Bradyrhizobium Populations Occur in Deep Soil under the Leguminous Tree Acacia albida

Soil cores were drilled under the leguminous tree Acacia albida growing in two different ecoclimatic zones of West Africa: the Sahelian area (100 to 500 mm of annual rainfall) and the Sudano-Guinean area (1,000 to 1,500 mm of annual rainfall). Soil samples were collected at different depths from the surface down to the water table level and analyzed for the presence of rhizobia able to nodulate A. albida. In both areas, population densities of rhizobia were substantially greater near the water table than near the surface. In the Sahelian area, rhizobia were present as deep as 34 m at a concentration of 1.3 × 10³/g of soil. In the Sudano-Guinean area, population densities at 0.5 to 4.5 m depth were higher than in the Sahelian area and, at several depths, comparable to that of temperate soils supporting legume crops (10⁴ rhizobia per g of soil). Surface and deep soil isolates from all four sites were found to be slow-growing rhizobia (Bradyrhizobium sp.). The proportion of effective isolates was almost the same within surface and deep soils.     

Bradyrhizobium sp. Strains that nodulate the leguminous tree Acacia albida produce fucosylated and partially sulfated nod factors

We determined the structures of Nod factors produced by six different Bradyrhizobium sp. strains nodulating the legume tree Acacia albida (syn. Faidherbia albida). Compounds from all strains were found to be similar, i.e., O-carbamoylated and substituted by an often sulfated methyl fucose and different from compounds produced by Rhizobium-Mesorhizobium-Sinorhizobium strains nodulating other species of the Acaciae tribe.     

Narrow- and Broad-Host-Range Symbiotic Plasmids of Rhizobium spp. Strains That Nodulate Phaseolus vulgaris

Agrobacterium transconjugants containing symbiotic plasmids from different Rhizobium spp. strains that nodulate Phaseolus vulgaris were obtained. All transconjugants conserved the parental nodulation host range. Symbiotic (Sym) plasmids of Rhizobium strains isolated originally from P. vulgaris nodules, which had a broad nodulation host range, and single-copy nitrogenase genes conferred a Fix⁺ phenotype to the Agrobacterium transconjugants. A Fix⁻ phenotype was obtained with Sym plasmids of strains isolated from P. vulgaris nodules that had a narrow host range and reiterated nif genes, as well as with Sym plasmids of strains isolated from other legumes that presented single nif genes and a broad nodulation host range. This indicates that different types of Sym plasmids can confer the ability to establish an effective symbiosis with P. vulgaris.     

Genetic Diversity and Phylogeny of Rhizobia That Nodulate Acacia spp. in Morocco Assessed by Analysis of rRNA Genes

Forty rhizobia nodulating four Acacia species (A. gummifera, A. raddiana, A. cyanophylla, and A. horrida) were isolated from different sites in Morocco. These rhizobia were compared by analyzing both the 16S rRNA gene (rDNA) and the 16S-23S rRNA spacer by PCR with restriction fragment length polymorphism (RFLP) analysis. Analysis of the length of 16S-23S spacer showed a considerable diversity within these microsymbionts, but RFLP analysis of the amplified spacer revealed no additional heterogeneity. Three clusters were identified when 16S rDNA analysis was carried out. Two of these clusters include some isolates which nodulate, nonspecifically, the four Acacia species. These clusters, A and B, fit within the Sinorhizobium lineage and are closely related to S. meliloti and S. fredii, respectively. The third cluster appeared to belong to the Agrobacterium-Rhizobium galegae phylum and is more closely related to the Agrobacterium tumefaciens species. These relations were confirmed by sequencing a representative strain from each cluster.     

Heat Curing of a Sym Plasmid in a Fast-Growing Rhizobium sp. That Is Able to Nodulate Legumes and the Nonlegume Parasponia sp

Genes involved in nodulation of both legumes and the nonlegume Parasponia sp., as well as nitrogenase genes, reside on a large plasmid in a fast-growing Rhizobium sp. from Lablab purpureus. This plasmid can be cured by incubation at elevated temperatures and can be mobilized by the P1 group plasmid RP1::Tn501.     

两株高效相思根瘤菌的抗药性研究及其质粒组成分析

目的:研究相思根瘤菌质粒与其抗药性之间的关系。方法:研究了相思根瘤菌MZ和AJ018在含不同浓度的抗生素的固体培养基和液体培养基的生长情况,并用碱裂解方法对其质粒组成进行检测。结果:两菌株对链霉素和卡那霉素均无抗性,而对其它抗生素都有不同程度的抗性。MZ菌株对实验中的氯霉素、氨苄青霉素、四环素三种抗生素的耐受范围与最大耐受值都比AJ018强,当平板培养基中氨苄青霉素、四环素、氯霉素的终浓度分别为250μg/ml、75μg/ml、150μg/ml时,AJ018在平板上无菌落生长,当三种抗生素终浓度分别为800μg/ml1、50μg/ml、400μg/ml时无菌落生长。两菌株都含有一个大约50kb的质粒。     

Inoculation of Acacia mangium with Alginate Beads Containing Selected Bradyrhizobium Strains under Field Conditions: Long-Term Effect on Plant Growth and Persistence of the Introduced Strains in Soil

The growth response of Acacia mangium Willd. to inoculation with selected Bradyrhizobium strains was investigated in two field trials in the Ivory Coast (West Africa). In the first trial (Anguededou), four provenances (i.e., trees originating from seeds harvested in different geographical areas) of A. mangium were inoculated with four Bradyrhizobium strains from different origins. Six months after being transplanted in the field, the heights of all inoculated trees showed a statistically significant increase of 9 to 26% compared with those of uninoculated trees, with the most effective strain being Aust 13c. After 19 months, the positive effect of inoculation on tree growth was confirmed. The effect of A. mangium provenance on tree growth was also highly significant. Trees from the Oriomo provenance of Papua New Guinea had a mean height that was 25% greater than those of other provenances. Analysis of variance showed a highly significant effect of interaction between strain and host provenance factors. Thus, most effective strain × provenance combinations could be proposed. Immunological identification of strains clearly showed that 90 to 100% of nodules from trees inoculated with three of the four Bradyrhizobium strains or from uninoculated trees contained exclusively Aust 13c 23 months after tree transplantation. This predominance of Aust 13c in nodules was still observed 42 months after tree transplantation. The second experiment (Port-Bouët), performed with a different soil, confirmed the long-term positive effect of Aust 13c on plant growth, its high competitive ability against indigenous strains, and its persistence in soil. Strain Aust 13c should thus be of great interest for inoculating A. mangium under a wide range of field conditions.     

Genetic Analysis in the Colonia Strain of PHYSARUM POLYCEPHALUM: Heterothallic Strains That Mate with and Are Partially Isogenic to the Colonia Strain

Amoebae of the Colonia (CL) strain, which normally produce plasmodia in clones, are shown to cross with mt3 and mt4 amoebae under appropriate conditions. Strains carrying mt3 and mt4 in a CL genetic background were constructed and used in the genetic analysis of mutants isolated in CL.     

Isolation and characterization of canthaxanthin biosynthesis genes from the photosynthetic bacterium Bradyrhizobium sp. strain ORS278

A carotenoid biosynthesis gene cluster involved in canthaxanthin production was isolated from the photosynthetic Bradyrhizobium sp. strain ORS278. This cluster includes five genes identified as crtE, crtY, crtI, crtB, and crtW that are organized in at least two operons. The functional assignment of each open reading frame was confirmed by complementation studies.     

Effect of the VAM fungus Glomus sp. on the growth and yield of soybean inoculated with Bradyrhizobium japonicum

The effect of two Bradyrhizobium japonicum strains (D344 and Urbana), on the frequency and intensity of infection by a VAM fungal Glomus sp. and the effect of VAM on biomass production by nodulating plants were tested in soybean growing in a soil containing low levels of accessible P and N. During the initial stage of vegetative growth, mycorrhiza frequency in roots inoculated with the two rhizobial strains did not differ. However, during flowering it was 178% higher in roots with the strain D344 than in the presence of the strain Ubrana. At final harvest (green pods) the VAM frequency did not differ in the presence of either strain. VAM positively affected biomass production, foliar concentrations of P, Zn and Cu, and number and dry matter yield of pods, but did not increase concentrations of total N and K. In nonmycorrhizal plants total nitrogenase activity (not nodule mass) and growth were higher with the rhizobial strain Urbana. The greatest nitrogenase activity, growth and yield occurred in the presence of the VAM fungus, and did not differ for plants with different strains of rhizobia.     

Isolation and Characterization of a Pseudomonas sp. That Mineralizes the s-Triazine Herbicide Atrazine

A bacterium that was capable of metabolizing atrazine at very high concentrations (>1,000 ppm) was isolated from a herbicide spill site. The organism was differentiated by observing clearing zones on indicator agar plates containing 1,000 ppm atrazine. Detailed taxonomic studies identified the organism as a Pseudomonas sp., designated ADP, that was dissimilar to currently known species. Pseudomonas sp. strain ADP metabolized atrazine as its sole nitrogen source. Nongrowing suspended cells also metabolized atrazine rapidly; for example, 9 x 10(sup9) cells per ml degraded 100 ppm of atrazine in 90 min. Atrazine was metabolized to hydroxyatrazine, polar metabolites, and carbon dioxide. When uniformly ring-labeled [(sup14)C]atrazine was used, 80% of the radioactivity was liberated as (sup14)CO(inf2). These data indicated the triazine ring was completely mineralized. The isolation and characterization of Pseudomonas sp. strain ADP may contribute to efforts on atrazine bioremediation, particularly in environments containing very high pesticide levels.     

Molecular and biochemical characterization of the 5-nitroanthranilic acid degradation pathway in Bradyrhizobium sp. strain JS329

Biodegradation pathways of synthetic nitroaromatic compounds and anilines are well documented, but little is known about those of nitroanilines. We previously reported that the initial step in 5-nitroanthranilic acid (5NAA) degradation by Bradyrhizobium sp. strain JS329 is a hydrolytic deamination to form 5-nitrosalicylic acid (5NSA), followed by ring fission catalyzed by 5NSA dioxygenase. The mechanism of release of the nitro group was unknown. In this study, we subcloned, sequenced, and expressed the genes encoding 5NAA deaminase (5NAA aminohydrolase, NaaA), 5NSA dioxygenase (NaaB) and lactonase (NaaC), the key genes responsible for 5NAA degradation. Sequence analysis and enzyme characterization revealed that NaaA is a hydrolytic metalloenzyme with a narrow substrate range. The nitro group is spontaneously eliminated as nitrite concomitant with the formation of a lactone from the ring fission product of 5NSA dioxygenation. The elimination of the nitro group during lactone formation is a previously unreported mechanism for denitration of nitro aliphatic compounds.     

Unusual methyl-branched alpha,beta-unsaturated acyl chain substitutions in the Nod Factors of an arctic rhizobium, Mesorhizobium sp. strain N33 (Oxytropis arctobia)

Mesorhizobium sp. strain N33 (Oxytropis arctobia), a rhizobial strain isolated in arctic Canada, is able to fix nitrogen at very low temperatures in association with a few arctic legume species belonging to the genera Astragalus, Onobrychis, and Oxytropis. Using mass spectrometry and nuclear magnetic resonance spectroscopy, we have determined the structure of N33 Nod factors, which are major determinants of nodulation. They are pentameric lipochito-oligosaccharides 6-O sulfated at the reducing end and exhibit other original substitutions: 6-O acetylation of the glucosamine residue next to the nonreducing terminal glucosamine and N acylation of the nonreducing terminal glucosamine by methyl-branched acyl chains of the iso series, some of which are alpha,beta unsaturated. These unusual substitutions may contribute to the peculiar host range of N33. Analysis of N33 whole-cell fatty acids indicated that synthesis of the methyl-branched fatty acids depended on the induction of bacteria by plant flavonoids, suggesting a specific role for these fatty acids in the signaling process between the plant and the bacteria. Synthesis of the methyl-branched alpha,beta-unsaturated fatty acids required a functional nodE gene.     

Oxygen and light effects on the expression of the photosynthetic apparatus in Bradyrhizobium sp. C7T1 strain

Photosynthetic bradyrhizobia are nitrogen-fixing symbionts colonizing the stem and roots of some leguminous plants like Aeschynomene. The effect of oxygen and light on the formation of the photosynthetic apparatus of Bradyrhizobium sp. C7T1 strain is described here. Oxygen is required for growth, but at high concentration inhibits the synthesis of bacteriochlorophyll (BChl) and of the photosynthetic apparatus. However, we show that in vitro, aerobic photosynthetic electron transport occurred leading to ADP photophosphorylation. The expression of the photosynthetic apparatus was regulated by oxygen in a manner which did not agree with earlier results in other photosynthetic bradyrhizobia since BChl accumulation was the highest under microaerobic conditions. This strain produces photosynthetic pigments when grown under cyclic illumination or darkness. However, under continuous white light illumination, a Northern blot analysis of the puf operon showed that, the expression of the photosynthetic genes of the antenna was considerable. Under latter conditions BChl accumulation in the cells was dependent on the oxygen concentration. It was not detectable at high oxygen tensions but became accumulated under low oxygen (microaerobiosis). It is known that in photosynthetic bradyrhizobia bacteriophytochrome photoreceptor (BphP) partially controls the synthesis of the photosystem in response to light. In C7T1 strain far-red light illumination did not stimulate the synthesis of the photosynthetic apparatus suggesting the presence of a non-functional BphP-mediated light regulatory mechanism.     

Protoplast Mutation and Genome Shuffling Induce the Endophytic Fungus Tubercularia sp. TF5 to Produce New Compounds

Tubercularia sp. TF5 is an endophytic fungal strain isolated from the medicinal plant Taxus mairei. Previously, taxol has been detected in the fermentation products of this strain. However, it lost the capability of producing taxol after long-term laboratory culture. Herein, we tried to reactivate the production of taxol by protoplast mutations and genome shuffling. The protoplasts of Tub. sp. TF5 were prepared from its mycelia, and mutated by UV and NTG. The mutant strains regenerated from the mutated protoplasts were selected and classified into four groups on the basis of their phenotypes, the profile of their metabolites analyzed by TLC, MS, and bioassay data. Then, genome shuffling was subsequently carried out with eight mutant strains, with two representatives from each protoplast mutant group, and genome shuffling mutant strains were obtained and screened using the same screening procedure. Although taxol has not been detected in any mutant, two important mutants, M-741 and G-444 were selected for metabolites isolation and determination due to their phenotypes, and differences in TLC analysis result from TF5 and other mutants. Three new sesquiterpenoids, namely tuberculariols A–C (1–3), and a known dihydroisocoumarin (4) were obtained from M-741. Eighteen novel compounds were isolated from G-444, including five new sesquiterpenoids (5-9), two new dihydroisocoumarins (10, 11), one new tetralone (12), together with 10 known compounds (13–20, 1, and 2). The compounds isolated from the M-741 and G-444 were different in structure types and substitutions from those of TF5 (15, 21–29). The results showed, for the first time, that protoplast mutations and genome shuffling are efficient approaches to mining natural products from endophytic fungi. Understanding the mechanisms of unlocking the biosynthesis of new metabolites will facilitate the manipulation of the secondary metabolism in fungi.     

Photoacclimation of Prochlorococcus sp. (Prochlorophyta) Strains Isolated from the North Atlantic and the Mediterranean Sea

Two Atlantic (SARG and NATL1) strains and one Mediterranean (MED) strain of Prochlorococcus sp., a recently discovered marine, free-living prochlorophyte, were grown over a range of "white" irradiances (lg) and under low blue light to examine their photoacclimation capacity. All three strains contained divinyl (DV) chlorophylls (Chl) a and b, both distinguishable from "normal" Chls by their red-shifted blue absorption maximum, a Chl c-like pigment at low concentration, zeaxanthin, and [alpha]-carotene. The presence of two phaeophytin b peaks in acidified extracts from both Atlantic strains grown at high lg suggests that these strains also had a normal Chl b-like pigment. In these strains, the total Chl b to DV-Chl a molar ratio decreased from about 1 at 7.5 [mu]mol quanta m-2 s-1 to 0.4 to 0.5 at 133 [mu]mol quanta m-2 s-1. In contrast, the MED strain always had a low DV-Chl b to DV-Chl a molar ratio, ranging between 0.13 at low lg and 0.08 at high lg. The discrepancies between the Atlantic and MED strains could result from differences either in the number of light-harvesting complexes (LHC) II per photosystem II or in the Chl b-binding capacity of the apoproteins constituting LHC II. Photosynthesis was saturated at approximately 5 fg C(fg Chl)-1 h-1 or 6 fg C cell-1 h-1, and growth was saturated at approximately 0.45 d-1 for both MED and SARG strains at 18[deg]C, but saturating irradiances differed between strains. Atlantic strains exhibited increased light-saturated rates and quantum yield for carbon fixation under blue light.     

Characterization of the Polyurethanolytic Activity of Two Alicycliphilus sp. Strains Able To Degrade Polyurethane and N-Methylpyrrolidone

Two bacterial strains (BQ1 and BQ8) were isolated from decomposed soft foam. These were selected for their capacity to grow in a minimal medium (MM) supplemented with a commercial surface-coating polyurethane (PU) (Hydroform) as the carbon source (MM-PUh). Both bacterial strains were identified as Alicycliphilus sp. by comparative 16S rRNA gene sequence analysis. Growth in MM-PUh showed hyperbolic behavior, with BQ1 producing higher maximum growth (17.8 ± 0.6 mg·ml⁻¹) than BQ8 (14.0 ± 0.6 mg·ml⁻¹) after 100 h of culture. Nuclear magnetic resonance, Fourier transform infrared (IR) spectroscopy, and gas chromatography-mass spectrometry analyses of Hydroform showed that it was a polyester PU type which also contained N-methylpyrrolidone (NMP) as an additive. Alicycliphilus sp. utilizes NMP during the first stage of growth and was able to use it as the sole carbon and nitrogen source, with calculated K_s values of about 8 mg·ml⁻¹. Enzymatic activities related to PU degradation (esterase, protease, and urease activities) were tested by using differential media and activity assays in cell-free supernatants of bacterial cultures in MM-PUh. Induction of esterase activity in inoculated MM-PUh, but not that of protease or urease activities, was observed at 12 h of culture. Esterase activity reached its maximum at 18 h and was maintained at 50% of its maximal activity until the end of the analysis (120 h). The capacity of Alicycliphilus sp. to degrade PU was demonstrated by changes in the PU IR spectrum and by the numerous holes produced in solid PU observed by scanning electron microscopy after bacterial culture. Changes in the PU IR spectra indicate that an esterase activity is involved in PU degradation.