Strain NZP2037 Research Articles

Assessment of Polygala paniculata (Polygalaceae) characteristics for evolutionary studies of legume\u2013rhizobia symbiosis

Root nodule (RN) symbiosis is a mutualistic interaction observed between nitrogen-fixing soil bacteria and nodulating plants, which are scattered in only four orders of angiosperms called nitrogen-fixing clade. Most of legumes engage in RN symbiosis with rhizobia. Molecular genetic analyses with legumes and non-leguminous nodulating plants revealed that RN symbiosis utilizes early signalling components that are required for symbiosis with arbuscular mycorrhizal (AM) fungi. However detailed evolutionary processes are still largely unknown. Comparative analyses with non-nodulating species phylogenetically related to legumes could be better strategies to study the evolution of RN symbiosis in legumes. Polygala paniculata is a non-leguminous species that belongs to a family different from legumes but that is classified into the same order, Fabales. It has appropriate characteristics for cultivation in laboratories: small body size, high fertility and short lifecycles. Therefore, we further assessed whether this species is suitable as a model species for comparative studies with legumes. We first validated that the plant we obtained in Palau was truly P. paniculata by molecular phylogenetic analysis using rbcL sequences. The estimated genome size of this species was less than those of two model legumes, Lotus japonicus and Medicago truncatula. We determined conditions for cultivation in vitro and for hairy root formation from P. paniculata seedlings. It would facilitate to investigate gene functions in this species. The ability of P. paniculata to interact with AM fungi was confirmed by inoculation with Rhizophagus irregularis, suggesting the presence of early signalling factors that might be involved in RN symbiosis. Unexpectedly, branching of root hairs was observed when inoculated with Mesorhizobium loti broad host range strain NZP2037, indicating that P. paniculata has the biological potential to respond to rhizobia. We propose that P. paniculata is used as a model plant for the evolutionary study of RN symbiosis.

Genome sequence of the Lotus spp. microsymbiont Mesorhizobium loti strain NZP2037.

Mesorhizobium loti strain NZP2037 was isolated in 1961 in Palmerston North, New Zealand from a Lotus divaricatus root nodule. Compared to most other M. loti strains, it has a broad host range and is one of very few M. loti strains able to form effective nodules on the agriculturally important legume Lotus pedunculatus. NZP2037 is an aerobic, Gram negative, non-spore-forming rod. This report reveals that the genome of M. loti strain NZP2037 does not harbor any plasmids and contains a single scaffold of size 7,462,792 bp which encodes 7,318 protein-coding genes and 70 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

Commonalities and Differences among Symbiosis Islands of Three <i>Mesorhizobium loti</i> Strains

To shed light on the breadth of the host range of Mesorhizobium loti strain NZP2037, we determined the sequence of the NZP2037 symbiosis island and compared it with those of strain MAFF303099 and R7A islands. The determined 533 kb sequence of NZP2037 symbiosis island, on which 504 genes were predicted, implied its integration into a phenylalanine-tRNA gene and subsequent genome rearrangement. Comparative analysis revealed that the core regions of the three symbiosis islands consisted of 165 genes. We also identified several NZP2037-specific genes with putative functions in nodulation-related events, suggesting that these genes contribute to broaden the host range of NZP2037.

Genetic Diversity and Host Range of Rhizobia Nodulating Lotus tenuis in Typical Soils of the Salado River Basin (Argentina)

A total of 103 root nodule isolates were used to estimate the diversity of bacteria nodulating Lotus tenuis in typical soils of the Salado River Basin. A high level of genetic diversity was revealed by repetitive extragenic palindromic PCR, and 77 isolates with unique genomic fingerprints were further differentiated into two clusters, clusters A and B, after 16S rRNA restriction fragment length polymorphism analysis. Cluster A strains appeared to be related to the genus Mesorhizobium, whereas cluster B was related to the genus Rhizobium. 16S rRNA sequence and phylogenetic analysis further supported the distribution of most of the symbiotic isolates in either Rhizobium or Mesorhizobium: the only exception was isolate BA135, whose 16S rRNA gene was closely related to the 16S rRNA gene of the genus Aminobacter. Most Mesorhizobium-like isolates were closely related to Mesorhizobium amorphae, Mesorhizobium mediterraneum, Mesorhizobium tianshanense, or the broad-host-range strain NZP2037, but surprisingly few isolates grouped with Mesorhizobium loti type strain NZP2213. Rhizobium-like strains were related to Rhizobium gallicum, Rhizobium etli, or Rhizobium tropici, for which Phaseolus vulgaris is a common host. However, no nodC or nifH genes could be amplified from the L. tenuis isolates, suggesting that they have rather divergent symbiosis genes. In contrast, nodC genes from the Mesorhizobium and Aminobacter strains were closely related to nodC genes from narrow-host-range M. loti strains. Likewise, nifH gene sequences were very highly conserved among the Argentinian isolates and reference Lotus rhizobia. The high levels of conservation of the nodC and nifH genes suggest that there was a common origin of the symbiosis genes in narrow-host-range Lotus symbionts, supporting the hypothesis that both intrageneric horizontal gene transfer and intergeneric horizontal gene transfer are important mechanisms for the spread of symbiotic capacity in the Salado River Basin.

Mutations conferring azide resistance enhance symbiotic nitrogen fixation in Rhizobium loti

Azide-resistant (AzR) mutants of Rhizobium loti strain NZP2037 were isolated. Mutations conferring azide resistance (azi) appeared at a frequency of 0.5 × 10-7. Nine AzR mutants of R. loti were characterised for their symbiotic behaviour with Lotus pedunculatus plants. In comparison to the wild type parent strain, AzR mutants exhibited either similar or higher symbiotic effectiveness. The azi mutations which enhanced nitrogen fixation as well as improving shoot dry weight of the inoculated plants also increased nodulation. Unlike several azi mutations in Escherichia coli, these azi mutations did not alter sensitivity of R. loti to phenethyl alcohol. One of the AzR mutants exhibited higher micro-aerobic, N′, N′, N′, N′-tetramethyl-p-phenylenediamine (TMPD) oxidase activity.

Comparative morphology and flavolan content of Rhizobium loti induced effective and ineffective root nodules on Lotus species, Leuceana leucocephala, Carmichaelia flagelliformis, Ornithopus sativus, and Clianthus puniceus

The morphology of Rhizobium loti induced root nodules and the flavolan content of nodulated roots of several Lotus species, Leuceana leucocephala, Carmichaelia flagelliformis, Ornithopus sativus, and Clianthus puniceus were examined. Rhizobium loti strain NZP2037 formed effective (Nod+Fix+) nodules on all legumes, but strain NZP2213 formed Nod+Fix+ nodules only on Lotus corniculatus var. cree and ineffective (Nod+Fix−) nodules on all other legumes. The Nod+Fix− nodules developed by NZP2213 showed morphologies ranging from the complete absence of bacteria within “tumour-like” structures to the development of nodules containing bacteria that were either not released or only incompletely released from infection threads. Within nodules formed by NZP2213 on Lotus corniculatus var. hirsutus and Carmichaelia flagelliformis the rhizobia had multiplied extensively within unwalled, plasma membrane bound, infection droplets. Flavolans rich in prodelphinidin, which is toxic towards NZP2213, were present in the roots of Lotus angustissimus, Lotus pedunculatus, Lotus subbiflorus, and Leuceana leucocephala, but only trace amounts of flavolan were found in the roots of Carmichaelia flagelliformis, Ornithopus sativus, and Clianthus puniceus.

Peptidoglycan-bound Polysaccharide Associated with Resistance of Rhizobium loti Strain NZP2037 to Lotus pedunculatus Root Flavolan

SUMMARY: Resistance of Rhizobium loti strain NZP2037 to the prodelphinidin-rich flavolan (condensed tannin) present in the roots of Lotus pedunculatus was associated with the presence of a peptidoglycan-bound flavolan-binding polysaccharide (FBP) in the outer cell membrane of this bacterium. The outer cell membrane of a flavolan-sensitive strain, NZP2213, did not contain this polysaccharide. FBP was extractable in highest concentration from NZP2037 cells during exponential growth of the bacteria. This correlated with exponential-phase cells of NZP2037 showing maximum resistance to flavolan. When added to cultures of NZP2213, FBP afforded a concentration-dependent protection of this strain to the bactericidal effect of the flavolan. ELISA and immunogold labelling of Rhizobium cells using FBP-specific antibodies confirmed the presence of FBP on the outer cell surface of NZP2037 cells and its absence from NZP2213 cells. Co-inoculation of L. pedunculatus with antibiotic-resistant mutants of NZP2037 and NZP2213 resulted in the formation of N2-fixing nodules containing both strains.

Symbiotic properties of Lotus pedunculitis root nodules induced by Rhizobium loti and Bradyrhizobium sp. (Lotus)

Vance, C. P., Reibach, P. H. and Pankhurst, C. E. 1987. Symbiotic properties of Lotus pedunculatus root nodules induced by Rhizobium loti and Bradyrhizobium sp. (Lotus).Symbiotic properties of root nodules were evaluated in glasshouse‐grown Lotus pedunculatus Cav. cv. Maku inoculated with either a fast‐growing Rhizobium loti strain NZP2037 or a slow‐growing Bradyrhizobium sp. (Lotus) strain CC814s. Although the nodule mass of plants inoculated with NZP2037 was twice that of plants inoculated with CC814s, the yield of NZP2037 shoots and roots was 50% that of CC814s shoots and roots. Nodules induced by Bradyrhizobium fixed substantially more N than nodules induced by R. loti. Glucose requirements [mol glucose (mol N2 fixed)‐1] of nodules induced by CC814s and NZP2037 were 7.1 and 16.6, respectively. Nodule enzymes of carbon and nitrogen assimilation reflected the disparity of the two sym‐bioses. Xylem sap of the symbiosis with the higher yield contained a higher concentration of asparagine [9.86 μmol (ml xylem sap)‘] than did the lower yielding symbiosis [5.80 umol (ml xylem sap)“’]. Nodule CO2 fixation was directly linked to nodule N assimilation in both symbioses. The results indicate that the difference between the two symbioses extend to nodule N and C assimilation and whole plant N transport. The data support a role for host plant modulation of bacterial efficiency and assimilation of fixed N.

Biosynthesis and degradation of nodule-specific Rhizobium loti compounds in Lotus nodules.

Two nodule-specific Rhizobium loti compounds were identified in Lotus tenuis and Lotus pedunculatus nodules induced by strain NZP2037. One, a silver nitrate-positive cation called rhizolotine, has been characterized as the riboside of a novel alpha-hydroxyimino acid containing a 1,4,5,6-tetrahydropyrimidine ring (G. J. Shaw, R. D. Wilson, G. A. Lane, L. D. Kennedy, D. B. Scott, and G. J. Gainsford, J. Chem. Soc. Chem. Commun., p. 180-181, 1986), and the other, yellow-1, stains yellow with ninhydrin. Both compounds were degraded by R. loti NZP2037 but not by strains of Rhizobium meliloti, Rhizobium trifolii, or Agrobacterium tumefaciens. Under the conditions tested neither compound was able to serve as a sole source of C or N for growth of R. loti NZP2037. Rhizolotine and yellow-1 were found in nodules from a range of different legumes inoculated with NZP2037, suggesting that the Rhizobium and not the host plant determines their synthesis. Neither compound was found in nodulelike structures of L. pedunculatus induced by transposon Tn5-induced noninfectious (Inf-) mutants of NZP2037 or in similar structures induced by a transconjugant of NZP2037 containing the symbiotic (Sym) cointegrate plasmid pPN1 of R. trifolii. Both compounds were also absent in the ineffective nodules induced by the bacterial-release-negative (Bar-) mutant, strain PN239. However, both compounds were present in nodules induced by the fixation-negative (Fix-) mutant PN235 and in Fix+ nodules formed by a plasmid-cured derivative of NZP2037. These results would suggest that infection and bacterial release from the infection thread are necessary for nodule (symbiotic) synthesis of these compounds.

Enhanced Nitrogen Fixation and Competitiveness for Nodulation of Lotus pedunculatus by a Plasmid-cured Derivative of Rhizobium loti

Summary: A plasmid-cured derivative of Rhizobium loti strain NZP2037, strain PN4010, was significantly more effective in N2 fixation and showed an improved capacity to compete with other root nodule bacteria for nodulation of Lotus pedunculatus, relative to that of strain NZP2037. Reintroduction of the plasmid pRlo2037 into PN4010 resulted in a return to the NZP2037 level of symbiotic effectiveness and competitiveness. The enhanced effectiveness and competitiveness of PN4010 for L. pedunculatus was associated with its increased capacity to form nodules. Strain PN4010 did not differ from NZP2037 in its growth characteristics in pure culture, in its resistance to L. pedunculatus root flavolan or in its ability to multiply in the L. pedunculatus rhizosphere in the presence of a competing strain of R. loti. An examination of 35 R. loti strains (including 25 field isolates) revealed two strains (NZP2014 and NZP2042) that did not contain a single large indigenous plasmid; both were significantly more effective in N2 fixation with L. pedunculatus than was NZP2037 or two other Nod+ Fix+ plasmid-containing strains of R. loti. Transfer of pRlo2037 into NZP2014 and NZP2042 reduced their effectiveness for L. pedunculatus.

The effect of bacterial strain and temperature changes on the nitrogenase activity of Lotus pedunculatus root nodules

After 40 days of growth at 25°C, Lotus pedunculatus cav., cv. Maku plants infected with Rhizobium loti strain NZP2037 displayed similar relative growth rates but had twice the nodule mass and only one third the whole plant dry weight of plants infected with Bradyrhizobium sp. (Lotus) strain CC814s. In the NZP2037 symbiosis, the rate of CO2 evolution (per g dry weight of nodulated root) was 1.6 times as high as that in the CC814s symbiosis while the rate of C2H2 reduction (per g dry weight of nodule) was only 48% of that in the CC814s symbiosis. Studies of the effect of short term temperature changes on the gas exchange characteristics (CO2 and H2 evolution, C2H2 reduction) of these symbioses revealed wide differences in the optima for C2H2 reduction. Nodules infected with NZP2037 displayed maximal C2H2 reduction rates [157 μmol (g dry weight nodule)−1 h−1] at 12°C, whereas nodules infected with CC814s were optimal at 30°C [208 μmol (g dry weight nodule)−1 h−1]. These short term studies suggested that differences in temperature optima for N2 may have partially accounted for the poorer effectivity, at 25°C, of strain NZP2037 when compared with strain CC‐814s.The relative efficiency [RE = 1 – (H2 evolution/C2H2 reduction)] of N2 fixation varied widely with temperature in the two symbioses, but there was a general trend toward higher RE with lower temperatures. The ratio of CO2 evolution: C2H2 reduction (mol/mol) in nodulated roots infected with CC814s was constant (ca 10 CO2/C2H2) between 5°C and 30°C, whereas in plants infected with NZP2037 it reached a minimal value of 3.3 CO2/C2H2 at 10°C and was 19 CO2/C2H2 at the growing temperature (25°C).

Transfer of an indigenous plasmid of Rhizobium loti to other rhizobia and Agrobacterium tumefaciens.

Rhizobium loti strains NZP2037 and NZP2213 were each found to contain a single large plasmid: pRlo2037a (240 MDal) and pRlo2213a (120 MDal), respectively. Plasmid DNA present in crude cell lysates of each strain and purified pRlo2037a DNA did not hybridize with pID1, a recombinant plasmid containing part of the nitrogen fixation (nif) region of R. meliloti, indicating that nif genes were not present on these plasmids. The transposon Tn5 was inserted into pRlo2037a and this plasmid was then transferred into R. leguminosarum, R. meliloti and Agrobacterium tumefaciens. All transconjugants failed to nodulate Lotus pedunculatus, suggesting that the ability to nodulate this legume was also not carried on pRlo2037a. Transfer of pRlo2037a to R. loti strain NZP2213 did not alter the Nod+ Fix- phenotype of this strain for L. pedunculatus. Determinants for flavolan resistance, believed to be necessary for effective nodulation of L. pedunculatus, were not carried on pRlo2037a. These data suggest that nodulation, nitrogen fixation and flavolan resistance genes are not present on the large plasmid in R. loti strain NZP2037.