Aromatic aminotransferase activity and indoleacetic acid production in Rhizobium meliloti.

Bacterial indoleacetic acid (IAA) production, which has been proposed to play a role in the Rhizobium-legume symbiosis, is a poorly understood process. Previous data have suggested that IAA biosynthesis in Rhizobium meliloti can occur through an indolepyruvate intermediate derived from tryptophan by an aminotransferase activity. To further examine this biosynthetic pathway, the aromatic aminotransferase (AAT) activity of Rhizobium meliloti 102F34 (F34) was characterized. At least four proteins were detected on nondenaturing gels of F34 protein extracts that exhibited AAT activity. All four of these AATs were constitutively produced and utilized the aromatic amino acids tryptophan, phenylalanine, and tyrosine as amino substrates. Two AATs were also capable of using aspartate. Plasmids from an F34 gene bank were identified that coded for the synthesis of at least three of these proteins, and the respective gene sequences were localized by transposon mutagenesis. Selected transposon insertions were recombined into the F34 genome to produce strains defective in two of these proteins (AAT1 and AAT2). Characterization of the mutants revealed that neither was essential for the biosynthesis of IAA in the absence of exogenous tryptophan, but that both contributed to IAA biosynthesis when high levels of exogenous tryptophan were present. AAT1 and AAT2 were also not required for the production of a minimal level of aromatic amino acids, but both were able to scavenge nitrogen from the aromatic amino acids during nitrogen deprivation. Neither AAT1 nor AAT2 was essential for symbiosis with alfalfa.     

The hisC1 gene, encoding aromatic amino acid aminotransferase-1 in Azospirillum brasilense Sp7, expressed in wheat

Background and aims

Production of indole-3-acetic acid (IAA) by Azospirillum brasilense is one of the most important mechanisms underlying the beneficial effects observed in plants after inoculation with this bacterium. This study determined the contribution of the hisC1 gene, which encodes aromatic amino acid aminotransferase-1 (AAT1), to IAA production, and analyzed its expression in the free-living state and in association with the roots of wheat.

Methods

We determined production of IAA and AAT activity in the mutant hisC::gusA-sm ^R . To study the expression of hisC1, a chromosomal gene fusion was analyzed by following β-glucuronidase (GUS) activity in vitro, in the presence of root exudates, and in association with roots.

Results

IAA production in the hisC mutant was not reduced significantly compared to the activity of the wild-type strain. AAT1 activity was reduced by 50% when tyrosine was used as the amino acid donor, whereas there was a 30% reduction when tryptophan was used, compared to the activity of the wild-type strain. Expression of the fusion protein was up-regulated in both logarithmic and stationary phases by several compounds, including IAA, tryptophan, tyrosine, and phenyl acetic acid. We observed the expression of hisC1 in bacteria associated with wheat roots. Root exudates of wheat and maize were able to stimulate hisC1 expression.

Conclusions

The expression data indicate that hisC1 is under a positive feedback control in the presence of root exudates and on plants, suggesting that AAT1 activity plays a role in Azospirillum–plant interactions.     

Isolation and characterization of diazotrophic growth promoting bacteria from rhizosphere of agricultural crops of Korea

Free-living nitrogen fixing bacteria were isolated from rhizosphere of seven different plant namely sesame, maize, wheat, soybean, lettuce, pepper and rice grown in Chungbuk Province, Korea. Five isolates with nitrogenase activity above 150nmol(-1) mg(-1) protein were identified based on, phenotypic and 16S rDNA sequences analysis. The strains were identified as Stenotrophomonas maltophilia (PM-1, PM-26), Bacillus fusiformis (PM-5, PM-24) and Pseudomonas fluorescens (PM-13), respectively. All the isolates produced indole-3-acetic acid (IAA), in the presence of tryptophan, ranging from 100.4 microg ml(-1) (PM-13) to 255 microg ml(-1) (PM-24). The isolate PM-24 (Bacillus fusiformis) exhibiting highest nitrogenase activity (3677.81 nmol h(-1) mg(-1) protein) and IAA production (255microg ml(-1)) has a promising potential for developing as a plant growth promoting rhizobacteria.     

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 indole-3-acetic acid, aromatic amino acid aminotransferase activities and plant growth promotion by Pantoea agglomerans rhizosphere isolates

The production of auxins, such as indole-3-acetic acid (IAA), by rhizobacteria has been associated with plant growth promotion, especially root initiation and elongation. Six indole-producing bacteria isolated from the rhizosphere of legumes grown in Saskatchewan soils and identified as Pantoea agglomerans spp. were examined for their ability to promote the growth of canola, lentil and pea under gnotobiotic conditions and for tryptophan (Trp)-dependent IAA production. Five of the isolates enhanced root length, root weight or shoot weight by 15–37% in at least one of the plant species, but isolates 3–117 and 5–51 were most consistent in enhancing plant growth across the three species. Indole concentrations in the rhizosphere of plants grown under gnotobiotic conditions increased in the presence of the rhizosphere isolates and when Trp was added 3 days prior to plant harvest. Isolates 3–117, 5–51 and 5–105 were most effective in increasing rhizosphere indole concentrations. Colony hybridization confirmed that all of the isolates possessed the ipdC gene which codes for a key enzyme in the Trp-dependent IAA synthetic pathway. The activity of amino acid aminotransferase (AAT), catalyzing the first step in the Trp-dependent synthetic pathway, was examined in the presence of Trp and other aromatic amino acids. All of the isolates accumulated Trp internally and released different amounts of IAA. The production of IAA from the isolates was greatest in the presence of Trp, ranging from 2.78 to 16.34 μg mg protein⁻¹ in the presence of 250 μg of Trp ml⁻¹. The specific activity of AAT was correlated with the concentration of IAA produced in the presence of Trp but not when tyrosine (Tyr), phenylalanine (Phe) or aspartate (Asp) was used as a sole nitrogen source. Isolate 3–117, which produced significant concentrations of IAA in the presence and absence of Trp, was able to use aromatic amino acids as sole sources of nitrogen and was most consistent in enhancing the growth of canola, lentil and pea may have potential for development as a plant growth-promoting inoculant. Responsible Editor: Peter A. H. Bakker.     

Auxin production by bacteria associated with orchid roots

Bacteria associated with the roots of greenhouse tropical orchids were shown to produce indole-3-acetic acid (IAA) and to excrete it into the culture liquid. The presence and activity of IAA were demonstrated colorimetrically, by thin-layer chromatography, and by biotests. The associated bacteria varied in their ability to excrete indole compounds (1-28 microg/ml nutrient broth). Addition of tryptophan to the growth medium enhanced phytohormone production. Upon addition of 200 microg/ml tryptophan, the bacteria isolated from Dendrobium moschatum roots (Sphingomonas sp. 18, Microbacterium sp. 23, Mycobacterium sp. 1, Bacillus sp. 3, and Rhizobium sp. 5) produced 50.2, 53.1, 92.9, 37.6, and 60.4 microg IAA/ml respectively, while the bacteria isolated from Acampe papillosa roots (Sphingomonas sp. 42, Rhodococcus sp. 37, Cellulomonas sp. 23, Pseudomonas sp. 24, and Micrococcus luteus) produced 69.4, 49.6, 53.9, 31.0, and 39.2 microg IAA/ml. Auxin production depended on cultivation conditions and on the growth phase of the bacterial cultures. Treatment of kidney bean cuttings with bacterial culture liquid promoted formation of a "root brush" with location height 7.4- to 13.4-fold greater than the one in the control samples. The ability of IAA-producing associated bacteria to act as stimulants of the host plant root development is discussed.     

Occurrence of Gluconacetobacter diazotrophicus in tropical and subtropical plants of Western Ghats, India

Endophytic bacteria were isolated from the tissues of surface sterilized roots, stems, and leaves of fifty different crop plants. Phenotypic, biochemical tests and species-specific PCR assay permitted identification of four isolates of Gluconacetobacter diazotrophicus from root tissues of carrot (Daucus carota L.), raddish (Raphanus sativus L.), beetroot (Beta vulgaris L.) and coffee (Coffea arabica L.). Further the plant growth promoting traits such as nitrogenase activity, production of phytohormone indole acetic acid (IAA), phosphorus and zinc solubilization were assessed. Significant nitrogenase activity was recorded among the isolates and all the isolates produced IAA in the presence of tryptophan. Though all the four isolates efficiently solubilized phosphorus, the zinc solubilizing ability differed among the isolates.     

Physiological evidence for differently regulated tryptophan-dependent pathways for indole-3-acetic acid synthesis in Azospirillum brasilense

Disruption of ipdC, a gene involved in indole-3-acetic acid (IAA) production by the indole pyruvate pathway in Azospirillum brasilense Sp7, resulted in a mutant strain that was not impaired in IAA production with lactate or pyruvate as the carbon source. A tryptophan auxotroph that is unable to convert indole to tryptophan produced IAA if tryptophan was present but did not synthesise IAA from indole. Similar results were obtained for a mutant strain with additional mutations in the genes ipdC and trpD. This suggests the existence of an alternative Trp-dependent route for IAA synthesis. On gluconate as a carbon source, IAA production by the ipdC mutant was inhibited, suggesting that the alternative route is regulated by catabolite repression. Using permeabilised cells we observed the enzymatic conversion of tryptamine and indole-3-acetonitrile to IAA, both in the wild-type and in the ipdC mutant. IAA production from tryptamine was strongly decreased when gluconate was the carbon source.     

Phytostimulatory effect of Azospirillum brasilense wild type and mutant strains altered in IAA production on wheat

Auxin production by Azospirillum is believed to play a major role in the observed plant growth promoting effect. By using different genetically modified strains, the contribution of auxin biosynthesis by A. brasilense in altering root morphology was evaluated in a plate assay. Inoculation with the wild type strains A. brasilense Sp245 and Sp7 resulted in a strong decrease in root length and increase in root hair formation. This effect was abolished when inoculating with an ipdC mutant of A. brasilense. The ipdC gene encodes a key enzyme in the IPyA pathway of IAA synthesis by A. brasilense. On the other hand, the observed auxin effect was further enhanced by adding tryptophan, a precursor of IAA, to the plates and could be mimicked by replacing the Azospirillum cells by a particular concentration of IAA. Furthermore, particular mutants (rpoN, scrp) and transconjugants (extra copy of ipdC) of A. brasilense were tested in the plate assay. Together, these results confirm the important role of IAA produced by Azospirillum in altering root morphology and illustrate the power of combining genetic tools and bioassays to elucidate the mechanism of a beneficial Azospirillum-plant interaction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inoculation Effects of Pseudomonas putida, Gluconacetobacter azotocaptans, and Azospirillum lipoferum on Corn Plant Growth Under Greenhouse Conditions

Alcohol production from corn is gaining importance in Ontario, Canada, and elsewhere. A major cost of corn production is the cost of chemical fertilizers and these continue to increase in price. The competitiveness of alcohol with fossil fuels depends on access to low-cost corn that allows growers to earn a sustainable income. In this study we set out to determine if we can identify root-associated microorganisms from Ontario-grown corn that can enhance the nutrient flow to corn roots, directly or indirectly, and help minimize the use of extraneous fertilizer. Bacteria were isolated from corn rhizosphere and screened for their capacity to enhance corn growth. The bacteria were examined for their ability to fix nitrogen, solubilize phosphate, and produce indole acetic acid (IAA) and antifungal substances on potato dextrose agar. Bacterial suspensions were applied to pregerminated seed of four corn varieties (39D82, 39H84, 39M27, and 39T68) planted in sterilized sand and unsterilized cornfield soil. The plants were grown under greenhouse conditions for 30 days. Three isolates were identified as having growth-promoting effect. These bacteria were identified as to species by biochemical tests, fatty acid profiles, and 16S rDNA sequence analysis. Corn rhizosphere isolates, Gluconacetobacter azotocaptans DS1, Pseudomonas putida CQ179, and Azospirillum lipoferum N7, provided significant plant growth promotion expressed as increased root/shoot weight when compared to uninoculated plants, in sand and/or soil. All strains except P. putida CQ179 were capable of nitrogen fixation and IAA production. Azospirillum brasilense, however, produced significantly more IAA than the other isolates. Although several of the strains were also able to solubilize phosphate and produce metabolites inhibitory to various fungal pathogens, these properties are not considered as contributing to growth promotion under the conditions used in this study. These bacteria will undergo field tests for their effect on corn growth.     

The characteristics of amino acid catabolism in bacteria of the genus Citrobacter

The comparison of the occurrence of enzymes effecting the deamination of dicarbon, aromatic and oxyamino acids, as well as transamination enzymes, in Citrobacter bacteria and the activity levels of these enzymes was made. The constant sign of such bacteria was the presence of serine and threonine dehydratase activity. 92% of the strains showed the presence of phenylalanine deaminase. No tryptophan activity was established. 96-98% of Citrobacter strains possessed phenylalanine, tyrosine and tryptophan aminotransferases with alpha-ketoglutaric acid functioning as the acceptor of the amino group.     

高产吲哚乙酸及高泌氨巴西固氨螺菌的筛选与鉴定

目的：从玉米根际和土壤中分离具有高产吲哚乙酸较强的泌氨能力的巴西固氮螺菌。方法：分别通过半固体NFb培养基、CR培养基、LB培养基分离培养固氮菌株,并经过一系列菌落菌体形态特征、生理生化特性和16S rDNA序列测定等试验对其进行鉴定。结果：经分离纯化获得10株固氮菌,并鉴定均为巴西固氮螺菌（Azospirillum brasilense）,其中菌株R7在甘油半固体培养基上能分泌约14mmol/L的氨,在添加了色氨酸的培养基中能够合成58.8μg/ml的吲哚-3-乙酸（IAA）。结论：成功筛选得到一株既高产吲哚乙酸又有较强的泌氨能力的巴西固氮螺菌。     

Content of indoleacetic acid and its metabolism in root nodules ofMelilotus alba

The root nodules ofMelilotus alba, a leguminous fodder herb, contain a high amount of indoleacetic acid (IAA). The tryptophan pool present in the nodule might serve as a source for the IAA production. Metabolism of IAA in the nodules was evidenced by the presence of IAA-metabolizing enzymes, IAA oxidase and peroxidase. A high amount of IAA was produced by the symbiont isolated from the nodules in culture, when supplemented with tryptophan. For IAA production, the bacteria preferred thel-isomer over thedl- ord-isomer of tryptophan. The possible role of nodular IAA production on the legume-Rhizobium symbiosis is discussed.     

Growth and indole-3-acetic acid biosynthesis of Azospirillum brasilense Sp245 is environmentally controlled

Batch and fed batch cultures of Azospirillum brasilense Sp245 were conducted in a bioreactor. Growth response, IAA biosynthesis and the expression of the ipdC gene were monitored in relation to the environmental conditions (temperature, availability of a carbon source and aeration). A. brasilense can grow and produce IAA in batch cultures between 20 and 38 degrees C in a standard minimal medium (MMAB) containing 2.5 gl(-1)l-malate and 50 microgml(-1) tryptophan. IAA synthesis requires depletion of the carbon source from the growth medium in batch culture, causing growth arrest. No significant amount of IAA can be detected in a fed batch culture. Varying the concentration of tryptophan in batch experiments has an effect on both growth and IAA synthesis. Finally we confirmed that aerobic growth inhibits IAA synthesis. The obtained profile for IAA synthesis coincides with the expression of the indole-3-pyruvate decarboxylase gene (ipdC), encoding a key enzyme in the IAA biosynthesis of A. brasilense.     

Involvement of IAA in the interaction betweenAzospirillum brasilense andPanicum miliaceum roots

The possible involvement of IAA in the effect thatAzospirillum brasilense has on the elongation and morphology ofPanicum miliaceum roots was examined by comparing in a Petri dish system the effects of inoculation with a wild strain (Cd) with those of an IAA-overproducing mutant (FT-326). Both bacterial strains produced IAA in culture in the absence of tryptophan. At the stationary growth phase, production of IAA by FT-326 wasca. 12 times greater than that of Cd. When inoculation was made with bacterial concentrations higher than, 10⁶ colony forming units ml^–1 (CFU ml^–1), both strains inhibited root elongation to the same extent. At lower concentrations Cd enhanced elongation, by 15–20%, while FT-326 was ineffective. Both strains promoted root-hair development, and root-hairs were produced nearer the root tip the higher the bacterial concentration (e. g. root elongation region was reduced). Effects of FT-326 on root-hair development were greater than those of Cd. Acidified ether extracts of Cd and FT-326 cultures had inhibitory or promoting effects on root elongation depending on the dilution applied. At low dilutions, extracts from FT-326 were more inhibitory for elongation than those from Cd. At higher dilutions root elongation was promoted, but FT-326 extracts had to be more diluted than those from Cd. Dilutions that promoted root elongation contained supra-optimal concentrations of IAA, 1–3 orders of magnitude higher than those required for optimal enhancement by synthetic IAA. It is suggested that the bacteria produce in culture an IAA-antagonist or growth inhibitor that decreases the effectiveness of IAA action. The large variability reported for the effects ofAzospirillum on root elongation could be the result of the opposite effects on root elongation of IAA and other compounds, produced by the bacteria.     

The enrichment of a ruminal bacterium (Megasphaera elsdenii YJ-4) that produces the trans-10, cis-12 isomer of conjugated linoleic acid

AIMS: To isolate predominant ruminal bacteria that produce trans-10, cis-12 conjugated linoleic acid (CLA) from linoleic acid (LA). METHODS AND RESULTS: Mixed bacteria from ruminal contents of a cow fed grain were enriched with DL-lactate and trypticase. They produced more trans-10, cis-12 CLA than those that were not enriched (7 vs 2 microg mg protein(-1), P < 0.05). Enrichments had an abundance of large cocci that produced trans-10, cis-12 CLA from LA. Strain YJ-4 produced the most trans-10, cis-12 CLA (approx. 7 microg mg protein(-1)) and 16S rDNA sequencing indicated that YJ-4 was a strain of Megasphaera elsdenii. Megasphaera elsdenii T81 produced approx. 4 microg trans-10, cis-12 CLA mg protein(-1) while strains B159, AW106 and JL1 produced < 0.5 microg mg protein(-1). The trans-10, cis-12 CLA production of YJ-4 was first order with respect to cell concentration (0-800 microg protein ml(-1)), but kinetics were not first order with respect to substrate concentration. CONCLUSIONS: Some M. elsdenii strains produce significant amounts of trans-10, cis-12 CLA. SIGNIFICANCE AND IMPACT OF THE STUDY: Trans-10, cis-12 CLA appears to cause milk fat depression in cattle fed diets supplemented with grain and polyunsaturated fatty acids, but predominant ruminal bacteria that produced trans-10, cis-12 CLA from LA had not previously been isolated.     

Acetylene reduction and indoleacetic acid production by Azospirillum isolates from Cactaceous plants

Azospirillum isolates were obtained from rhizosphere soil and roots of three cactaceae species growing under arid conditions. All Azospirillum isolates from rhizosphere and roots ofStenocereus pruinosus andStenocereus stellatus were identified asA. brasilense; isolates of surface-sterilized roots fromOpuntia ficus-indica were bothA. brasilense andA. lipoferum. Azospirilla per g of fresh root in the three species ranged from 70×10³ to 11×10³. The most active strains in terms of C₂H₂ reduction (25–49.6 nmol/h·ml) and indoleacetic acid (IAA) production (36.5–77 μg/ml) were those identified asA. brasilense and isolated from Stenocereus roots.A. lipoferum isolated from Opuntia roots produced low amounts of IAA (6.5–17.5 μg/ml) and low C₂H₂-reduction activity (17.8–21.2 nmol/h·ml).     

INVOLVEMENT OF INDOLE‐3‐ACETIC ACID PRODUCED BY THE GROWTH‐PROMOTING BACTERIUM AZOSPIRILLUM SPP. IN PROMOTING GROWTH OF CHLORELLA VULGARIS1

Involvement of indole‐3‐acetic acid (IAA), produced by the microalgae‐growth‐promoting bacteria Azospirillum brasilens and A. lipoferum, in promoting growth of the microalga Chlorella vulgaris Beij. was studied. Four wildtype strains of Azospirillum and their IAA‐deficient mutants were co‐immobilized with C. vulgaris in alginate beads. Cultures were grown in synthetic growth medium supplemented with tryptophan. Growth promotion of microalgae and production of exogenous IAA by Azospirillum spp. were monitored. All wildtype Azospirillum spp. produced significant but varying amounts of IAA, while their mutant forms produced significantly less. The results demonstrated a significant growth promotion in Chlorella cultures when immobilized with the four wildtype strains of Azospirillum, while very low or no enhanced growth was induced by the four IAA‐deficient mutants, compared to when C. vulgaris is immobilized alone. A complementation experiment, where an IAA‐attenuated mutant (A. brasilense SpM7918) was supplemented with IAA produced by its parental wildtype strain (A. brasilense Sp6), restored growth promotion in the microalgae‐mutant culture.     

Antagonism among Gluconacetobacter diazotrophicus strains in culture media and in endophytic association

In this study the antagonistic activity among 55 Gluconacetobacter diazotrophicus strains, belonging to 13 electrophoretic types (ETs), in culture media was analyzed. Antagonistic effects were seen only in strains belonging to two ETs named ET-1 and ET-3. Two out of 29 ET-1 strains, and 3 out of 7 ET-3 strains of G. diazotrophicus showed antagonistic effects against many other strains belonging to all the ETs of this species analyzed, and against closely related strains of Gluconacetobacter species, including Gluconacetobacter johannae, Gluconacetobacter azotocaptans and Gluconacetobacter liquefaciens but not against other phylogenetically distant bacterial species. Results showed that the substance responsible of such antagonistic activity is a low molecular mass molecule (approximately 3400 Da), stable from pH 3.5 to 8.5, and very stable at 4 degrees C for 10 months. This substance was sensitive to proteases, and the antagonistic activity was lost after 2 h at 95 degrees C. All of these features show that the substance is related to bacteriocin-like molecules. The antagonistic substance should be chromosomally encoded because ET-3 strains of G. diazotrophicus do not harbor any plasmids. The antagonistic ability of ET-3 strains of G. diazotrophicus could be an advantage for the natural colonization of the sugarcane environment, as was observed in experiments with micropropagated sterile sugarcane plantlets co-inoculated with a bacteriocin-producer strain and a bacteriocin-sensitive strain of G. diazotrophicus. In these experiments, both in the rhizosphere as well as inside the roots, the bacteriocin-sensitive population decreased drastically. In addition, this study shows that inside the plants there may exist antagonistic interactions among endophytic bacteria like to those described among the rhizospheric community.     

Growth behaviour and bioproduction of indole acetic acid by a Rhizobium sp. isolated from root nodules of a leguminous tree Dalbergia lanceolaria

The Rhizobium sp. isolated from healthy and mature root nodules of a leguminous tree, Dalbergia lanceolaria Linn. f., preferred mannitol and KNO3 for growth as carbon and nitrogen sources, respectively. The bacterium produced a high amount (22.3 microg/ml) of indole acetic acid (IAA) from L-tryptophan supplemented basal medium. Growth and IAA production started simultaneously. IAA production was maximum at 20 hr when the bacteria reached the stationary phase of growth. Cultural requirements were optimized for maximum growth and IAA production. The IAA production by the Rhizobium sp. was increased by 270.8% over control when the medium was supplemented with mannitol (1%,w/v), SDS (1 microg/ml), L-asparagine (0.02%,w/v) and biotin (1 microg/ml) in addition to L-tryptophan (2.5 mg/ml). The possible role of IAA production in the symbiosis is discussed.