Structural complexity of the symbiotic plasmid of Rhizobium leguminosarum bv. phaseoli.

The complete physical map of the symbiotic plasmid of Rhizobium leguminosarum bv. phaseoli strain CFN42 was established. The data support the concept that Rhizobium symbiotic genes are part of a complex genomic structure which contains a large amount of reiterated DNA sequences. This plasmid is a circular structure of 390 kb with approximately 10 families of internally reiterated DNA sequences of two to three elements each. One family includes two directly oriented nitrogenase operons situated 120 kb apart. We also found several stretches of pSym that are reiterated in other replicons of the cell. Localization of symbiotic gene sequences by heterologous hybridization revealed that nodABC sequences are separated in two regions, each of which contains a nod boxlike element, and it also suggested the presence of two copies of the nifA and nodD gene sequences. We propose that the complex structure of the symbiotic plasmid allows interactions between repeated DNA sequences which, in turn, might result in frequent rearrangements.     

Response of Rhizobium leguminosarum bv phaseoli to acidity

Soil acidity constraints grain legume production in tropical soils, both limiting Rhizobium survival and reducing nodulation. Strains of rhizobia with greater tolerance to hydrogen-ion concentration have been identified, but the basis for strain differences in pH tolerance has yet to be determined. In this study, strains of Rhizobium leguminosarum by phaseoli which differed in their tolerance to acidity were exposed to acid pH, then cell levels of potassium and calcium determined, and specific ‘acid-shock’ proteins identified. Lowering the external pH to 4.6–4.7 resulted in an immediate efflux of calcium from the cell of both acid tolerant and sensitive bean strains. Change in cell potassium levels on exposure to acidity varied with the strain. Strain UMR 1899 and an acid-sensitive mutant derived from it maintained high cytoplasmic potassium at acid pH, whereas an acid-sensitive strain UMR 1632 underwent a marked decline in cell potassium at pH 4.6. Exposure of these strains to pH 4.5 in the presence of [³⁵S]-labeled methionine enhanced production of a number of proteins, while synthesis of other proteins at this pH was significantly reduced. Differences in banding pattern were also evident between UMR1899 and the Tn5-induced pH-sensitive mutant UMR5005 derived from it, and between cells grown in the presence and absence of calcium and phosphorus.     

Different plasmids of Rhizobium leguminosarum bv. phaseoli are required for optimal symbiotic performance.

Rhizobium leguminosarum bv. phaseoli CFN42 contains six plasmids (pa to pf), and pd has been shown to be the symbiotic plasmid. To determine the participation of the other plasmids in cellular functions, we used a positive selection scheme to isolate derivatives cured of each plasmid. These were obtained for all except one (pe), of which only deleted derivatives were recovered. In regard to symbiosis, we found that in addition to pd, pb is also indispensable for nodulation, partly owing to the presence of genes involved in lipopolysaccharide synthesis. The positive contribution of pb, pc, pe, and pf to the symbiotic capacity of the strain was revealed in competition experiments. The strains that were cured (or deleted for pe) were significantly less competitive than the wild type. Analysis of the growth capacity of the cured strains showed the participation of the plasmids in free-living conditions: the pf- strain was unable to grow on minimal medium, while strains cured of any other plasmid had significantly reduced growth capacity in this medium. Even on rich medium, strains lacking pb or pc or deleted for pe had a diminished growth rate compared with the wild type. Complementation of the cured strains with the corresponding wild-type plasmid restored their original phenotypes, thus confirming that the effects seen were due only to loss of plasmids. The results indicate global participation of the Rhizobium genome in symbiotic and free-living functions.     

Effect of glutamate transport and catabolism on symbiotic effectiveness in Rhizobium leguminosarum bv. phaseoli

Seven Tn5 induced mutants unable to use glutamate as sole carbon and nitrogen source were isolated from the effective Rhizobium leguminosarum bv. phaseoli strain P121-R. As indicated by restriction and hybridisation analysis, all the mutants arose from a single Tn5 insertion in the chromosome. The ¹⁴C-glutamate uptake rate of the mutants was 76 to 88% lower than that of strain P121-R. Inoculation of Phaseolus vulgaris cv. Labrador with these mutants significantly decreased shoot dry matter yield and the total nitrogen content respectively, as compared to inoculation with the parental strain P121-R. All the mutants formed nodules, however they were smaller, white to greenish and approximately 30% less numerous than those formed by strain P121-R. These observations suggest that glutamate transport and catabolism in R. leguminosarum bv. phaseoli P121-R may play an important role in the establishment of an effective symbiosis in field bean. None of the mutants isolated was an auxotroph. All mutants were unable to grow on aspartate suggesting that glutamate and aspartate, probably have the same transporter as indicated in Rhizobium meliloti and in Bacillus subtilis. All mutants readily used glutamine, proline, arginine as sole carbon and nitrogen source, but grew more slowly than the wild type strain. On the other hand, all the mutants were impaired in growth on histidine and -aminobutyrate as sole carbon and nitrogen source. As the catabolism of these amino acids occurs predominantly through glutamate, our results indicate that mutants are also impaired in their ability to use histidine and -aminobutyrate as a nitrogen source. Our results also suggest that other amino acids catabolized through the glutamate pathways may be an additional important carbon source for bacteroids in nodules.     

High-frequency rearrangements in Rhizobium leguminosarum bv. phaseoli plasmids.

High-frequency genomic rearrangements affecting the plasmids of Rhizobium leguminosarum bv. phaseoli CFN42 were analyzed. This strain contains six large plasmids ranging in size from 200 to 600 kb. In the absence of any selective pressure, we found 11 strains from 320 analyzed colonies that presented different kinds of plasmid-borne rearrangements, including sequence amplification, deletion, cointegration, and loss of plasmids. These data support the concept that the R. leguminosarum bv. phaseoli genome is a dynamic structure and imply that strains are mixtures of similar but not identical cells.     

Rhizobium phaseoli cytochrome c-deficient mutant induces empty nodules on Phaseolus vulgaris L.

A Rhizobium phaseoli cytochrome mutant, unable to oxidize N,N,N′,N′ -tetramethyl-p-phenylend(amine (TMPD), was isolated after Mu-dl (Kan lac) mutagenesis of the wild-type strain CE-3. Mutant strain CFN4202 had sixfold less haem-c but similar levels of b type, o and aa₃ cytochromes than the wild-type strain. CFN402 strain also showed reduced NADH- and TMPD-oxidase activity than the wild-type strain. Succinate-oxidase activities were very similar. Western blot experiments, using antiserum against bovine c₁ and c cytochromes, revealed that both proteins were present in CFN4202 membranes, suggesting a defect of haem binding to cytochrome c. Nodules formed by this strain in Phaseolus vulgaris did not contain bacteroids. These data suggest that the cytochrome c-aa₃ chain or some other respiratory chain, containing c-type cytochromes in R. phaseoli, is essential for bacterial division during the early steps of the symbiotic interaction with the legume-host.     

Competition among Rhizobium leguminosarum bv. phaseoli strains for nodulation of common bean.

Six effective Rhizobium leguminosarum bv. phaseoli strains were examined for nodulation competitiveness on common bean (Phaseolus vulgaris L.), using all possible two-strain combinations of inoculum. Nodule occupancy was determined with strain-specific fluorescent antibodies. The strains were divided into three groups according to their overall competitive abilities on pole bean cv. Kentucky Wonder and bush bean cv. Bountiful. Strains TAL 182 and TAL 1472 were highly competitive (greater than 70% nodule occupancy); strains KIM-5, Viking 1, and CIAT 899 were moderately competitive (approximately 50% nodule occupancy); and strain CIAT 632 was poorly competitive (less than 5% nodule occupancy). The competitiveness of the six strains was similar on the two host cultivars. The proportion of competing strains in the inoculum influenced the nodule occupancy of the highly competitive and moderately competitive strains, but not that of the poorly competitive strain. Two outstanding strains (TAL 182 and TAL 1472) were identified as ideal model strains for molecular and genetic studies on nodulation competitiveness.     

Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions.

Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant. When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-). The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes. The other mutants had normal plasmid content. In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology. The insertions of the other Ndv mutants were apparently in the chromosome. They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45. In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype. However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype. When plasmid pJB4JI was transferred to two other R. phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained.     

Genetics of thermotolerance in ciprofloxacin resistant mutant of Rhizobium leguminosarum bv phaseoli

A ciprofloxacin resistant mutant (Cf(R)) of Rhizobium leguminossarum bv phaseoli USDA 2695 which nodulates common bean plants (Phaseolus vulgaris L) was isolated after nitrous acid mutagenesis. Another mutant resistant to nalidixic acid (Nal(R)) was isolated spontaneously. Both mutants showed thermotolerance as evident by their ability to grow at elevated (40 degrees C) temperature, although the wild type (USDA 2695) failed to grow at this temperature. Transformation and plasmid curing experiments suggested the gene(s) controlling thermotolerance (TrR) and resistance to nalidixic acid or ciprofloxacin were located on the main chromosome and not on the plasmids. High frequency of co-transfer of TrR-Cr(R) and Tr(R)-Nal(R) during transformation experiments indicated a close association of these gene(s). Role of DNA gyrase and supercoiling in these thermotolerant mutants has been discussed.     

The dnaJ (hsp40) locus in Rhizobium leguminosarum bv. phaseoli is required for the establishment of an effective symbiosis with Phaseolus vulgaris

P121R25 is a Tn5-induced mutant of the effective Rhizobium leguminosarum bv. phaseoli strain P121R that is unable to use glutamate as the sole carbon and nitrogen source and is defective in symbiotic nitrogen fixation. Enzymatic analysis showed that three enzymes implicated in glutamate metabolism (glutamate dehydrogenase, 2-oxoglutarate dehydrogenase, and glutamate synthase) were affected by this mutation. Sequencing of the chromosomal locus bordering the Tn5 in P121R25 indicated the presence of the dnaK and dnaJ genes in an arrangement similar to that described in R. leguminosarum bv. viciae (GenBank accession number Y14649). The mutation was located in the dnaJ (hsp40) gene.     

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

Aims

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

Methods

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

Results

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

Conclusions

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

Tolerance ofRhizobium leguminosarum bv.phaseoli to acidity and drought

Ten strains ofRhizobium leguminosarum bv.phaseoli isolated from soils of Morocco were more tolerant than three culture collection strains to acid conditions in culture media or in sterile soil. The survival rate of a tolerant strain in a sandy acid soil was greater than a sensitive strain at different humidity levels. These properties should give locally selected strains an advantage in nodulatingPhaseolus vulgaris roots in soils similar to those used here.     

Speed of nodulation and competitive ability among strains of Rhizobium leguminosarum bv phaseoli

This study examines the speed of nodulation of 20 strains of Rhizobium leguminosarum bv phaseoli, and relates this trait to the competitive performance of these strains with Phaseolus vulgaris L. At 25/20°C day/night temperature, and with 10⁷ cells applied per growth pouch, there was a strong positive correlation between the speed of nodulation and the competitiveness of strains with the nod ⁺ fix^– reference strain UMR 1116. Strains UMR 1084, 1125, 1165, 1173 and 1384 combined good competitive performance with extensive nodulation in the uppermost root regions. When inoculant levels in the RTM studies were reduced to 10³ cells per pouch no correlation between the apparent competitiveness of strains and their speed in nodulation was evident, presumably because cells had to undergo multiplication before infection. Nodulation was also delayed when growth temperatures were raised to 31/26°C, but a correlation was still evident between competitive performance and nodulation in the region 0.1 to 5.0 mm below the RTM at the time of inoculation. From these results speed of nodulation can be used to estimate the competitive potential of Rhizobium strains, but only under carefully regulated conditions. The effects of inoculation level and temperature on the relationship between speed of nodulation and strain competitiveness could explain the inconsistent results obtained in earlier studies on this topic.Journal paper No. 16962, Agricultural Experiment Station, University of Minnesota, St. Paul, MN 55108, USA     

Transposon mediation allows a symbiotic plasmid of Rhizobium leguminosarum bv. trifolii to become a symbiosis island in Agrobacterium and Rhizobium

The symbiotic plasmid (pSym) of Rhizobium leguminosarum bv. trifolii 4S5, which carries Tn5-mob, was successfully transferred into Agrobacterium tumefaciens A136 by using a conjugation method. The resulting transconjugants induced the development of ineffective nitrogen-fixing nodules on the roots of white clover seedlings. Depending on the manner in which the pSym was retained, the transconjugants were divided into two groups of strains, Afp and Afcs. pSym was retained as a plasmid in the Afp strains but was integrated into the int gene encoding a phage-related integrase on the linear chromosome of A. tumefaciens A136 in strain Afcs1 (one of the Afcs strains) to form a symbiosis island. Conjugation was performed between strain Afcs1 and R. leguminosarum bv. trifolii H1 (a pSym-cured derivative of wild-type strain 4S), and the Rhizobium H1tr strains were screened as transconjugants. Eighteen of the H1tr strains induced effective nitrogen-fixing nodules on the roots of the host plants. pSym was transferred into all of the transconjugants, except for strain H1tr1, at the same size as pSym of strain 4S5. In strain H1tr1, pSym was integrated into the chromosome as a symbiosis island. These data suggest that pSym can exist among Rhizobium and Agrobacterium strains both as a plasmid and as a symbiosis island with transposon mediation.     

Molecular characterization and symbiotic importance of prsD gene of Rhizobium leguminosarum bv. trifolii TA1.

The prsD, prsE and orf3 genes of Rhizobium leguminosarum bv. trifolii strain TA1 encode the proteins which are significantly related to the family of bacterial ABC transporters type I secretion systems. The prsD:Km(r) mutant of strain TA1 induced non-nitrogen-fixing nodules on Trifolium pratense. Microscopic analysis of the nodules induced by prsD mutant did not reveal major abberations in the bacteroid appearance. The exopolysaccharide of prsD mutant was produced in increased amount and its level of polymerization was changed. SDS/PAGE of the proteins from the culture supernatants showed a lack of the 47-kDa protein in the culture of prsD mutant. Thus, PrsD may play a role in the export of this protein.     

Biochemical and symbiotic properties of histidine-requiring mutants of Rhizobium leguminosarum biovar trifolii

A perturbation of the histidine biosynthetic pathway in legume microsymbionts can abolish their symbiotic competence. Twenty-one histidine-requiring (His⁻) mutants were isolated from berseem clover-nodulating, symbiotically-competent (Nod⁺, Fix⁺) Rhizobium leguminosarum bv. ' trifolii ' strain RTH 48 Sm^r by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis followed by enrichment. These mutants were analysed for their biochemical defect and the corresponding effect, if any, on their symbiotic abilities. Cross-feeding, supplementation and enzymatic studies identified three types of mutants. Group 1 mutants, His-2 and His-12, grew with histidine supplementation but not with the addition of either L -histidinol or L -histidinol phosphate to the medium ; they lacked histidinol dehydrogenase (EC 1.1.1.23) activity and consequently formed only ineffective, or 'non-fixing' nodules. Group 2 mutant, His-17, grew when supplemented with either L -histidinol or L -histidine, had low histidinol phosphate phosphatase (EC 3.1.3.15) activity (37% of wild-type), and consequently failed to nodulate berseem clover. Group 3, the remaining 18 mutants, grew when supplemented with L -histidinol phosphate, L -histidinol or histidine, and did not nodulate. Typically, reversion rates were between 10⁻⁷ and 10⁻⁸. Defects in early steps of the pathway abolished nodulating ability, whereas lesions in the last step did not. The last step, however, was required for symbiotic nitrogen fixation. It is hypothesized that histidine may be supplied by the host in sufficient quantity for nodulation by histidinol dehydrogenase mutants to occur, whereas the amount provided in the nodule may be insufficient to support bacteroid development and nitrogen fixation.     

Effect of divalent cations on succinate transport in Rhizobium tropici,R. leguminosarum bv phaseoli and R. loti

Rhizobium tropici, R. leguminosarum bv phaseoli and R. loti each have an active C₄-dicarboxylic acid transport system dependent on an energized membrane. Free thiol groups are probably involved at the active site. Since EDTA inhibited succinate transport in R. leguminosarum bv phaseoli and R. loti, divalent cations may participate in the process; the activity was reconstituted by the addition of Ca²⁺ or Mg²⁺. However, EDTA had no effect on succinate transport in R. tropici, R. meliloti or R. trifolii strains. Ca²⁺ or Mg²⁺ had a similar effect on the growth rates of R. tropici and R. leguminosarum bv phaseoli; R. tropici did not require Ca²⁺ to grow on minimal medium supplemented with succinate but R. leguminosarum bv phaseoli required either or both of the divalent cations Ca²⁺ and Mg²⁺. A R. tropici Mu-dI (lacZ) mutant defective in dicarboxylic acid transport, was isolated and found unable to form effective bean nodules.The authors are with the Division of Biochemistry, Instituto de Investigaciones Biológicas Clemente Estable, Avda, Italia 3318, 11.600 Montevideo, Uruguay