin-oligosaccharides (LCOs) produced by strain A1 after induction by the flavonoid naringenin or various pea root exudates. The structural analysis of the LCOs by mass spectrometry revealed that strain A1 synthesizes a family of at least 23 different LCOs. The use of exudates instead of naringenin resulted only in quantitative differences in the ratios of various LCOs produced.

Determination of plasmid-encoded functions in Rhizobium leguminosarum biovar trifolii using proteome analysis of plasmid-cured derivatives.

We have used proteome analysis of derivatives of R. leguminosarum biovar trifolii strain ANU843, cured of indigenous plasmids by a direct selection system, to investigate plasmid-encoded functions. Under the conditions used, the plasmid-encoded gene products contributed to only a small proportion of the 2000 proteins visualised in the two-dimensional (2-D) protein map of strain ANU843. The level of synthesis of thirty-nine proteins was affected after curing of either plasmid a, c or e. The differences observed upon plasmid curing included: protein loss, up/down-regulation of specific proteins and novel synthesis of some proteins. This suggests that a complex interplay between the cured plasmid and the remaining replicons is occurring. Twenty-two proteins appeared to be absent in the cured strains and these presumably are encoded by plasmid genes. Of these, a small heat shock protein, a cold shock protein, a hypothetical YTFG-29.7 kDa protein, and the alpha and beta subunits of the electron transfer flavoprotein were identified by N-terminal microsequencing and predicted to be encoded by plasmid e. Four of the sequenced proteins putatively encoded on plasmid e and two encoded on plasmid c were novel. In addition, curing of plasmid e and c consistently decreased the levels of 3-isopropylmalate dehydratase and malate dehydrogenase, respectively, suggesting that levels of these proteins may be influenced by plasmid-encoded functions. A protein with homology to 4-oxalocrotonate tautomerase, which is involved in the biodegradation of phenolic compounds, was found to be newly synthesised in the strain cured of plasmid e. Proteome analysis provides a sensitive tool to examine the functional organisation of the Rhizobium genome and the global gene interactions which occur between the different replicons.

Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides.

The expression of the auxin responsive reporter construct, GH3:gusA, was examined in transgenic white clover plants to assess changes in the auxin balance during the earliest stages of root nodule formation. Reporter gene expression was monitored at marked locations after the application of bacteria or signal molecules using two precise inoculation techniques: spot-inoculation and a novel method for ballistic microtargeting. Changes in GH3:gusA expression were monitored after the inoculation of Rhizobium leguminosarum biovar trifolii, non-host rhizobia, lipo-chitin oligosaccharides (LCOs), chitin oligosaccharides, a synthetic auxin transport inhibitor (naphthylphthalamic acid; NPA), auxin, the ENOD40-1 peptide or different flavonoids. The results show that clover-nodulating rhizobia induce a rapid, transient and local downregulation of GH3:gusA expression during nodule initiation followed by an upregulation of reporter gene expression at the site of nodule initiation. Microtargeting of auxin caused a local and acropetal upregulation of GH3:gusA expression, whereas NPA caused local and acropetal downregulation of expression. Both spot-inoculation and microtargeting of R. l. bv. trifolii LCOs or flavonoid aglycones induced similar changes to GH3:gusA expression as NPA. O-acetylated chitin oligosaccharides caused similar changes to GH3:gusA expression as R. l. bv. trifolii spot-inoculation, but only after delivery by microtargeting. Non-O-acetylated chitin oligosaccharides, flavonoid glucosides or the ENOD40-1 peptide failed to induce any detectable changes in GH3:gusA expression. GH3:gusA expression patterns during the later stages of nodule and lateral root development were similar. These results support the hypothesis that LCOs and chitin oligosaccharides act by perturbing the auxin flow in the root during the earliest stages of nodule formation, and that endogenous flavonoids could mediate this response.

New Rhizobium leguminosarum flavonoid-induced proteins revealed by proteome analysis of differentially displayed proteins.

Proteome analysis was used to establish the first two-dimensional protein map of Rhizobium. R. leguminosarum bv. trifolii strain ANU843 was grown in defined medium in the presence and absence of the flavonoid 7,4'-dihydroxyflavone. Over 1,700 constitutive proteins were resolved, representing about 30% of the estimated genomic output. Proteome analysis of flavonoid-treated cells was done to reveal differentially displayed proteins. The results showed that while the global expression pattern of proteins was largely unaltered by the treatment, four inducible proteins were observed. The four inducible proteins and 20 constitutively expressed proteins were subjected to sequence analysis to provide internal standards for the construction of a two-dimensional Rhizobium protein data base. The identity of 12 proteins, including NodE and NodB, was established. NodE was present throughout the growth of the cells but was diminished in amount in stationary phase cells whereas NodB was not detected in the later stages of growth. Two of the induced proteins sequenced did not match any known nodulation gene product, with one of these being present in mid-late log and stationary phase cells and possessing four consecutive His residues at the N-terminal sequencing was successful with 100 to 200 fmol of protein. Proteome analysis provides a sensitive new tool to examine plant-microbe interactions.

Transgenic white clover. Studies with the auxin-responsive promoter, GH3, in root gravitropism and lateral root development.

We report improved method for white clover (Trifolium repens) transformation using Agrobacterium tumefaciens. High efficiencies of transgenic plant production were achieved using cotyledons of imbibed mature seed. Transgenic plants were recovered routinely from over 50% of treated cotyledons. The bar gene and phosphinothricin selection was shown to be a more effective selection system than nptII (kanamycin selection) or aadA (spectinomycin selection). White clover was transformed with the soybean auxin responsive promoter, GH3, fused to the GUS gene (beta-glucuronidase) to study the involvement of auxin in root development. Analysis of 12 independent transgenic plants showed that the location and pattern of GUS expression was consistent but the levels of expression varied. The level of GH3:GUS expression in untreated plants was enhanced specifically by auxin-treatment but the pattern of expression was not altered. Expression of the GH3:GUS fusion was not enhanced by other phytohormones. A consistent GUS expression pattern was evident in untreated plants presumably in response to endogenous auxin or to differences in auxin sensitivity in various clover tissues. In untreated plants, the pattern of GH3:GUS expression was consistent with physiological responses which are regarded as being auxin-mediated. For the first time it is shown that localised spots of GH3:GUS activity occurred in root cortical tissue opposite the sites where lateral roots subsequently were initiated. Newly formed lateral roots grew towards and through these islands of GH3:GUS expression, implying the importance of auxin in controlling lateral root development. Similarly, it is demonstrated for the first time that gravistimulated roots developed a rapid (within 1 h) induction of GH3:GUS activity in tissues on the non-elongating side of the responding root and this induction occurred concurrently with root curvature. These transgenic plants could be useful tools in determining the physiological and biochemical changes that occur during auxin-mediated responses.

Efficient callus formation and plant regeneration from leaves of oats (Avena sativa L.).

An efficient plant regeneration system from leaf-derived callus in 6 genotypes of Avena sativa L. has been established. Regenerable callus was induced in the basal 1-2 mm region of 2 to 5 day old seedlings. Plants were regenerated from the regenerable callus and grown to maturity. The frequency of regenerable callus formation and plant regeneration was correlated with the position, developmental stage and genotype of the expiant. The regeneration capacity of the first one mm of the leaf basal region from three day old seedlings was comparable to that of immature embryos. Furthermore, the leaf regenerable calli were subcultured for 8 months without loss of their regeneration capabilities.

Rhizobium inoculation and physical wounding result in the rapid induction of the same chalcone synthase copy in Trifolium subterraneum.

The gene or genes encoding chalcone synthase (CHS) in the legume Trifolium subterraneum (subterranean clover) were induced within 6 hr after inoculation with Rhizobium leguminosarum bv. trifolii strain ANU843. No induction was found in uninoculated controls or plants inoculated with either the nodulation-deficient R. l. bv. trifolii strain ANU845 (pSym-) or R. meliloti strain 1021, which is capable of nodulating alfalfa but not clover. Morphological examination of the interaction between the legume and bacteria in this system showed that root hair distortion (a marker of the early events in the interaction) was apparent within 10 hr after inoculation. This indicated that CHS induction could occur before any detectable sign of rhizobial penetration of root hairs. The addition of a crude preparation of R. l. bv. trifolii lipooligosaccharide signals (Nod metabolites) to the plant growth medium had no effect on the expression of CHS over 36 hr, although root hair distortion was apparent over this time. These treatments were then contrasted with physical wounding. Wounding the plants led to a rapid induction of CHS, occurring within 2 hr. Sequence analysis of cloned CHS cDNA from pools sampled after Rhizobium inoculation or wounding treatments showed the gene designated CHS5 was the major CHS species in both treatments. Conserved sequences were found in promoters of CHS5 and soybean Gmchs7, a gene which has overlapping expression patterns. These findings support the view that the induction of the phenylpropanoid pathway is involved in the very early events of the Rhizobium infection of legumes.

In Trifolium subterraneum, chalcone synthase is encoded by a multigene family.

Chalcone synthase (CHS) catalyzes the first and key regulatory step in flavonoid biosynthesis. We report the existence and characterization of a CHS multigene family present in Trifolium subterraneum L. cultivar Karridale. The CHS family consists of at least four members, which are tightly clustered in a 15-kb region. The complete sequences of two of these genes (CHS1 and CHS2) are presented. The putative promoters of these genes have sequences which are homologous to those known, or implicated, in regulation of the expression of phenylpropanoid-encoding genes.

Studies of the Physiological and Genetic Basis of Acid Tolerance in Rhizobium leguminosarum biovar trifolii.

Acid-tolerant Rhizobium leguminosarum biovar trifolii ANU1173 was able to grow on laboratory media at a pH as low as 4.5. Transposon Tn5 mutagenesis was used to isolate mutants of strain ANU1173, which were unable to grow on media at a pH of less than 4.8. The acid-tolerant strain ANU1173 maintained a near-neutral intracellular pH when the external pH was as low as 4.5. In contrast, the acid-sensitive mutants AS25 and AS28 derived from ANU1173 had an acidic intracellular pH when the external pH was less than 5.5. The acid-sensitive R. leguminosarum biovar trifolii ANU794, which was comparatively more sensitive to low pH than mutants AS25 and AS28, showed a more acidic internal pH than the two mutants when the three strains were exposed to medium buffered at a pH of less than 5.5. The two acid-sensitive mutants had an increased membrane permeability to protons but did not change their proton extrusion activities. However, the acid-sensitive strain ANU794 exhibited both a higher membrane permeability to protons and a lower proton extrusion activity compared with the acid-tolerant strain ANU1173. DNA hybridization analysis showed that mutants AS25 and AS28 carried a single copy of Tn5 located in 13.7-kb (AS25) and 10.0-kb (AS28) EcoRI DNA fragments. The wild-type DNA sequences spanning the mutation sites of mutants AS25 and AS28 were cloned from genomic DNA of strain ANU1173. Transfer of these wild-type DNA sequences into corresponding Tn5-induced acid-sensitive mutants, respectively, restored the mutants to their acid tolerance phenotypes. Mapping studies showed that the AS25 locus was mapped to a 5.6-kb EcoRI-BamHI megaplasmid DNA fragment, whilst the AS28 locus was located in an 8.7-kb BglII chromosomal DNA fragment.

Involvement of Genes on a Megaplasmid in the Acid-Tolerant Phenotype of Rhizobium leguminosarum Biovar Trifolii.

The acid-tolerant Rhizobium leguminosarum biovar trifolii strain ANU1173 exhibited several new phenotypes when cured of its symbiotic (Sym) plasmid and the second largest megaplasmid. Strain P22, which has lost these two plasmids, had reduced exopolysaccharide production and cell mobility on TY medium. The parent strain ANU1173 was able to grow easily in laboratory media at pH 4.5, whereas the derivative strain P22 was unable to grow in media at a pH of <4.7. The intracellular pH of strain ANU1173 was 6.8 when the external pH was 4.5. In contrast, strain P22 had an acidic intracellular pH of <6.4 when the external pH was <5.5. Strain P22 had a dramatically increased membrane permeability to protons and decreased proton extrusion activity. Analysis with sodium dodecyl sulfate-polyacrylamide gels showed that strain P22 lacked a slow-migrating lipopolysaccharide (LPS) banding group which was present in the parent strain. Mobilization of the second largest megaplasmid of strain ANU1173 back into strain P22 restored the altered LPS structure and physiological characteristics of strain P22. Mobilization of the Sym plasmid of strain ANU1173 into strain P22 showed that the second largest megaplasmid of strain ANU1173 was required for the establishment of nitrogen-fixing nodules on Trifolium repens and Trifolium subterraneum. Furthermore, an examination of a large number of specific exopolysaccharide- or LPS-deficient Rhizobium mutants did not show a positive correlation between exopolysaccharide or LPS synthesis and acid tolerance.

Construction of an Acid-Tolerant Rhizobium leguminosarum Biovar Trifolii Strain with Enhanced Capacity for Nitrogen Fixation.

Strain ANU1173 is an acid-tolerant Rhizobium leguminosarum biovar trifolii strain that is able to nodulate subterranean clover plants growing in agar culture at pH 4.4 At pH 6.5, its symbiotic effectiveness in association with Trifolium subterraneum cv. Mt. Barker was 80% relative to that of strain ANU794, a Sm derivative of the commercial inoculant R. leguminosarum bv. trifolii TA1. Strain ANU1173 contained four indigenous megaplasmids, the smallest of these being the symbiotic (Sym) plasmid. The critical pH requirement for growth of strain ANU1173 in laboratory media was shown not to be associated with this plasmid. When the Sym plasmid of strain ANU1173(pSym-1173) was mobilized into a Nod strain of R. leguminosarum bv. viciae, the plasmid conferred to the transconjugant a level of symbiotic effectiveness in association with T. subterraneum that was similar to that observed with ANU1173. The symbiotic effectiveness of strain ANU1173 was improved by first curing pSym-1173 (generating strain ANU1184) and replacing it with another R. leguminosarum bv. trifolii Sym plasmid, pBR1AN. Subterranean clover plants inoculated with strain ANU1184 (pBR1AN) exhibited a 35 or 53% increase in acetylene reduction activity and a 20 or 17% increase in dry weight when grown at pH 6.5 and pH 4.4, respectively, compared with plants inoculated with strain ANU1173 and grown under the same pH conditions. It was further shown that pBR1AN was stably maintained in strain ANU1184 under free-living and symbiotic conditions. These results indicate that it is possible to construct an acid-tolerant strain of R. leguminosarum bv. trifolii with an enhanced capacity for nitrogen fixation.

Heterologous exopolysaccharide production in Rhizobium sp. strain NGR234 and consequences for nodule development.

Rhizobium sp. strain NGR234 produces large amounts of acidic exopolysaccharide. Mutants that fail to synthesize this exopolysaccharide are also unable to nodulate the host plant Leucaena leucocephala. A hybrid strain of Rhizobium sp. strain NGR234 containing exo genes from Rhizobium meliloti was constructed. The background genetics and nod genes of Rhizobium sp. strain NGR234 are retained, but the cluster of genes involved in exopolysaccharide biosynthesis was deleted. These exo genes were replaced with genes required for the synthesis of succinoglycan exopolysaccharide from R. meliloti. As a result of the genetic manipulation, the ability of these hybrids to synthesize exopolysaccharide was restored, but the structure was that of succinoglycan and not that of Rhizobium sp. strain NGR234. The replacement genes were contained on a cosmid which encoded the entire known R. meliloti exo gene cluster, with the exception of exoB. Cosmids containing smaller portions of this exo gene cluster did not restore exopolysaccharide production. The presence of succinoglycan was indicated by staining with the fluorescent dye Calcofluor, proton nuclear magnetic resonance spectroscopy, and monosaccharide analysis. Although an NGR234 exoY mutant containing the R. meliloti exo genes produced multimers of the succinoglycan repeat unit, as does the wild-type R. meliloti, the deletion mutant of Rhizobium sp. strain NGR234 containing the R. meliloti exo genes produced only the monomer. The deletion mutant therefore appeared to lack a function that affects the multiplicity of succinoglycan produced in the Rhizobium sp. strain NGR234 background. Although these hybrid strains produced succinoglycan, they were still able to induce the development of an organized nodule structure on L. leucocephala. The resulting nodules did not fix nitrogen, but they did contain infection threads and bacteroids within plant cells. This clearly demonstrated that a heterologous acidic exopolysaccharide structure was sufficient to enable nodule development to proceed beyond the developmental barrier imposed on mutants of Rhizobium sp. strain NGR234 that are unable to synthesize any acidic exopolysaccharide.