Culture growth and IAA production by a microbial diazotropic symbiont of stem-nodules of the legumeAeschynomene aspera

The stem nodules of the legumeAeschynomene aspera contain indoleacetic acid and a high amount of tryptophan. TheAzorhizobium caulinodans isolated from the stem nodules of the leguminous emergent hydrophyte produced a high amount of IAA (14.8 mg/L) inl-tryptophan-supplemented basal medium. The IAA yields paralleled the culture growth rate and increased up to 52 h. No separate growth and production phase was observed. The IAA production was increased 344% when the medium was supplemented withl-tryptophan, sucrose, FeSO₄·7H₂O, NaNO₃, ascorbic acid and sodium dodecyl sulfate. The possible role of the IAA production in the legume-bacterium symbiosis is discussed.     

News & Notes Indole Acetic Acid and Its Metabolism in Root Nodules of a Monocotyledonous Tree Roystonea regia

A monocotyledonous tree, Roystonea regia, was found to bear root nodules. The root nodules contained a high amount (16.9 μg/g fresh mass) of indole acetic acid (IAA). A big tryptophan pool (1555.1 μg/g fresh mass) was found in the root nodules, which might serve as a source of IAA production. The presence of IAA-metabolizing enzymes IAA oxidase and peroxidase indicated metabolism of IAA in the root nodules. The symbiont isolated from the root nodules of R. regia, a Rhizobium sp., produced high amount of IAA in culture when supplemented with tryptophan. The possible role of this IAA production in the monocotyledonous tree–Rhizobium symbiosis is discussed. Received: 31 December 1997 / Accepted: 5 February 1998     

Production of indole acetic acid in root nodules and culture by a Rhizobium species from root nodules of the fodder legume Melilotus alba DESR.

The root nodules of Melilotus alba DESR ., a fodder legume, contained high amounts of IAA. A tryptophan pool present in the nodule might serve as a source of IAA production. Presence of IAA oxidase and peroxidase in the nodules indicated the metabolism of IAA, at least in part, in the nodules. The Rhizobium species isolated from the root nodules produced a high amount of IAA (190 μg/ml) from L-tryptophan supplemented basal medium. IAA production and microbial growth were coincident. The production of IAA by the Rhizobium sp. was increased by 315% when the medium was supplemented with lactose (1%), NiCl₂ (10 μg/ml), cetyl pyridinium chloride (0.5 μg/ml) and glutamic acid (0.4%), in addition to L-tryptophan (3 mg/ml). The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is discussed.     

Screening and Optimization of Indole-3-Acetic Acid Production and Phosphate Solubilization from Rhizobacteria Aimed at Improving Plant Growth

A total of 216 bacterial strains were isolated from rice rhizospheric soils in Northern Thailand. The bacterial strains were initially tested for solubilization of inorganic phosphate, indole acetic acid (IAA) production, selected strains were then tested for optimized conditions for IAA production and whether these caused stimulatory effects on bean and maize seedling growth. It was found that all strains had solubilized inorganic phosphate (P), but only 18.05% produced IAA. The best IAA producer was identified by biochemical testing and 16S rDNA sequence analysis as Klebsiella SN 1.1. In addition to being the best IAA producer, this strain was a high P-solubilizer and produced the highest amount of IAA (291.97 ± 0.19 ppm) in culture media supplemented with l-tryptophan. The maximum production of IAA was achieved after 9 days of incubation. The culture requirements were optimized for maximum IAA production. The tested of IAA production by selected isolates was studied in a medium with 0, 0.1, 0.2, 0.5, 0.7, and 0.9% (v/v) l-tryptophan. Low levels (12.6 ppm) of IAA production was recorded without tryptophan addition. Production of IAA in Klebsiella SN 1.1 increased with an increase to 0.2% (v/v) tryptophan concentration. The production of IAA was further confirmed by extraction of crude IAA from this isolate and subsequent Thin Layer Chromatography (TLC) analysis. A specific spot from the extracted IAA production was found to correspond with a standard spot of IAA with the same R _f value. The Klebsiella strain SN 1.1 also demonstrated stimulatory effects on bean seedlings in vivo.     

Production and metabolism of indole acetic acid in roots and root nodules of Phaseolus mungo

The mature root nodules of Phaseolus mungo (L.), a leguminous pulse, contain higher amount of indole acetic acid (IAA) than non-nodulated roots. The tryptophan pool present in the mature nodule and young roots might serve as a precursor for the IAA production. Presence of IAA metabolising enzymes – IAA oxidase and peroxidase – indicate the metabolism of IAA in the nodules and roots. In culture, the symbiont, isolated from the nodules, produced a high amount of IAA, when tryptophan was supplied in the medium as a precursor. The symbiont preferred l-isomer over the dl- or d-isomer of tryptophan for IAA production.The important physiological implication of the IAA production in the legume-Rhizobium symbiosis is discussed.     

Studies of the root nodules of leguminous plants IV: Production of indole acetic acid by a bradyrhizobium sp. from the root nodules of a leguminous shrub,crotalaria retusa L

Bradyrhizobium sp. isolated from the root nodules of a leguminous shrub, Crotalaria retusa L., produced a high amount of indole acetic acid (IAA) from tryptophan in the culture. The bacteria preferred D-isomer to the DL- or L-isomer of tryptophan for the IAA production. The IAA production could be increased up to 153.6% over control by supplementing the medium with arabinose (0.5%), ZnSO₄(0.01 μg/ml), KNO₃ (0.1%), thiamine-HCl (0.01 μg/ml) and EDTA (5 μg/ml). The possible role of the rhizobial production of IAA with the rhizobia-legume 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.     

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.     

Stimulation of indoleacetic acid production in a <Emphasis Type="Italic">Rhizobium</Emphasis> isolate of <Emphasis Type="Italic">Vigna mungo</Emphasis> by root nodule phenolic acids

The influence of endogenous root nodules phenolic acids on indoleacetic acid (IAA) production by its symbiont (Rhizobium) was examined. The root nodules contain higher amount of IAA and phenolic acids than non-nodulated roots. Presence of IAA metabolizing enzymes, IAA oxidase, peroxidase, and polyphenol oxidase indicate the metabolism of IAA in the nodules and roots. Three most abundant endogenous root nodule phenolic acids (protocatechuic acid, 4-hydroxybenzaldehyde and p-coumaric acid) have been identified and their effects on IAA production by the symbiont have been studied in l-tryptophan supplemented yeast extract basal medium. Protocatechuic acid (1.5 μg ml⁻¹) showed maximum stimulation (2.15-fold over control) of IAA production in rhizobial culture. These results indicate that the phenolic acids present in the nodule might serve as a stimulator for IAA production by the symbiont (Rhizobium). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Some tryptophan pathways in the phytopathogen Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv.oryzae, the causal or-ganism of bacterial blight of rice which produces leaf blight as well as kresek (wilt) symptoms in plants were tested for indole, auxin production in culture supplemented withl-tryptophan. On the basis of indoleacetic acid (IAA) production the isolates were grouped into IAA-positive and IAA-negative. Out of 17 isolates, 11 were IAA-positive while 6 were IAA-negative. The isolates metabolized tryptophan through two different routes and the isolates vary in the pathway of tryptophan utilization. The IAA-positive isolates converted tryptophan to IAA as the end product, whereas the IAA-negative isolates formed anthranilate as an intermediate metabolite and finally produced pyrocatecholvia the kynurenine pathway. Quantification of tryptophan metabolism revealed that the maximum production of IAA and pyrocatechol in culture occurred during 2-d incubation at 30±2°C.     

Production of Indole Acetic Acid in Culture by a Rhizobium Species from the Root Nodules of a Monocotyledonous Tree,Roystonea regia

The study of the rhizobial root nodules of the monocotyledonous tree Roystonea regia revealed that the Rhizobium sp. isolated from the root nodules produced high amounts (45.6 μg/ml) of indole acetic acid (IAA) from L‐tryptophan supplemented basal medium. The IAA production reached its optimum using 3 mg/ml of L‐tryptophan. The preferred carbon and nitrogen sources were glucose and KNO₃ and the optimum concentrations 1% and 0.02%, respectively. FeSO₄ × 7 H₂O was found to be the only metal ion that increased IAA production. An optimum IAA production was also achieved when the basal medium was supplemented with glucose (1%), FeSO₄ × 7 H₂O (10 μg/ml), KNO₃ (0.02%) as well as EDTA (5 μg/ml) and L‐tryptophan (3 mg/ml). The possible role of IAA production in the monocotyledonous tree‐Rhizobium symbiosis is discussed. Hormone production is shown to be the beneficial aspect of this symbiosis as shown earlier in dicotyledonous plants.     

Production of 3-indoleacetic acid by aRhizobium sp. fromMimosa pudica

ARhizobium species isolated from the root nodules of the sensitive plant,Mimosa pudica, produced 60 mg/L of 3-indoleacetic acid (IAA) froml-tryptophan in culture. The production of IAA started simultaneously with the growth and had no different growth or production phase. The stationary phase of growth was reached after 55 h, but the production of IAA increased gradually up to 80 h, and then remained constant. The IAA production could be promoted in the culture medium up to 365% by supplementing the medium with maltose, CuSO₄ and Triton X-100.     

Studies on seasonal variation of plant hormones,nitrogen flixation and indole acetic acid metabolism in root nodulus ofButea monosperma (Lam.) Taub.

Summary Mature nodules ofButea monosperma (Lam.) Taub. (B. monosperma) contained higher amounts of indole acetic acid-like (IAA), cytokinin-like (CK), gibberellic acid-like (GA) and abscisic acid-like (ABA) substances, and were more active in IAA metabolism and nitrogen fixation. Roots contained lower amount of all the hormones. The higher level of IAA in the nodules than the roots was due to more synthesis from tryptophan and simultaneously less destruction by IAA oxidase. IAA oxidase, in turn, was regulated by phenols.Regardless of the hormone levels, the nodules of a particular age attained a fixed size indicating that regulation of formation and growth of the nodules are not the sole function of the nodular hormones. Seasonal studies on the mature nodules showed positive relation of nitrogenase (N₂ase) with IAA and CK, and negative relation with GA and ABA.     

Growth behaviour and indole acetic acid (IAA) production by a Rhizobium isolated from root nodules of Alysicarpus vaginalis DC

From the root nodules of Alysicarpus vaginalis DC, the symbiont was isolated and identified as a Rhizobium sp. The bacteria produced a high amount (107 microg/ml) of indole acetic acid (IAA) in culture from tryptophan supplemented yeast extract mannitol medium. The isolate preferred L-isomer of tryptophan for maximum IAA production. The production was maximum when the bacteria reached its stationary phase of growth. The production of IAA could be increased up to 70% over yeast extract glucose medium by supplementing ZnSO4, 7H2O (0.5 microg/ml). L-asparagine (0.2%) and sodium dodecyl sulfate (1.0 microg/ml). The possible relationship between the rhizobial IAA production and legume-rhizobia symbiosis is discussed.     

Production of 3-indolylacetic acid in root nodules and culture by a <Emphasis Type="Italic">Rhizobium</Emphasis> species isolated from root nodules of the leguminous pulse <Emphasis Type="Italic">Phaseolus mungo</Emphasis>

The root nodules of Phaseolus mungo (a herbaceous leguminous pulse) contained a high amount of 3-indolylacetic acid (IAA). A tryptophan pool present in the nodule might play the role of precursor for IAA production. From the root nodule a Rhizobium sp. was isolated. The symbiont produced a large amount of IAA (142 mug/mL) from L-tryptophan-supplemented basal medium. The production of IAA by the symbiont was much increased over the control when a L: -tryptophan (2 mg/mL) supplemented C-free mineral medium was enriched with mannitol (1 %), L: -asparagine (0.3 %) and thiamine hydrochloride (1 mug/mL). The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is discussed.     

d-Alanine production from dl-alanine by Candida maltosa with asymmetric degrading activity

Summary To develop a practical process for d-alanine production from dl-alanine, we screened 107 yeasts for their asymmetric degrading activity against dl-alanine. Candida maltosa JCM1504 degraded the l-isomer ten times more rapidly than the d-isomer. The cells of this strain were used as a biocatalyst for eliminating the l-isomer. However, when the degradation reaction was conducted in the presence of a high concentration of dl-alanine, the pH of the reaction mixture was rapidly increased by the liberation of ammonia from l-alanine, and consequently the reaction stopped. This hindrance was overcome by controlling the pH value at 6.0 with H₂SO₄ during the reaction. Additionally, we found that the maximum rate of l-isomer degradation was obtained at 30° C and pH 6.0 under conditions of high aeration (1.0 vvm) and agitation (1200 rpm). Under the optimal conditions, the l-isomer of 200 g dl-alanine/l was completely degraded within 40 h and 90 g d-alanine/l remained in the reaction mixture. d-Alanine was easily isolated from the reaction mixture. The chemical and optical purity of the d-isomer product so obtained was 99.0% and 99.9% enantiomeric excess, respectively.Offprint requests to: I. Umemura     

Biosynthesis of indole-3-acetic acid in tomato shoots: Measurement,mass-spectral identification and incorporation of −2H from −2H2O into indole-3-acetic acid,d- and l-tryptophan,indole-3-pyruvate and tryptamine

Indole-3-acetic acid (IAA) and its putative precursors, l- and d-tryptophan, indole-3-pyruvate, and tryptamine were isolated from tomato (Lycopersicon esculentum (L.) Mill.) shoots, identified by mass spectrometry, and measured using capillary gas chromatography with an electron capture detector and radioactive internal standards. Average amounts present were 7.9ng · (g FW)^–-1 IAA, 5.7ng · (g FW)^–-1 indole-3-pyruvate, 132 ng · (g FW)^–-1 tryptamine, 103 ng · (g FW)^–-1 d-tryptophan, and 2250 ng · (g FW)^–-1 l-tryptophan. Indole-3-acetaldoxime was not found; detection limits were less than 1ng · (g FW)^–-1. When tomato shoots were incubated for 6, 10 and 21 h in 30% ^–2H₂O, up to four positions in IAA, l- and d-tryptophan, tryptamine and indole-3-pyruvate became labelled with ^–2H. Compounds became labelled rapidly with 10% of IAA molecules containing ^–2H after 6 h. The percentage of labelled molecules of IAA and l-tryptophan increased up to 10 h but then decreased again, correlating with an increase in the total shoot tryptophan and presumably a result of protein hydrolysis in the excised, slowly senescing tissue. The amount of ^–2H in d-tryptophan also showed an increase followed by a decrease, but the proportion of labelled molecules was much less than in l-tryptophan and IAA. Tryptamine became labelled initially at a similar rate to IAA but continued to accumulate ^–2H up to 21 h. We conclude that tryptamine is synthesized from a different pool of tryptophan from that used in IAA synthesis, and is not a major endogenous precursor of IAA in tomato shoots. Indole-3-pyruvate was the most heavily labelled compound after 6 and 10 h incubation (21-h data not available). Furthermore, the proportion of ^–2H-labelled indole-3-pyruvate molecules was quantitatively consistent with the amount of label in IAA. On the other hand, a quantitative comparison of the IAA turnover rate and the rate of ^–2H incorporation into both l- and d-tryptophan indicates that IAA is not made from the total shoot pool of either l- or d-tryptophan. Instead IAA appears to be synthesized from a restricted pool which is turning over rapidly and which has access to both newly synthesized tryptophan and that from protein hydrolysis.Abbreviations GC-ecd gas chromatography with electroncapture detector - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - IAOX indole-3-acetaldoxime - IPyA indole-3-pyruvate - PFB pentafluorobenzyl - R_T retention time - TNH₂ tryptamine - Trp tryptophan - SIM selected ion monitoring We wish to thank Ms. Sue Alford for running the mass spectra and Dr Harry Young for advice with the mass spectrometry. The work was supported by grants from the University of Auckland Research Committee and the C. Alma Baker Trust fund. The mass spectrometer was supported jointly by the University Grants Commitee (NZ) and the DSIR Division of Horticulture and Processing.     

d-Methionine preparation from racemic methionines by Proteus vulgaris IAM 12003 with asymmetric degrading activity

The microbial degradation of l-methionine was investigated in order to develop a practical process for d-methionine production from racemic methionines. Among the 1000 culture strains tested, microorganisms belonging to the Achromobacter, Bacillus, Micrococcus, Morganella, Proteus, Providencia, Pseudomonas and Sarcina genera exhibited a high l-methionine-degrading activity. Proteus vulgaris IAM 12003 was determined to be the best strain and was used as a biocatalyst for eliminating the l-isomer. The degradation of l-isomer in this P. vulgaris IAM 12003 cell was assured by the action of l-amino acid oxidase. The maximum rate of l-isomer degradation was obtained at 30 °C and pH 8.0. Under these optimal conditions, the l-isomer in a 100 g/l mixture of racemic methionines was almost degraded within 20 h, with 46.5 g d-methionine/l remaining in the reaction mixture. Crystalline d-methionine, with a chemical purity greater than 99% and optical purity of 99.9% enantiomeric excess, was obtained at a yield of 30% from the reaction mixture by simple purification. Received: 17 June 1996 / Received last revision: 11 September 1996 / Accepted: 29 September 1996     

Amino acid transport by<Emphasis Type="Italic"> Sphingomonas</Emphasis> sp. strain Ant 17 isolated from oil-contaminated Antarctic soil

The Antarctic bacterial isolate Sphingomonas sp. strain Ant 17 utilized a wide range of L-isomer amino acids as the sole carbon and energy source for growth. The pH and temperature optima for growth on amino acids were pH 7.0 and 15°C, respectively. Growth on serine and tryptophan was inhibited by uncouplers and inhibitors of oxidative phosphorylation, but not by monensin, a Na⁺/H⁺ antiporter, suggesting that sodium gradients were not specifically required for growth on these amino acids. Serine transport was via a high-affinity (apparent K_m of 8 M) permease specific for both the L- and D-isomer. Tryptophan transport exhibited biphasic kinetics with both high-affinity (apparent K_m of 2.5 M) and low-affinity (non-saturable) uptake systems detected. The high-affinity system was specific for L-tryptophan, L-tyrosine, and L-phenylalanine whereas the low-affinity permease was specific for L-tryptophan and L-phenylalanine, but not L-tyrosine. Neither orthovanadate nor sodium arsenate, inhibitors of ATP-dependent permeases, had any significant inhibitory effect on the rate of serine and tryptophan transport. The protonophore carbonyl cyanide m-chlorophenylhydrazone completely abolished serine and tryptophan transport; maximum rates of solute uptake were observed at acidic pH values (pH 4.0–5.0) for both amino acids. These results suggest that an electrochemical potential of protons is the driving force for serine and tryptophan transport by Ant 17. These high-affinity proton-driven permeases function over environmental extremes (e.g. broad temperature and pH range) that are likely to prevail in the natural habitat of this bacterium.     

Overexpression of the gene forN-acylamino acid racemase fromAmycolatopsis sp. TS-1-60 inEscherichia coli and continuous production of optically active methionine by a bioreactor

A DNA sequence encodingN-acylamino acid racemase (AAR) was inserted downstream from the T7 promoter in pET3c. The recombinant plasmid was introduced intoEscherichia coli MM194 lysogenized with a bacteriophage having a T7 RNA polymerase gene. The amount of AAR produced by theE. coli transformant was 1100-fold more than that produced byAmycolatopsis sp. TS-1-60, the DNA donor strain. The AAR was purified to homogeneity from the crude extract of theE. coli transformant by two steps: heat treatment and Butyl-Toyopearl column chromatography. Bioreactors for the production of optically active amino acids were constructed with DEAE-Toyopearl-immobilized AAR andd- orl-aminoacylase.d- orl-methionine was continuously produced with a high yield fromN-acetyl-dc-methionine by the bioreactor.