Peanut agglutinin induced alterations in capsular and extracellular polysaccharide synthesis andex -planta nitrogenase activity of cowpea rhizobia

The root exudate ofArachis hypogea (groundnut) and its seed lectin peanut agglutinin were found to stimulate the synthesis of exopolysaccharide and capsular polysaccharide of the microsymbiont cowpeaRhizobium strain JLn (c). The synthesis of capsular polysaccharide was enhanced 1.5-fold and 2-fold in the presence of peanut agglutinin and root exudate, respectively. The synthesis of capsular polysaccharide was suppressed in the presence of different forms of combined nitrogen. Quantitative differences were also detected between the exopolysaccharide of cells grown in the presence and absence of root exudate. Electron microscopic examination of negatively stained lectin-treated JLn (c) cells showed an increased deposition of capsular polysaccharide surrounding the cells. Hurthermore,ex planta nitrogenase activity of JLn(c) cells in the presence of lectin was found to be enhanced by 63% in correlation with the increased synthesis of polysaccharides. Part of this work was presented at the colloquium session of the 4th Hederation of Asian and Oceanian Biochemists Congress, held at Singapore, in November 1986.     

Extracellular polysaccharides of cowpea rhizobia: compositional and functional studies

Polysaccharides excreted by cowpea Rhizobium strains JLn(c) and RA-1 were mixtures of complex acidic exopolysaccharides and low molecular weight neutral glucans. These polymers were fractionated using gel filtration chromatography. Purified fractions of the acidic heteropolymer reacted with peanut agglutinin to give precipitin bands when subjected to Ouchterlony gel diffusion. The acidic exopolysaccharide was found to contain mainly glucose, galactose, glucuronic acid, mannose and fucose. The non-carbohydrate substituents of the acidic heteropolymer were pyruvate, acetate and uronate which were identified by infrared and proton nuclear magnetic resonance spectroscopy as well as by chemical analysis.Abbreviations EPS Extracellular polysaccharide - YEM yeast extract mannitol - PNA peanut agglutination - ¹H-NMR proton nuclear magnetic resonance     

Peanut agglutinin-induced structural changes in cowpea Rhizobia as revealed by freeze-etching

The ultrastructure of two strains of cowpeaRhizobium species, JLn(c) and NC-92, was studied especially with regards to the effect of peanut agglutinin on the cell envelope. Freezeetching electron microscopy revealed the structural details of the outer and the cytoplasmic membrane. The topology of both membranes resembled that of other Gram-negative bacteria. The cells displayed marked polarity, expressed in the form of an extracellular polar body (EPB) near one end and a periplasmic bay at the opposite end. The EPB was seen as a polar outgrowth of the outer membrane and resembled in structure the outer membrane. The existence of a polar periplasmic bay as a characteristic feature of cowpea rhizobia is reported here for the first time and has been implicated as a probable site for polysaccharide synthesis. Ruthenium red staining showed a polar mode of capsule initiation in the early exponential phase. Peanut agglutinin was found to enhance the incidence of EPB which is necessary for adhesion of rhizobia to root hairs. This suggests that lectins serve as an important biological stimulus which preconditions the symbiont for successful attachment to root hairs.     

Isolation and characterization of siderophore from cowpeaRhizobium (peanut isolate)

In an iron-depleted broth culture of cowpeaRhizobium (a peanut isolate), phenolate type of compounds were detected. Chemical characterization showed the presence of 2,3-dihydroxy benzoic acid (DHBA) and 3,4-DHBA in the siderophore extract. Lysine and alanine were identified as conjugated amino acids of the siderophore. Maximum concentration of the siderophore in the culture supernatant was found after 24 h of growth. The compounds in the extracted siderophore induced growth ofRhizobium in a medium containing EDTA. Addition of lysine and alanine in the growth medium (20 mM each) led to a fourfold increase in siderophore production.     

Effect of Drought on the Growth and Survival of the Stress-Tolerant Bacterium <Emphasis Type="Italic">Rhizobium</Emphasis> sp. NBRI2505 <Emphasis Type="Italic">sesbania</Emphasis> and Its Drought-Sensitive Transposon Tn<Emphasis Type="Italic">5</Emphasis> Mutant

Studies were conducted to elucidate the nature of drought tolerance in the bacterium Rhizobium sp. NBRI2505 sesbania and its transposon Tn5 induced mutant to assess the role of salt, pH, and temperature stresses in contributing to drought tolerance, and to correlate drought tolerance and symbiotic effectiveness. Rhizobium sp. NBRI2505 sesbania tolerated yeast extract mannitol broth (YEB) containing 28% salt (NaCl; wt/vol) for up to 18 h of incubation at 30°C, survived a 2-h incubation in YEB at 65°C, and when subjected to drought stress, tolerated YEB containing 45% polyethylene glycol 6000 (PEG; wt/vol) for up to 5 days of incubation at 30°C. One drought-sensitive mutant Rhizobium sp. NBRI2505 sesbania T112 (T112) containing a single Tn5 insertion was selected after screening about 10,000 clones. T112 was specifically defective in its tolerance for drought: when subjected to drought stress, it tolerated YEB containing 45% PEG for up to 2 days of incubation at 30°C. T122 mutant was also more sensitive to the heat and desiccation stresses, compared with Rhizobium sp. NBRI2505 sesbania in the presence of 45% PEG. Our results demonstrated a positive effect of calcium on the survival of Rhizobium sp. sesbania under acidic stress conditions. The observed enhanced survival at pH 3 of Rhizobium sp. NBRI2505 sesbania and T112 in the presence of 5% CaCO₃ suggests the requirement of calcium for growth and survival, which may have an ecological significance in acidic soils. Mutant strain T112 produced ineffective symbiosis with the plant host in the presence of 2.5 and 5% PEG, indicating that drought tolerance is required for effective symbiosis. Received: 11 January 2002 / Accepted: 18 February 2002     

Diverse effects of formate on the assimilatory metabolism of nitrate and nitrite inRhizobium

Two strains ofRhizobium, cowpeaRhizobium 32H1 andRhizobium japonicum CB 1809, showed a marked stimulation in growth on addition of formate to the minimal medium containing nitrate as the sole source of nitrogen. The amount of accumulated nitrite and specific nitrate reductase activity was much higher in cultures supplemented with formate than in the control medium. In contrast, growth, consumption of nitrite and specific nitrite reductase activity in minimal medium + nitrite was greatly reduced by the addition of formate. A chlorate resistant mutant (Chl-16) was isolated spontaneously which contained a nitrite reductase which was not inhibited by formate. The results suggest that formate serves as an electron donor for nitrate reductase and inhibits nitrite assimilation inRhizobium     

Crossing the Limits of Rhizobium Existence in Extreme Conditions

An ecological survey was conducted to characterize 5000 Rhizobium sp. sesbania strains of diverse geographical origin, isolated from the root nodules of Sesbania aculeata growing in neutral (pH 7) and alkaline (pH 8.5 and above) soils. The rhizobia from the alkaline soil showed significantly higher salt tolerance than those isolated from neutral soil. Upper limits of stress survival of rhizobial isolates, Rhizobium sp. NBRI0102 sesbania selected from neutral soil, and Rhizobium sp. NBRI2505 sesbania selected from alkaline soil, were studied under free living conditions. Rhizobium sp. NBRI0102 sesbania and Rhizobium sp. NBRI2505 sesbania tolerated yeast extract mannitol broth (YEB) containing 10% and 28% salt (NaCl, wt/vol) for up to 18 h of incubation at 30°C. Growth of Rhizobium sp. NBRI0102 sesbania and Rhizobium sp. NBRI2505 sesbania at pH 7, 11, and 12 was identical, except for a lag period of about 10 h in the growth of Rhizobium sp. NBRI0102 sesbania at pH 11 and 12, as compared with pH 7. Rhizobium sp. NBRI0102 sesbania and Rhizobium sp. NBRI2505 sesbania survived at 50°C and 65°C, in YEB at pH 7 for up to 4 and 2 h, respectively. To our knowledge, this is the first report of rhizobia demonstrating survival of Rhizobium sp. NBRI2505 sesbania, estimated by counting viable cells, to such extreme conditions of salt and temperature, individually. In contrast to Rhizobium sp. NBRI0102 sesbania, high temperature was tolerated efficiently by Rhizobium sp. NBRI2505 sesbania, in the presence of salt at higher pH. Our results suggest that the possession of the trait of high salt tolerance might be of some evolutionary significance for the survival of rhizobia in alkaline soils, at high pH and temperature. Received: 23 May 2000 / Accepted: 26 June 2000     

Estimation of root nodule development by <Emphasis Type="Italic">Rhizobium</Emphasis> sp. Using hydroponic culture

A nitrogen-fixing bacterium isolated from the root nodules of a cultivated leguminous plant, soybean (Glycine max L.), was cultivable and was identified as Rhizobium sp. Bacterial species isolated from root nodules of wild leguminous plants including -bush clover, white dutch clover, wisteria, and false acacia were identified as Burkholderia cepacia, Pseudomonas migulae, Pseudomonas putida, and Flavobacterium sp, respectively, all of which are heterotrophic bacteria that grow in the rhizosphere. Temperature gradient gel electrophoresis (TGGE) 16S-rDNA bands extracted directly from the bacterial population within the root nodules of the wild leguminous plants were identified as Rhizobium sp, Mesorhizobium sp, and Bradyrhizobium sp. none were cultivable. Rhizobium sp. isolated from soybean root nodule generated approximately 48 and 19 mg/L of ammonium in glucose- and starch-defined medium, respectively, during 8 days of growth. The growth rate of Rhizobium sp. was increased by the addition of yeast extract but not by the addition of ammonium. K _m and V _max for starch saccharification measured with the extracellular crude enzyme of Rhizobium sp. were 0.7556 mg/L and 0.1785 mg/L/min, respectively. The inoculation of Rhizobium sp. culture into a hydroponic soybean plant culture activated root nodule development and soybean plant growth. The inoculated Rhizobium sp. survived for at least 4 weeks, based on the TGGE pattern of 16S-rDNA. The 16S-rDNA of Rhizobium sp. isolated from newly developed root nodules was homologous with the inoculated species.     

Studies on root nodules of leguminous plants bioproduction of indole acetic acid by a Rhizobium sp. from a twiner Clitoria ternatea L.

The Rhizobium sp. isolated from the root nodules of Clitoria ternatea L., a leguminous twiner, produced a high amount of IAA (16.4 μg/ml) from tryptophan in an unsupplemented basal medium. The production of IAA started simultaneously with the growth and had no different growth and production phase. The growth and production were parallel and increased up to 45–50 h. The IAA production by the Rhizobium sp. was increased by 520% when the medium was supplemented with fructose (1.5%), MnSO₄ (1.0 μg/ml), riboflavin (0.10 μg/ml) and Triton X-100 (0.01%). The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is discussed.     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

Effects of Salt and pH Stress on Temperature-Tolerant Rhizobium sp. NBRI330 Nodulating Prosopis juliflora

A study was conducted to examine the growth response of a rhizobial strain Rhizobium sp. NBRI330 isolated from root nodules of Prosopis juliflora growing in alkaline soil. The strain had the ability to nodulate P. juliflora. Nursery grown plants inoculated with Rhizobium sp. NBRI330 had 60.6% higher plant dry weight, as compared with uninoculated plants. The individual stress survival limit of a rhizobial strain Rhizobium sp. NBRI330 isolated from alkaline soil in a medium containing 32% (wt/vol) salt was 8 h, and at 55°C up to 3 h. The length of Rhizobium sp. NBRI330 in salt-stressed cells increased significantly to 3.04 μm from 1.75 μm of non-stressed control cells. On the contrary, the length of pH-stressed cells declined to 1.40 μm. Compared with non-stressed control rod-shaped cells, the shape of temperature-stressed cells changed to spherical, of 0.42 μm diameter. High temperature (45°C) was tolerated efficiently by Rhizobium sp. NBRI330 in the presence of salt at pH 12, as compared with pH 7. Received: 13 September 1999 / Accepted: 14 October 1999     

Isolation of the replication DNA region from a Rhizobium plasmid and examination of its potential as a replicon for Rhizobiaceae cloning vectors

The DNA region essential for replication and stability of a native plasmid (pTM5) from Rhizobium sp. (Hedysarum) has been identified and isolated within a 5.4-kb PstI restriction fragment. The isolation of this region was accomplished by cloning endonuclease-restricted pTM5 DNA into a ColE1-type replicon and selecting the recombinant plasmids containing the pTM5 replicator (pTM5 derivative plasmids) by their ability to replicate in Rhizobium. DNA homology studies revealed that pTM5-like replicons are present in cryptic plasmids from some Rhizobium sp. (Hedysarum) strains but not in plasmids from strains of other Rhizobium species or Agrobacterium tumefaciens. The pTM5 derivative plasmids were able to replicate in Escherichia coli and A. tumefaciens and in a wide range of Rhizobium species. On the basis of stability assays in the absence of antibiotic selective pressure, the pTM5 derivative plasmids were shown to be highly stable in both free-living and symbiotic cells of Rhizobium sp. (Hedysarum). The stability of these plasmids in other species of Rhizobium and in A. tumefaciens varied depending on the host and on the plasmid. Most pTM5 derivative plasmids tested showed significantly higher symbiotic stability than RK2 derivative plasmids pRK290 and pAL618 in Rhizobium sp. (Hedysarum), R. meliloti, and R. leguminosarum bv. phaseoli. Consequently, we consider that the constructed pTM5 derivative plasmids are potentially useful as cloning vectors for Rhizobiaceae.     

Diazotrophic bacteria associated with banana (Musa spp.).

Nitrogen-fixing bacteria were isolated from surface sterilized banana (Musa spp.) plants and constituted a minor proportion of banana endophytic bacteria. Some isolates were characterized by alloenzyme profiles, biochemical tests, 16S rRNA and rpoB partial gene sequences, plasmid profiles and plant colonization. A large group of enterobacterial isolates that could not be clearly affiliated, most of them ascribed to group I (with characteristics of Enterobacter cloacae) were the diazotrophs most frequently found in banana. Different Klebsiella spp. and Rhizobiumsp. were identified as well. Klebsiella spp. were isolated from inside the roots and stems of plants grown in the two geographical regions sampled and from tissue culture-derived plantlets. Rhizobium sp. isolates were obtained only from Colima where bananas are grown extensively. Group I isolates and Rhizobium sp. could be re-isolated from surface-sterilized banana derived from tissue culture at five months after inoculation and significant increases in stem and leave fresh weight were obtained with some of the isolates.     

Production of Exo-polysaccharide by <Emphasis Type="Italic">Rhizobium</Emphasis> sp.

Two fold increase in the yield of glucose and maltose containing exo-polysaccharide (EPS) by Rhizobium sp. was observed during its growth in modified YEMB. EPS production, plant growth promotion activity and root colonization of Rhizobium sp. studies showed enhanced EPS synthesis, more seed germination and over all improvement in plant growth over control and R. meliloti treatment. Groundnut seeds bacterized with Rhizobium sp. resulted in 69.75% more root length, 49.51% more shoot height, 13.75% more number of branches and 13.60% more number of pods over the control and R. meliloti treatment. Bacterization of wheat seeds increased the dry matter yield of roots (1.7-fold), and roots adhering soil (RAS) (1.5) and shoot mass (1.9-fold). Rhizobium sp. inoculation also increased the population density of EPS-producing bacteria on the rhizoplane. Roots of plants inoculated with Rhizobium sp. maintained a higher K⁺/Na⁺ ratio and K⁺–Na⁺ selectivity.     

Factors affecting growth of Bdellovibrio on Rhizobium

The extent of decline in the population density of Rhizobium sp. exposed to Bdellovibrio was markedly reduced in the presence of montmorillonite, kaolinite or vermiculite but not by a soil clay fraction. Increasing levels of montmorillonite reduced the numbers of vibrios that appeared in a two-membered culture and allowed for greater survival of the rhizobia. Bdellovibrio and not Rhizobium sp. was retained when mixed with the three clay minerals, but no appreciable retention was evident with the soil clay fraction. Suspensions of colloidal soil organic matter protected the hosts from parasitism, although aqueous extracts of soil did not affect the relationship. Cells from old Rhizobium sp. cultures were attacked only after a lag phase, but rhizobia that had been stored were more rapidly lysed than cells tested immediately after removal from the growth medium. The possible significance of these findings to the survival of rhizobia in soils containing Bdellovibrio is discussed.     

Studies on Endophytic Colonization Ability of Two Upland Rice Endophytes, <Emphasis Type="Italic">Rhizobium</Emphasis> sp. and <Emphasis Type="Italic">Burkholderia</Emphasis> sp., Using Green Fluorescent Protein Reporter

Colonization ability of the two endophytic bacteria, isolated from surface sterilized roots of upland cultivated rice viz., Rhizobium sp. and Burkholderia sp., was compared after genetically tagging them with a constitutively expressing green fluorescent protein gene (gfp/gusA). Confocal laser scanning microscopy (CLSM) of gnotobiotically grown seedlings of Narendradhan 97, inoculated with gfp/gusA-tagged endophytes, revealed that both Rhizobium sp. and Burkholderia sp. colonized the intercellular spaces in the root cortex when inoculated separately. Colonization by gfp/gusA-tagged Rhizobium sp. was severely inhibited when co-inoculated with an equal number (10⁶ cfu ml⁻¹) of wild type Burkholderia sp. Burkholderia sp. was a more aggressive endophytic colonizer of rice than Rhizobium sp. The potential of using gfp/gusA reporter and CLSM as tools in evaluating competitive ability of colonization among endophytes is demonstrated in this study.     

Production of extracellular polysaccharides by a Rhizobium species from root nodules of Cajanus cajan

The ability of the Rhizobium sp., isolated from the root nodules of the leguminous pulse yielding shrub Cajanus cajan, to produce extracellular polysaccharides (EPS) was checked. A large amount of EPS (1, 128 μg/ml) was produced by the bacteria in yeast extract mannitol medium. Growth and EPS production started simultaneously, but the production reached its maximum level in the stationary phase of growth at 28 h. The EPS production by this Rhizobium sp. was much higher than by many other strains from nodules of Cajanus cajan which took a much longer time to reach maximum EPS production than this strain. The maximum EPS production (2,561 μg/ml) was obtained when the medium was supplemented with mannitol (1%), cetyl pyridinium chloride (2 μg/ml) and KNO₃ (0.2%), in which the production was increased by 276% compared to the control. The EPS production rose in the period up to 65 h with increased mannitol concentration. The EPS contained arabinose, xylose and rhamnose monomers. The possible role of rhizobial EPS production in root nodule symbiosis is discussed.     

Studies on the root nodules of leguminous plants: The production of indole acetic acid in culture by a Rhizobium sp. from a leguminous climbing shrub Derris scandens benth

The Rhizobium sp. When isolated form the root nodules of a leguminous climbing shrub Derris scandens produced a high amount of indole acetic acid (IAA) (135.2 μg/ml) from the tryptophan-supple-mented basal medium. Growth and IAA production started simultaneously, and the maximum amount of IAA was produced as a secondary metabolite in the stationary phase of growth. The IAA production by the Rhizobium sp. was increased by 503% when the medium was supplemented with mannitol (2%), KNO₃ (0.2%), nicotinic acid (0.1 μg/ml) and MnSO₄ (1 μg/ml) in addition to tryptophan (4 mg/ml)/ The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is also discussed.     

Control of the initial steps in heme biosynthesis in free-livingRhizobium sp. by culture conditions

Cell-free extracts obtained from free-livingRhizobium sp. in early stationary phase had three times as much 5-aminolevulinate synthase activity as did similar extracts from log phase cells. The level of 5-aminolevulinate dehydratase was also elevated at this point. The presence of 0.1 mM hemin in the culture medium prevented the transitory increase in enzyme activities during this early stationary phase. The effect of hemin was counteracted by 1 mg bovine serum albumin per milliliter medium. This control of the development of 5-aminolevulinate synthase and 5-aminolevulinate dehydratase activities by free hemin suggests a mechanism by which heme and globin formation might be coordinated for the synthesis of leghemoglobin in legume root nodules.     

Evidence for the involvement of iron siderophore in the transport of molybdenum in cowpeaRhizobium

The production of a catechol type of siderophore by an iron-depleted culture of cowpeaRhizobium decreased with the increase in the concentration of molybdenum in the growth medium above 1 mM. In vitro addition of molybdenum at pH 5 and 7 changed the absorbance maxima of siderophore, indicating the interaction of molybdenum with siderophore. Tungsten, which is a competitive inhibitor of molybdenum, was unable to dissociate the molybdenum-siderophore conjugate. In the presence of iron, siderophore increased the uptake of molybdenum. Under these conditions, the addition of 2,3-dihydroxybenzoic acid did not show an increase in the uptake. This suggests that an entire siderophore molecule is involved in the transport of molybdenum.