Mechanism of Nitrate Tolerance in Tn5 Mutants of Cicer-Rhizobium Strains

The activities of nitrate reductase (NR) and nitrite reductase (NiR) and production of indole-3-acetic acid (IAA) by symblotic nitrate tolerant Tn5 mutant AC-10 of Cicer-Rhizobium strain F-75 and mutants BC-35 and BC-46 of strain G36-84 developed earlier, have been studied under ex planta condition. The rhizobiaI mutants and their parental strains were grown with nitrate (0.0, 0.5, 1, 2 or 4 mM), aerobically and microaerobically. The overall activities of NR were 70–91% lower in aerobically grown and 78–87% lower in microaerobically grown mutant cells compared to their parental strains. Similarly, the overall activities of NiR were 36–55% and 27–37% lower in aerobically and microaerobically grown mutant cells, respectively, compared to their parental strains. On the contrary, the overall production of IAA in the culture medium by aerobically grown mutant cells was significantly higher compared to their parental strains. Based on these results, it has been suggested that impaired NR activity and a favourable NiR/NR ratio preventing nitrite accumulation in the rhizobial mutants, may be responsible for imparting nitrate tolerance to chickpea - Rhizobium symbiotic system.     

Bradyrhizobium japonicum mutants defective in root-nodule bacteroid development and nitrogen fixation

The genome of the slow-growing Bradyrhizobium japonicum (strain 110) was mutagenized with transposon Tn5. A total of 1623 kanamycin/streptomycin resistant derivatives were screened in soybean infection tests for nodulation (Nod) and symbiotic nitrogen fixation (Fix). In this report we describe 14 strains possessing a stable, reproducible Nod⁺Fix^- phenotype. These strains were also grown under microaerobic culture conditions to test them for free-living nitrogen fixation activity (Nif). In addition to strains having reduced Fix and Nif activities, there were also strains that had reduced symbiotic Fix activity but were Nif⁺ ex planta.Analysis of the genomic structure revealed that the majority of the strains had a single Tn5 insertion without any further apparent physical alteration. A few strains had additional insertions (by Tn5 or IS50), or a deletion, or had cointegrated part of the vector used for Tn5 mutagenesis. One of the insertions was found in a known nif gene (nifD) whereas all other mutations seem to affect different, hitherto unknown genes or operons.Several mutant strains had an altered nodulation phenotype, inducing numerous, small, widely distributed nodules. Light and electron microscopy revealed that most of these mutants were defective in different stages of bacteroid development and/or bacteroid persistence. The protein patterns of the mutants were inspected by two-dimensional gel electrophoresis after labelling microaerobic cultures with l-(³⁵S)methionine. Of particular interest were mutants lacking a group of proteins the synthesis of which was known to be under oxygen control. Such strains can be regarded as potential regulatory mutants.     

A Medicago truncatula Tobacco Retrotransposon Insertion Mutant Collection with Defects in Nodule Development and Symbiotic Nitrogen Fixation

A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation. Mutants were sorted into six distinct phenotypic categories: 72 nonnodulating mutants (Nod-), 51 mutants with totally ineffective nodules (Nod+ Fix-), 17 mutants with partially ineffective nodules (Nod+ Fix+/-), 27 mutants defective in nodule emergence, elongation, and nitrogen fixation (Nod+/- Fix-), one mutant with delayed and reduced nodulation but effective in nitrogen fixation (dNod+/- Fix+), and 11 supernodulating mutants (Nod++Fix+/-). A total of 2,801 flanking sequence tags were generated from the 156 symbiotic mutant lines. Analysis of flanking sequence tags revealed 14 insertion alleles of the following known symbiotic genes: NODULE INCEPTION (NIN), DOESN'T MAKE INFECTIONS3 (DMI3/CCaMK), ERF REQUIRED FOR NODULATION, and SUPERNUMERARY NODULES (SUNN). In parallel, a polymerase chain reaction-based strategy was used to identify Tnt1 insertions in known symbiotic genes, which revealed 25 additional insertion alleles in the following genes: DMI1, DMI2, DMI3, NIN, NODULATION SIGNALING PATHWAY1 (NSP1), NSP2, SUNN, and SICKLE. Thirty-nine Nod- lines were also screened for arbuscular mycorrhizal symbiosis phenotypes, and 30 mutants exhibited defects in arbuscular mycorrhizal symbiosis. Morphological and developmental features of several new symbiotic mutants are reported. The collection of mutants described here is a source of novel alleles of known symbiotic genes and a resource for cloning novel symbiotic genes via Tnt1 tagging.     

Production and Excretion of Nod Metabolites by Rhizobium leguminosarum bv. trifolii Are Disrupted by the Same Environmental Factors That Reduce Nodulation in the Field

Lipooligosaccharides (Nod metabolites) have been shown to be essential for the successful nodulation of legumes. In strains of Rhizobium leguminosarum bv. trifolii, Nod metabolites were detected predominantly within the cell and to a lesser extent in the periplasmic space and the growth medium. The production, and in particular the excretion, of Nod metabolites was restricted by a range of environmental conditions which are associated with poor nodulation in the field. Lowering the medium pH from 7.0 to 5.0, reducing the phosphate concentration from 1 mM to 5 μM KH₂PO₄, and lowering the incubation temperature from 28 to 18°C affected the number and relative concentrations of the Nod metabolites made. The form and concentration of the nitrogen source affected the relative concentrations of the Nod metabolites produced and excreted. KNO₃ concentrations of >10 mM did not affect cell growth rate but substantially reduced the number of Nod metabolites released. Environmental stresses differentially altered Nod metabolite production and excretion in the same strain carrying different introduced nod regions. Strain ANU845(pWLH1) produced and excreted comparatively fewer Nod metabolites at pH 5.0 and at 18°C than strain ANU845(pRI4003). The excretion but not the production of Nod metabolites by strain ANU845(pRtO32) was dependent on the presence of both nodI and nodJ. Tn5-induced nodI and nodJ mutants did not accumulate any metabolites either outside the cell or within the outer membrane or periplasmic space. Recognition that Nod metabolite accumulation is a complex system of production and excretion, with each component responding differently to changes in environmental conditions, has many consequences, both at the molecular level and in the field. The ability of different strains to produce and release Nod metabolites is likely to be a major determinant of nodule occupancy and should be considered when screening strains suitable for adverse environments.     

Plasmids and stability of symbiotic properties of Rhizobium trifolii.

A conjugal plasmid which encodes both peak nodulation genes and nitrogenase genes, and which is labeled with the transposon Tn5, was transferred to a wild-type Rhizobium trifolii strain to examine the stability and expression of the host range and fixation (Fix+) phenotypes. Transconjugates were isolated which were shown to initially form nitrogen-fixing nodules (Nod+ Fix+) on both clovers and peas. These hybrid strains were then repeatedly passaged through either pea or clover nodules or onto a solid agar medium to determine whether these broadened-host-range characteristics were stably maintained. An instability was noted in the capacity of some of these hybrids to form nitrogen-fixing nodules on all of the host plants used. The broadened nodulation ability was, however, more readily maintained. In some cases, the changes in the Nod+ Fix+ phenotype could be attributed to demonstrable changes in the plasmid profile of the hybrid strains, whereas in other cases no demonstrable plasmid alterations could be detected.     

Modification of symbiotic interaction of pea (Pisum sativum L.) andRhizobium leguminosarum by induced mutations

Summary In pea (Pisum sativum L.), mutants could be induced, modified in the symbiotic interaction withRhizobium leguminosarum. Among 250 M₂-families, two nodulation resistant mutants (K₅ and K₉) were obtained. In mutant K₅ the nodulation resistance was monogenic recessive and not Rhizobium strain specific. Out of 220 M₂-families one mutant nod₃ was found which could form nodules at high nitrate concentrations (15 mM KNO₃). This mutant nodulated abundantly with severalRhizobium strains, both in the absence and presence of nitrate. Probably as the result of a pleiotropic effect, its root morphology was also changed. Among 1800 M₂-families, five nitrate reductase deficient mutants were obtained and one of them (mutant E₁) was used to study the inhibitory effect of nitrate on nodulation and nitrogen fixation.The results of the present investigation show that pea mutants which are modified in their symbiosis withRhizobium leguminosarum, can readily be obtained. The significance of such mutants for fundamental studies of the legume-Rhizobium symbiosis and for applications in plant breeding is discussed.     

Hybridization of DNA from Actinomycetes of the genus Frankia with nitrogenase structural genes (nifHDK) of Klebsiella pneumoniae and nod-genes of Rhizobium melioti

DNA sequence homology with the plasmid pSA30 carrying the cloned nifHDK genes from Klebsiella pneumoniae was revealed in ten Frankia spp. strains, nitrogen-fixing symbionts of non-legumes, irrespective of the strain phenotype (Nod+Fix+, Nod+ Fix- or Nod- Fix?). None of the Frankia spp. DNAs exhibited homology with the plasmid pRmSL26 harbouring the Rhizobium meliloti nod-genes encoding for early symbiotic functions.     

Genetic and functional analysis of the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI

Thirty Tn5- or Tn1831-induced nodulation (nod) mutants of Rhizobium leguminosarum were examined for their genetic and symbiotic properties. Thirteen mutants contained a deletion in Sym plasmid pRL1JI. These deletions cover the whole nod region and are 50 kb in size. All remaining seventeen mutations are located in a 6.6 kb EcoRI nod fragment of the Sym plasmid. Mutations in a 3.5 kb part on the right hand side of this 6.6 kb fragment completely prevent nodulation on Vicia sativa. All mutants in this 3.5 kb area are unable to induce marked root hair curling and thick and short roots.Mutations in a 1.5 kb area on the left hand side of the 6.6 kb nod fragment generate other symbiotic defects in that nodules are only rarely formed and only so after a delay of several days. Moreover, infection thread formation is delayed and root hair curling is more excessive than that caused by the parental strain. Their ability to induce thick and short roots is unaltered.Mutations in this 1.5 kb region are not complemented by pRmSL26, which carries nod genes of R. meliloti, whereas mutations in the 3.5 kb region are all complemented by pRmSL26.Abbreviations Rps repression of production of small bacteriocin - Mep medium bacteriocin production - Nod nodulation - Fix fixation - Tsr thick and short roots - Flac root hair curling - Hsp host specificity - Flad root hair deformation - Tc tetracycline - Km kanamycin - Cm chloramphenicol - Sp spectinomycin - Sm streptomycin - R resistant     

Candidatus Frankia Datiscae Dg1, the Actinobacterial Microsymbiont of Datisca glomerata,Expresses the Canonical nod Genes nodABC in Symbiosis with Its Host Plant

Frankia strains are nitrogen-fixing soil actinobacteria that can form root symbioses with actinorhizal plants. Phylogenetically, symbiotic frankiae can be divided into three clusters, and this division also corresponds to host specificity groups. The strains of cluster II which form symbioses with actinorhizal Rosales and Cucurbitales, thus displaying a broad host range, show suprisingly low genetic diversity and to date can not be cultured. The genome of the first representative of this cluster, Candidatus Frankia datiscae Dg1 (Dg1), a microsymbiont of Datisca glomerata, was recently sequenced. A phylogenetic analysis of 50 different housekeeping genes of Dg1 and three published Frankia genomes showed that cluster II is basal among the symbiotic Frankia clusters. Detailed analysis showed that nodules of D. glomerata, independent of the origin of the inoculum, contain several closely related cluster II Frankia operational taxonomic units. Actinorhizal plants and legumes both belong to the nitrogen-fixing plant clade, and bacterial signaling in both groups involves the common symbiotic pathway also used by arbuscular mycorrhizal fungi. However, so far, no molecules resembling rhizobial Nod factors could be isolated from Frankia cultures. Alone among Frankia genomes available to date, the genome of Dg1 contains the canonical nod genes nodA, nodB and nodC known from rhizobia, and these genes are arranged in two operons which are expressed in D. glomerata nodules. Furthermore, Frankia Dg1 nodC was able to partially complement a Rhizobium leguminosarum A34 nodC::Tn5 mutant. Phylogenetic analysis showed that Dg1 Nod proteins are positioned at the root of both α- and β-rhizobial NodABC proteins. NodA-like acyl transferases were found across the phylum Actinobacteria, but among Proteobacteria only in nodulators. Taken together, our evidence indicates an Actinobacterial origin of rhizobial Nod factors.     

Isolation and characterization of a gene associated with sulfate assimilation in Sinorhizobium fredii WGF03

Sulfur is an essential element for rhizobia, such as sulfated modified Nod factors and nitrogenase. To investigate the role of sulfur metabolism in Rhizobium-Soybean symbiosis, a transponson random insertional mutants’ library was constructed and a sulfur assimilation-related gene was isolated and characterized. A mutant strain unable to utilized sulfate was screened from 11,000 random insertional mutants of Sinorhizobium fredii WGF03. Sequencing analysis showed that a sulfate assimilation-related gene (cysDN) was inserted by the Tn transponson. Mutants inactivated in cysD and cysN (SMcysDF and SMcysNF) were constructed by homologous recombination using the suicide plasmid pK18mob. The mutants SMcysDF and SMcysNF could no longer utilize sulfate as sulfur source. Phenotype analysis revealed that mutation of cysDN had multiple effects on S. fredii WGF03. Root hair deformation assay showed that the activity of Nod factors secreted by mutants SMcysDR and SMcysNR elicited minimal hair initiation only. Soybean plant tests indicated that the mutant strains delayed 1–2 days to nodulate and exhibited lower nodulation efficiency and symbiotic efficiency than the wild-type strain. The complementary strain of cysD and cysN (HcysDF and HcysNF) could restore the nodulation efficiency.     

Isolation and characterization of Rhizobium (IC3342) genes that determine leaf curl induction in pigeon pea.

Nodulation by the Rhizobium strain IC3342 causes a leaf curl syndrome in certain tropical legumes such as pigeon pea (Cajanus cajan) (N.M. Upadhyaya, J.V.D.K. Kumar Rao, D.S. Letham, and P.J. Dart, Physiological and Molecular Plant Pathology 39:357-373, 1991). Transposon (Tn5) mutagenesis of this leaf curl-inducing (Curl+) Rhizobium strain yielded two Curl- Fix- and three Curl- Fix+ mutants. Plasmid visualization and subsequent Southern blot hybridization analyses with Tn5, nif and nod gene probes showed that the Tn5 element had inserted into the symbiotic (Sym) plasmid in three of the mutants. Restriction endonuclease analyses indicated that none of the Tn5 insertions were closely linked. Tn5-containing EcoRI fragments were cloned from each mutant and used as probes to isolate the corresponding wild-type DNA fragments from a cosmid (pLAFR3) genomic library. Fix+ and/or Curl+ phenotypes were restored in each mutant by the introduction of cosmids containing the corresponding wild-type DNA. A closely related but Curl- Rhizobium strain ANU240 was shown, by Southern hybridization, to contain conserved DNA sequences of all but one of the identified genetic regions of the Curl+ Rhizobium strain IC3342. Cosmids containing the genetic region unique to the strain IC3342, designated lcr1, conferred a Curl+ phenotype on the strain ANU240. DNA sequence analysis of the cloned lcr1 region revealed five open reading frames (ORFs). The ORF2 showed homology with the Escherichia coli regulatory gene ompR, and ORF4 showed homology with E. coli and Rhizobium meliloti regulatory genes fnr and fixK, respectively.     

Cytochrome mutants of bradyrhizobium induced by transposon tn5

Transposon Tn5 was used to mutate Bradyrhizobium japonicum USDA 61N. From over 5000 clones containing Tn5, 12 were selected and purified using a chemical reaction to identify oxidase-deficient clones. Four classes of mutants were identified based on the alterations in cytochromes. Most of the mutants had alterations in more than one cytochrome. Southern hybridization analysis of restricted genomic DNA of a representative strain of each class demonstrated that each mutant had a single Tn5 insert. Thus a single Tn5 insert produced pleiotropic effects on cytochromes. One class, which was totally deficient in cytochromes aa₃ and c, produced ineffective nodules on soybeans. Most of the strains representing the other classes produced effective nodules but exceptions were observed in each class. Bacteroids of the wild-type strain contained cytochrome aa₃. Bacteroids from one class of mutants were totally devoid of cytochrome aa₃. Several of these strains produced effective symbioses indicating that cytochrome aa₃ is not required for an effective symbiosis in this DNA homology group II strain which normally has this terminal oxidase in bacteroids.     

Transconjugants of Agrobacterium radiobacter harbouring sym genes of Rhizobium galegae can form an effective symbiosis with Medicago sativa

It is known that the Rhizobium galegae genomes contain megaplasmids. The suicide vector pSUP2111 with nifH gene of R. meliloti was introduced into the strains CIAM 0703 and CIAM 0711 of R. galegae inducing effective nodules on Galega orientalis plants. The formation of self-transmissible megaplasmids was observed. The megaplasmid transfer into non-nodulating R. meliloti mutants resulted in partial complementation of the nodulation defect in recipient strains though only one transconjugant showed the nitrogen-fixing activity in symbiosis with alfalfa and another one in symbiosis with G. orientalis plants. Among the Agrobacterium strains harbouring R. galegae megaplasmids there were four classes of transconjugants: (1) Nod+ Fix- in symbiosis with goat's rue plants (three strains); (2) Nod+ Fix- on Medicago sativa (two strains); (3) Nod+ Fix+ on M. sativa (five strains); (4) Nod- with both plant hosts (11 strains).     

Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis

We have physically and genetically characterized 20 symbiotic and 20 auxotrophic mutants of Rhizobium meliloti, the nitrogen-fixing symbiont of alfalfa (Medicago sativa), isolated by transposon Tn5 mutagenesis. A "suicide plasmid" mutagenesis procedure was used to generate TN-5-induced mutants, and both auxotrophic and symbiotic mutants were found at a frequency of 0.3% among strains containing random TN5 insertions. Two classes of symbiotic mutants were isolated: 4 of the 20 formed no nodules at all (Nod-), and 16 formed nodules which failed to fix nitrogen (Fix-). We used a combination of physical and genetic criteria to determine that in most cases the auxotrophic and symbiotic phenotypes could be correlated with the insertion of a single Tn5 elements. Once the Tn5 element was inserted into the R. meliloti genome, the frequency of its transposition to a new site was approximately 10-8 and the frequency of precise excision was less than 10-9. In approximately 25% of the mutant strains, phage Mu DNA sequences, which originated from the suicide plasmid used to generate the Tn5 transpositions, were also found in the R. meliloti genome contiguous with Tn5. These later strains exhibited anomalous conjugation properties, and therefore we could not correlate the symbiotic phenotype with a Tn5 insertion. In general, we found that both physical and genetic tests were required to fully characterize transposon-induced mutations.     

Effect of N and P nutrition on extracellular secretion of lumichrome, riboflavin and indole acetic acid by N2-fixing bacteria and endophytes isolated from Psoralea nodules

Soils of the Cape Fynbos in South Africa are very low in nutrients, especially N and P, which affect bacterial growth and metabolism. In this study, the effect of supplying nitrate (14.8 and 59.3?mM NO ₃ ^? ), ammonium (28.1 and 112.0?mM NH ₄ ⁺ ) and phosphorus (1.4 and 5.7?mM P) to five N₂-fixing and 11 non-nodulating bacterial strains isolated from root nodules of Psoralea species in the Cape Fynbos was assessed. The data revealed marked variation in the secretion of lumichrome, riboflavin and IAA into culture filtrate. There was generally greater production of lumichrome, riboflavin and IAA by the N₂-fixing bacteria than those unable to nodulate P. pinnata and siratro, with much greater concentrations of lumichrome and riboflavin in culture filtrate at high P than low P. At low and high P, symbiotic strain TUT57pp produced 2.2-fold and 3.2-fold more IAA than TUT65prp and TUT33pap respectively, (two non-nodulating strains also with greater IAA production). Although ammonium nutrition has no effect on riboflavin production, it altered lumichrome concentrations in culture filtrate. While ammonium application had no effect, supplying bacterial cells with high nitrate concentration significantly decreased cellular production of lumichrome and riboflavin, two important symbiotic signal molecules. The observed nitrate inhibition of lumichrome and riboflavin biosynthesis and release is in addition to its depressive effect on nodulation and N₂ fixation in symbiotic legumes.     

Generation of Bradyrhizobium japonicum Mutants with Increased N2O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N₂O) reductase (N₂OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N₂O respiration. Mutation of dnaQ, which encodes the epsilon subunit of DNA polymerase III, gives a strong mutator phenotype in Escherichia coli. pKQ2 introduction into B. japonicum USDA110 increased the frequency of occurrence of colonies spontaneously resistant to kanamycin. A series of repeated cultivations of USDA110 with and without pKQ2 was conducted in anaerobic conditions under 5% (vol/vol) or 20% (vol/vol) N₂O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N₂OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 to 12 times the N₂OR activity of the wild-type USDA110. Although two mutants had a weak or null fix phenotype for symbiotic nitrogen fixation, the remaining two (5M09 and 5M14) had the same symbiotic nitrogen fixation ability and heterotrophic growth in culture as wild-type USDA110.     

Isolation and Characterization of an Oxygen Sensitive Mutant of Azorhizobium caulinodans

An oxygen sensitive mutant of Azorhizobium caulinodans strain IRBG 46 was isolated by NTG mutagenesis. It was defective in N₂ fixation under 3% O₂ level, while under 1% O₂ it was almost as active as the parent strain IRBG 46. The mutant was also found to be a slow grower with reduced respiratory activity, low azide tolerance and no catalase activity. However, it did not differ from its parent strain with respect to nitrate respiration. Under symbiotic condition the mutant formed smaller, light green nodules as compared to bigger, dark green nodules formed by the wild type strain. The mutant was also defective in N₂ fixation under symbiotic condition. Complementation analysis showed that the mutation might be in either fixL or fixJ gene which are involved in O₂ regulation of nif/fix gene expression. A possible role of all these factors in conferring a highly O₂ tolerant nitrogen fixing system in the organism, has been discussed.     

Identification of genes involved in salt tolerance and symbiotic nitrogen fixation in chickpea rhizobium Mesorhizobium ciceri Ca181

In an attempt to find the genes involved in salt tolerance of the highly adaptable chickpea rhizobium strain, Mesorhizobium ciceri Ca181, a Tn5 transposon insertion library was generated and screened to identify five mutants with inability to survive in the presence of 0.1 M NaCl. The genes disrupted in these mutants due to insertion of the transposon were identified by sequencing of Tn5 flanking sequences after inverse PCR. One of the mutants had a disruption in diguanylate cyclase gene which is involved in bacterial biofilm formation and persistence. The second mutant had a disruption in an ABC transporter membrane protein gene, which is involved in the uptake of nutrients and cellular osmoprotection. The third mutant had a disruption in a gene showing homology with rhamnulose 1-phosphate aldolase which has an important role in the central metabolism of L-rhamnulose. The fourth mutant had a disruption in a capsule synthesis gene and the fifth mutant had an insertion in an oxidoreductase gene. When these mutants were inoculated into the host chickpea plant under normal non-saline conditions, they formed symbiotic nodules but with severely reduced nitrogenase activity. Hence, it appears that bacterial ability to adapt to hyper-osmotic salt stress conditions is also important for its nitrogen fixing ability in the chickpea root nodules. Allele mining for variant forms of the identified genes in the germplasm resources of M. ciceri may help in the development of highly adaptive and efficient nitrogen fixing strains of the chickpea rhizobium.