Characterization of ‘Schizokinen’; a dihydroxamate-type siderophore produced by Rhizobium leguminosarum IARI 917

The Rhizobia comprise one of the most important groups of beneficial bacteria, which form nodules on the roots (rarely on the stems) of leguminous plants. They live within the nodules and reduce atmospheric nitrogen to ammonia, which is further assimilated by plants into required nitrogenous compounds. The Rhizobia in return obtain nutrition from the plant. Rhizobia are free-living soil bacteria and have to compete with other microorganisms for the limited available iron in the rhizosphere. In order to acquire iron Rhizobia have been shown to express siderophore-mediated iron transport systems. Rhizobium leguminosarum IARI 917 was investigated for its ability to produce siderophore. It was found to produce a dihydroxamate type siderophore under iron restricted conditions. The siderophore was purified and chemically characterized. The ESMS, MS/MS and NMR analysis indicate the dihydroxamate siderophore to be ‘schizokinen’, a siderophore reported to be produced by Bacillus megaterium that shares a similar structure to ‘rhizobactin 1021’ produced by Sinorhizobium meliloti 1021. This is the first report of production of schizokinen by a strain of R. leguminosarum, therefore it was carefully investigated to confirm that it is indeed ‘schizokinen’ and not a degradation product of ‘rhizobactin 1021’. Since ferric–siderophore complexes are transported across the outer membrane (OM) into the periplasm via an OM receptor protein, R. leguminosarum IARI 917 was investigated for the presence of an OM receptor for ‘ferric–schizokinen’. SDS PAGE analysis of whole cell pellet and extracted OM fractions indicate the presence of a possible iron-repressible OM receptor protein with the molecular weight (MW) of approximately 74 kDa.     

Isolation and partial characterization of phenolate siderophore from Rhizobium leguminosarum IARI 102

Abstract Rhizobium leguminosarum IARI 102 produced 2,3-dihydroxy benzoic acid, a type of phenolate siderophore, under iron-starved conditions. Hydroxamic acids were not detected. Maximum production of siderophore was found at 26 h of growth in a chemically defined medium at 28°C with shaking. Threonine was detected as the amino acid conjugate of the siderophore. Addition of Fe³⁺ to the culture medium increased the growth yield significantly, but depressed the production of the iron chelating compound.     

Studies on the respiration of efficient and inefficient strains of Rhizobium

Summary In a pot culture experiment using sterilised soil, growth and nitrogen content of Berseem (Trifolium alexandrinum) inoculated with 15 strains of Rhizobium trifolii and of pea (Pisum sativum) inoculated with 10 strains of Rhizobium leguminosarum were found to vary considerably depending on the strains of the respective Rhizobium used. Out of the 15 strains of Rhizobium trifolii, 6 strains were found to be highly efficient (increasing the nitrogen content by more than 70 per cent over the control-uninoculated) and the rest as either moderately efficient (increase in N by 30–50 per cent) or inefficient (increase in N by 4 to 20 per cent) strains. Similarly, out of the 10 strains of Rhizobium leguminosarum 5 strains were found to be highly efficient, 1 moderately efficient and the rest were more or less inefficient strains.The respiration rate (l O₂ consumed per hour per mg dry cells) of efficient and inefficient strains of Rhizobium trifolii and Rhizobium leguminosarum in glucose, maltose and mannitol substrate did not bear any relation to their efficiency. However, the stimulation of the respiratory rate of the Rhizobium strains due to the addition of glycine to the glucose substrate was found to be significantly more in case of efficient strains of Rhizobium trifolii and Rhizobium leguminosarum than those of the inefficient ones.The data presented in the paper is taken from the thesis submitted by the Senior author, to the P. G. School, IARI, N. Delhi, in 1968, for Ph. D. degree.     

Specificity of siderophore-mediated transport of iron in rhizobia

The trihydroxamate siderophore, hydroxamate K, has been purified from culture filtrates of iron-deficient Rhizobium leguminosarum biovar viciae MNF710. The iron complex has a molecular weight of 828 and an absorption maximum at 443 nm (_M=1510). ⁵⁵Fe complexed to purified hydroxamate K was taken up by MNF710, its hydroxamate-negative mutant MNF7102 and Rhizobium leguminosarum biovar trifolii WU95 via an iron-regulated transport system, but Rhizobium meliloti U45 failed to take up the iron-siderophore complex under any conditions. A similar pattern of iron uptake was observed with ferrioxamine B. MNF710, MNF7102, U45 and WU95 all transported ⁵⁵Fe-ferrichrome but only the first three strains took up ⁵⁵Fe-ferrichrome A. All these ⁵⁵Fe-trihydroxamate uptake systems were ironregulated in MNF710, MNF7102 and WU95. In contrast, uptake of ⁵⁵Fe-rhodotorulate, a dihydroxamate, was essentially constitutive in all four organisms. Similarly, uptake of ⁵⁵Fe-citrate and ⁵⁵Fe-nitrilotriacetic acid was constitutive. None of the strains took up ⁵⁵Fe complexed with enterobactin or with pyoverdins from Pseudomonas aeruginosa ATCC15692 (PAO1) and Pseudomonas fluorescens ATCC17400.     

Expression of Escherichia coli tryptophan operon in Rhizobium leguminosarum.

Summary RP4-trp hybrid plasmid containing Escherichia coli whole tryptophan operon was conjugatively transferred from E. coli to Rhizobium leguminosarum strains carrying mutations in different trp genes, converting their Trp^– phenotype to Trp⁺. That the phenotype change of the R. leguminosarum cells was due to the presence of the E. coli tryptophan operon was verified by the isolation of RP4-trp hybrid plasmid from the R. leguminosarum conjugant cells, and by re-transfer of RP4-trp plasmid by conjugation back to E. coli trp and Pseudomonas putida trp strains. Enzymatic activities of anthranilate synthetase and subunit of tryptophan synthetase in crude extracts of R. leguminosarum cells containing RP4-trp plasmid were much higher than that of the wild-type cells and were not repressed by the presence of tryptophan in the culture medium.     

Siderophore-Producing <Emphasis Type="Italic">Alcaligenes feacalis</Emphasis> Exhibited More Biocontrol Potential Vis-à-Vis Chemical Fungicide

We report the in vitro phytopathogen suppression activity of siderophoregenic preparations of Alcaligenes feacalis vis-à-vis the oraganochlorine fungicide, bavistin. Siderophore-rich culture broth, siderophore-rich supernatant, and purified siderophore preparation exerted antifungal activity against Aspergillus niger NCIM 1025, A. flavus NCIM 650, Fusarium oxysporum NCIM 1008, and Alternaria alternata IARI 715. Among all the preparations, siderophore-rich broth exhibited potent antifungal activity. The minimum fungicidal concentration required was 75 μl for A. niger and F. oxysporum and 50 μl for A. flavus and A. alternata.     

Influence of the ionic form of mineral salts on growth of faba bean andRhizobium leguminosarum

Summary The nodulation and growth of faba bean plants (Vicia faba cultivar Giza 2) at 2 months' age were studied in pot trials using an alluvial clay soil (pH 7.9). Chlorides, carbonates and sulphates of Na⁺, K⁺, Ca²⁺, and Mg²⁺ were added at 20 mEq/100 g soil and the tolerance ofRhizobium leguminosarum (biovar.viceae, Strain F-15) grown on pure culture medium, salinized with the same salts, was then tested. Salts were generally inhibitory towards plant and bacterium. Number and characteristics of plant root-nodules, dry weight of plants (shoots and roots), N content in plants and the bacterial colony count decreased. Chlorides were the most inhibitory in soil and carbonates the most in culture medium, whilst sulphates were the least inhibitory in either case. Na⁺ gave maximum inhibition, whereas Mg²⁺ was the least detrimental. Formation of effective nodules on roots of the plants grown in the salinized soil was very poor, butR. leguminosarum showed possible tolerance when grown on salinized culture medium.

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Influence de la forme ionique des sels minéraux sur la croissance de la fève faba et de Rhizobium leguminosarum

Résumé La nodulation et la croissance des plants de fève faba (Vicia faba cultivar Giza 2) agés de 2 mois ont été étudiés dans des essais en pots en utilisant un sol argileux alluvial (pH 7.9). On a ajouté les chlorures, carbonates et sulfates de Na⁺, K⁺ et Mg²⁺ à la dose de 20 mEq par 100 g de sol. On a testé ensuite la tolérance deRhizobium leguminosarum (biovar.viceae, souche F-15), cultivé en milieu de culture pure, salinée avec les mêmes sels. Les sels sont d'une manière générale inhibiteurs vis à vis de la plante et de la bactérie. Le nombre et les caractéristiques des nodules radiculaires de la plante, le poids sec des plantes (pousses aériennes et racines), le contenu azoté dans les plantes et le nombre de colonies bactériennes décroissent. Les chlorures sont le plus inhibiteurs dans le sol et les carbonates le sont dans le milieu de culture tandis que les sulfates sont le moins inhibiteurs dans les deux cas. Na⁺ est le plus inhibiteur, tandis que Mg²⁺ est le moins nocif. La formation de nodules effectifs sur les racines de plants cultivés dans le sol saliné est très pauvre, maisR. leguminosarum démontre une tolérance possible lorsqu'il est précultivé dans un milieu de culture saliné.

     

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     

Growth regulators,Rhizobium and nodulation in peas

The cytokinin content of roots and nodules of pea and the culture supernatants from two strains of Rhizobium leguminosarum has been examined. Roots, nodules and wild-type Rhizobium culture medium contained very little cytokinin as indicated by bioassay. Chemical ionisation gas chromatography-mass spectrometric analysis of the isopentenyladenine content of the culture medium from the Rhizobium strains confirmed that the content of the wild-type was low (approx. 1 ng dm^-3) but that it was increased by the introduction of the Agrobacterium Ti plasmid into the Rhizobium strain.Abbreviations CI chemical ionisation - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - iPAde isopentenyladenine - MIM multiple ion monitoring     

A Catecholic Siderophore Produced by the Thermoresistant Bacillus licheniformis VK21 Strain

Thermophilic and thermoresistant strains of bacilli were screened on a medium containing Chrome Azurol S for the producers of siderophores. It was found that the Bacillus licheniformis VK21 strain dramatically increases secretion of the metabolite, a chelator of Fe³⁺, in response to addition of manganese(II) salts. The growth of the producer on a minimal medium containing MnSO₄ under the conditions of iron deficiency is accompanied by the accumulation of a catecholic product, the content of which reaches maximum at the beginning of the stationary growth phase of culture. In the presence of FeCl₃, the amount of the catecholic product in the medium considerably decreases. The siderophore, called S_VK21, was isolated from the cultural medium and purified by reversed phase HPLC, and its siderophore function was confirmed by the test for the restoration of growth of producer cells in a medium containing EDTA. The UV spectrum of the siderophore has absorption maxima at 248 and 315 nm. According to the amino acid analysis and NMR spectrometry, the metabolite S_VK21 is 2,3-dihydroxybenzoyl-glycyl-threonine.     

Removal of chromium using Rhizobium leguminosarum

The chromium (Cr^III and Cr^VI) removal capability of Rhizobium leguminosarum was checked by estimating the amount of chromium in the medium before and after inoculation. To determine the efficiency of R. leguminosarum in removal of chromium, the influence of physical and chemical parameters such as temperature, pH and different concentrations (0.1–1.0 mM) of trivalent (Cr^III) and hexavalent (Cr^VI) chromium were studied. The chromium removal in aqueous solution by different size of active and inactivated biomass and immobilized cells of R. leguminosarum in a packed-bed column was also carried out. Results showed that in a medium containing up to 0.5 mM concentration of both Cr^III and Cr^VI, R. leguminosarum showed optimal growth. The maximum chromium removal was at pH 7.0 and 35°C. Active biomass removed 84.4 ± 3.6% of Cr^III and 77.3 ± 4.3% of Cr^VI in 24 h of incubation time. However, inactivated biomass removed maximum chromium after 36 h of incubation. Immobilized bacterial cells in a packed-bed column removed 86.4 ± 1.7% of Cr^III and 83.8 ± 2.2% of Cr^VI in 16 and 20 h of incubation time, respectively.     

Effect of Aluminum on the Production of Siderophore by Rhizobium sp. (Cicer arietinum)

Rhizobium sp. strain BICC 651 in the presence of 100 μM Al³⁺ produced a threefold higher level of siderophore than in the control culture under iron limitation during the stationary phase. Al³⁺ in increasing concentrations resulted in decreased growth, and the effect was alleviated by the addition of iron. Siderophore production decreased gradually in Al³⁺-treated culture as well as in the control with the addition of increasing concentrations of Fe³⁺, and at 50 μM Fe³⁺ the level of siderophore was practically undetectable. The siderophore binds Fe³⁺ and also Al³⁺. The outer membrane protein profiles of the bacteria grown in the presence or absence of Al³⁺ were indistinguishable. Received: 15 November 1999 / Accepted: 21 December 1999     

Growth of Rhizobium leguminosarum in a periodic pressure oscillating, solid-state fermentation of wheat straw

Wheat straw impregnated with a nutrient solution was used to culture Rhizobium leguminosarum. The fermentation was carried out in a periodic pressure, oscillating, solid-state fermenter. At 30 °C and 3 atm, Rhizobium leguminosarum grew to 5.3×10¹⁰ c.f.u. g^–1 substrate dry matter in about 36 h, while only 1.8×10¹⁰ c.f.u. g^–1 substrate dry matter was obtained in a conventional static tray fermenter.     

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.     

Hydroxamate siderophore synthesis by Phialocephala fortinii, a typical dark septate fungal root endophyte

The siderophore production of various isolates of Phialocephala fortinii was assessed quantitatively as well as qualitatively in batch assays under pure culture conditions at different pH values and iron(III) concentrations. We found a distinct effect of both of these parameters on siderophore synthesis and as well as on fungal growth. In comparative analyses of two of the isolates, maximum siderophore production was found at a pH in the range of pH 4.0 to 4.5 while, under the experimental conditions employed, the optimal concentration of ferric iron was determined to be between 20–40 g iron (III) l^–1 (0.36–0.72 M, respectively). HPLC analysis of the culture filtrate of most of the isolates of P. fortinii revealed the excretion of ferricrocin as main hydroxamate siderophore, followed by ferrirubin and ferrichrome C. The pattern of release of these three substances proved to be dependent on pH and iron(III) concentration of the culture medium, and to be specific for each isolate under investigation.     

Effect of iron on the production of pyoverdine and outer membrane proteins ofPseudomonas aeruginosa PA01

Pseudomonas aeruginosa PA01 grown under iron-deficient conditions excreted a yellowish-green pigment, pyoverdine, which has been reported as a siderophore of this bacterium. Addition of iron to the culture medium increased bacterial growth but repressed the production of pyoverdine. Iron levels of the culture medium also had a significant role in the composition of outer membrane proteins ofP. aeruginosa PA01.     

Tn5 insertion mutants of Pseudomonas sp. 267 defective in siderophore production and their effect on clover (Trifolium pratense) nodulated with Rhizobium leguminosarum bv. trifolii

Pseudomonas sp. strain 267 isolated from soil promoted growth of different plants under field conditions and enhanced symbiotic nitrogen fixation in clover under gnotobiotic conditions. This strain produced pyoverdine-like compound under low-iron conditions and secreted vitamins of the B group. The role of fluorescent siderophore production in the beneficial effect of strain 267 on nodulated clover plants was investigated. Several non-fluorescent (Pvd^-) Tn5 insertion mutants of Pseudomonas sp. strain 267 were isolated and characterized. The presence of Tn5 insertions was confirmed by Southern analysis of EcoRI digested genomic DNA of each derivative strain. The siderophore-negative mutants were compared to the parental strain with respect to their growth promotion of nodulated clover infected with Rhizobium leguminosarum bv. trifolii 24.1. We found that all isolated Pvd^- mutants stimulated growth of nodulated clover plants in a similar manner to the parental strain. No consistent differences were observed between strain 267 and Pvd^- derivatives strains with respect to their plant growth promotion activity under gnotobiotic conditions.Dr Deryto died in august 1994     

Incorporation of l-lysine and 2,2′-dipyridyl in the growth media promotes desferrioxamine E production by an actinobacterium

The study describes the use of the chelating agent 2,2′-dipyridyl in conjunction with lysine to increase the production of the siderophore desferrioxamine E by a previously described actinobacterium 23F. Desferrioxamine E is a type of siderophore known to be produced by Streptomycete species. Lysine is a precursor of the siderophore and its presence in the culture medium is known to promote desferrioxamine E synthesis. The further addition of 2,2′-dipyridyl was found to enhance production of the siderophore in the presence of lysine (5 g l^?1) nearly twofold when incorporated at a concentration of 200 μM. Increasing the concentration of the chelating agent above 200 μM resulted in a decrease in siderophore production. The role of the chelating agent was thought to be in creating iron-limiting conditions in the culture medium and so promoting the induction of the desferrioxamine E biosynthetic pathway. This medium is likely to be a useful tool in the screening for producers of desferrioxamine E.     

Influence of growth conditions on production of capsular and extracellular polysaccharides byRhizobium leguminosarum

The influence of growth rate and medium composition on exopolymer production byRhizobium leguminosarum was studied. When grown in medium containing 10g/l mannitol and 1g/l glutamic acid,Rhizobium leguminosarum biovartrifolii TA-1 synthesized up to 2.0g/l of extracellular polysaccharide (EPS), and up to 1.6g/l of capsular polysaccharide (CPS). Under non-growing cell conditions in medium without glutamic acid, CPS synthesis by strain TA-1 could proceed to 2.1g/l, while EPS-production remained relatively low (0.8g/l). Maximal CPS-yield was 2.9g CPS/l medium in a medium containing 20g/l mannitol and 2g/l glutamic acid. TheEPS-deficient strain R. leguminosarum RBL5515,exo4::Tn5 was able to produce CPS to similar levels as strain TA-1, but CPS-recovery was easier because of the low viscosity of the medium and growth of the cells in pellets. With strain TA-1 in nitrogen-limited continuous cultures with a constant biomass of 500mg cell protein/l, EPS was the most abundant polysaccharide present at every dilution rate D (between 0.12 and 0.02 h^–1). The production rates were 50–100mg/g protein/h for EPS and 15–20mg/g protein/h for CPS. Only low amounts of cyclic -(1,2)-glucans were excreted (10–30 mg/l) over the entire range of growth rates.Abbreviations bv biovar - CPS capsular polysaccharide - EPS extracellular polysaccharide - HM_r high molecular mass - LM_r low molecular mass - YEMCR Yeast Extract-Mannitol-Congo Red agar     

Bacteroid-encoded proteins are secreted into the peribacteroid space by Rhizobium leguminosarum

Bacteroids of Rhizobium leguminosarum in root nodules of Pisum sativum are enclosed by a plant-derived peribacteriod membrane (PBM). The contents of the interstitial peribacteroid space (PBS) between bacteroid membrane and PBM were isolated by a controlled osmotic shock of PBM-enclosed bacteroids and analysed by two-dimensional gel electrophoresis. Silver staining revealed approximately 40 PBS polypeptides. Ex planta ³⁵S-methionine labeling of PBM-enclosed bacteroids revealed that about 90% of the PBS proteins are synthesized by the bacteroid. Approximately 30% of the PBS polypeptides are common between the PBS and the periplasmic space of free-living bacteria; one (38kDa) PBS protein is also excreted by free-living bacteria in the bacterial culture medium. At least four bacteroid-encoded PBS polypeptides were clearly identified as symbiosis-specific.