nitrogenase and indoleacetic-acid

This review is devoted to the mechanisms and genetic control of processes underlying the formation and efficiency of associative relationships between bacteria and plants. The role of different polysaccharides and cellular fibrils in the appearance of associative relations and the biosynthetic pathway of these compounds and structures is considered. The molecular mechanisms of bacterial systems responsible for stimulating plant growth and development--nitrogen fixation and synthesis of a plant hormone, indoleacetic acid--are presented. The properties of associative bacteria are discussed in comparison with the relevant characteristics of the most studied free-living or symbiotic model species of bacteria.

Topics: Bacteria; Bacterial Physiological Phenomena; Indoleacetic Acids; Nitrogenase; Plants

Phosphorus-solubilizing microorganisms is a microbial fertilizer with broad application potential. In this study, 7 endophytic phosphate solubilizing bacteria were screened out from Chinese fir, and were characterized for plant growth-promoting traits. Based on morphological and 16S rRNA sequence analysis, the endophytes were distributed into 5 genera of which belong to Pseudomonas, Burkholderia, Paraburkholderia, Novosphingobium, and Ochrobactrum. HRP2, SSP2 and JRP22 were selected based on their plant growth-promoting traits for evaluation of Chinese fir growth enhancement. The growth parameters of Chinese fir seedlings after inoculation were significantly greater than those of the uninoculated control group. The results showed that PSBs HRP2, SSP2 and JRP22 increased plant height (up to 1.26 times), stem diameter (up to 40.69%) and the biomass of roots, stems and leaves (up to 21.28%, 29.09% and 20.78%) compared to the control. Total N (TN), total P (TP), total K (TK), Mg and Fe contents in leaf were positively affected by PSBs while showed a significant relationship with strain and dilution ratio. The content of TN, TP, TK, available phosphorus (AP) and available potassium (AK) in the soil increased by 0.23-1.12 mg g

Topics: Agricultural Inoculants; Bacteria; Bacterial Proteins; Carbon-Carbon Lyases; Cunninghamia; Endophytes; Indoleacetic Acids; Nitrogenase; Phosphates; Phosphorus; RNA, Ribosomal, 16S; Seedlings; Siderophores; Soil

Rahnella aquatilis HX2 was isolated from Beijing vineyard soil and used as a plant growth-promoting rhizobacterium in the field. Previous studies have shown that it has a broad in vitro antimicrobial spectrum and could inhibit a variety of plant pathogenic bacteria and fungi. In this study, a gene, acdS, encoding 1-aminocyclopropane-1-carboxylic acid-deaminase was disrupted by in-frame deletion in the HX2 strain. Compared to the wild-type, the acdS-mutant had higher rates of nitrogen fixation, reduced indole-3-acetic acid production, lowered efficacy as a biological control agent against the grape crown gall pathogen Agrobacterium vitis. Under saline stress conditions, plant height, above-ground fresh weight, root fresh weight of corn plants were increased by treatment with HX2 but this increase was compromised by the disruption of acdS gene. Our data confirmed the function of HX2 on plant growth promoting and demonstrated that acdS gene plays a major role in its PGPR activities.

Topics: Agrobacterium; Anti-Bacterial Agents; Bacterial Proteins; Carbon-Carbon Lyases; Germination; Indoleacetic Acids; Mutation; Nitrogenase; Rahnella; Salt Tolerance; Zea mays

This study was aimed at selecting purple non-sulfur bacteria (PNSB) isolated from various paddy fields, including Cd- and Zn-contaminated paddy fields, based on their biofertilizer properties. Among 235 PNSB isolates, strain TN110 was most effective in plant growth-promoting substance (PGPS) production, releasing 3.2 mg/L of [Formula: see text] , 4.11 mg/L of 5-aminolevulinic acid (ALA) and 3.62 mg/L of indole-3-acetic acid (IAA), and reducing methane emission up to 80%. This strain had nifH, vnfG and anfG, which are the Mo, V and Fe nitrogenase genes encoded for key enzymes in nitrogen fixation under different conditions. This strain provided 84% and 55% removal of Cd and Zn, respectively. Another isolate, TN414, not only produced PGPS (1.30 mg/L of [Formula: see text] , 0.94 mg/L of ALA and 0.65 mg/L of IAA), but was also efficient in removing both Cd and Zn at 72% and 74%, respectively. Based on 16S rDNA sequencing, strain TN110 was identified as Rhodopseudomonas palustris, while strain TN414 was Rubrivivax gelatinosus. A combination of TN110 and TN414 could potentially provide a biofertilizer, which is a greener alternative to commercial/chemical fertilizers and an agent for bioremediation of heavy metals and greenhouse gas mitigation in paddy fields.

Topics: Agriculture; Aminolevulinic Acid; Bacteria; Biodegradation, Environmental; Cadmium; Indoleacetic Acids; Metals, Heavy; Nitrogen Fixation; Nitrogenase; Oryza; Plant Development; Rhodopseudomonas; RNA, Ribosomal, 16S; Soil Microbiology; Soil Pollutants; Zinc

Rice plants are selective with their associations with bacteria that are beneficial for growth, nutrient uptake, exhibit induced resistance or antagonism towards pathogens. Cyanobacteria as bioinoculants are known to promote the growth and health of rice plants. The present investigation was aimed at understanding whether and how cyanobacterial (Calothrix elenkinii) inoculation influenced the rice plant growth and the culturable bacterial populations and identifying the dominant culturable "microbiome" members. The plant tissue extracts were used to enumerate populations of the culturable microbiome members using selected enrichment media with different nutrient levels. About 10-fold increases in population densities of culturable microbiome members in different media were recorded, with some isolates having metabolic potential for nitrogen fixation and phosphorus solubilization. Fatty acid methyl ester (FAME) analysis and 16S rRNA sequencing of selected microbial morphotypes suggested the predominance of the members of Bacillaceae. Significant increases in plant growth attributes, nitrogenase activity and indole acetic acid production, and activities of hydrolytic and defense enzymes were recorded in the Calothrix inoculated plants. The PCR-based analysis and scanning electron microscopic (SEM) observations confirmed the presence of inoculated cyanobacterium inside the plant tissues. This investigation illustrated that cyanobacterial inoculation can play significant roles in improving growth and metabolism of rice directly and interact with the beneficial members from the endophytic microbiome of rice seedlings synergistically.

Topics: Biodiversity; Biological Evolution; Chlorophyll; Cyanobacteria; DNA Fingerprinting; Fatty Acids; Indoleacetic Acids; Metagenome; Microbiota; Nitrogenase; Oryza; Phylogeny; Plant Roots; Plant Shoots; RNA, Ribosomal, 16S; Symbiosis

Endophytic bacteria that have plant growth promoting traits are of great interest in green biotechnology. The previous thought that the Azoarcus genus comprises bacteria that fit into one of two major eco-physiological groups, either free-living anaerobic biodegraders of aromatic compounds or obligate endophytes unable to degrade aromatics under anaerobic conditions, is revisited here.. Light, confocal and electron microscopy reveal that Azoarcus sp. CIB, a facultative anaerobe β-proteobacterium able to degrade aromatic hydrocarbons under anoxic conditions, is also able to colonize the intercellular spaces of the rice roots. In addition, the strain CIB displays plant growth promoting traits such nitrogen fixation, uptake of insoluble phosphorus and production of indoleacetic acid. Therefore, this work demonstrates by the first time that a free-living bacterium able to degrade aromatic compounds under aerobic and anoxic conditions can share also an endophytic lifestyle. The phylogenetic analyses based on the 16S rDNA and nifH genes confirmed that obligate endophytes of the Azoarcus genus and facultative endophytes, such as Azoarcus sp. CIB, locate into different evolutionary branches.. This is the first report of a bacterium, Azoarcus sp. CIB, able to degrade anaerobically a significant number of aromatic compounds, some of them of great environmental concern, and to colonize the rice as a facultative endophyte. Thus, Azoarcus sp. CIB becomes a suitable candidate for a more sustainable agricultural practice and phytoremediation technology.

Topics: Anaerobiosis; Azoarcus; Biodegradation, Environmental; Colony Count, Microbial; Endophytes; Hydrocarbons, Aromatic; Indoleacetic Acids; Nitrogen Fixation; Nitrogenase; Oryza; Phosphates; Plant Roots; Solubility

This study was conducted to evaluate selected biomolecular characteristics of rice root-associated diazotrophs isolated from the Tanjong Karang rice irrigation project area of Malaysia. Soil and rice plant samples were collected from seven soil series belonging to order Inceptisol (USDA soil taxonomy). A total of 38 diazotrophs were isolated using a nitrogen-free medium. The biochemical properties of the isolated bacteria, such as nitrogenase activity, indoleacetic acid (IAA) production and sugar utilization, were measured. According to a cluster analysis of Jaccard's similarity coefficients, the genetic similarities among the isolated diazotrophs ranged from 10% to 100%. A dendogram constructed using the unweighted pair-group method with arithmetic mean (UPGMA) showed that the isolated diazotrophs clustered into 12 groups. The genomic DNA rep-PCR data were subjected to a principal component analysis, and the first four principal components (PC) accounted for 52.46% of the total variation among the 38 diazotrophs. The 10 diazotrophs that tested highly positive in the acetylene reduction assay (ARA) were identified as Bacillus spp. (9 diazotrophs) and Burkholderia sp. (Sb16) using the partial 16S rRNA gene sequence analysis. In the analysis of the biochemical characteristics, three principal components were accounted for approximately 85% of the total variation among the identified diazotrophs. The examination of root colonization using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) proved that two of the isolated diazotrophs (Sb16 and Sb26) were able to colonize the surface and interior of rice roots and fixed 22%-24% of the total tissue nitrogen from the atmosphere. In general, the tropical soils (Inceptisols) of the Tanjong Karang rice irrigation project area in Malaysia harbor a diverse group of diazotrophs that exhibit a large variation of biomolecular characteristics.

Topics: Cyanobacteria; Indoleacetic Acids; Malaysia; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Nitrogenase; Soil Microbiology

Brachiaria brizantha is considered one of the preferred fodders among farmers for having high forage yield and large production of root mass. The association of beneficial bacteria with these grasses can be very valuable in the recovery of the pasture areas with nutritional deficiency. With the aim of studying this possibility, we carried out the sampling of soil and roots of B. brizantha in three areas (Nova Odessa-SP, São Carlos-SP and Campo Verde-MT, Brazil). Seventy-two bacterial strains were isolated and used in tests to evaluate their biotechnological potential. Almost all isolates presented at least one positive feature. Sixty-eight isolates produced analogues of indole-3-acetic acid, ten showed nitrogenase activity when subjected to the method of increasing the concentration of total nitrogen (total N) in the culture medium and sixty-five isolates showed nitrogenase activity when subjected to acetylene reduction technique. The partial sequencing of 16S rRNA of these isolates allowed the identification of seven main groups, with the prevalence of those affiliated to the genus Stenotrophomonas (69 %). At the end, this work elected the strains C4 (Pseudomonadaceae) and C7 (Rhodospirillaceae) as promising organisms for the development of inoculants due to their higher nitrogenase activity.

Topics: Bacteria; Brachiaria; Indoleacetic Acids; Nitrogen; Nitrogenase; Plant Development; Plant Growth Regulators; Plant Roots; Poaceae; RNA, Ribosomal, 16S; Soil; Soil Microbiology

Endosulfan is a lypophilic persistent organic pollutant (POP) that has caused widespread concern due to its persistence in the environment, toxicity and bioaccumulation in living organisms. The aim of this study is to isolate endosulfan-degrading bacteria taken from five coffee farms historically exposed to this insecticide which could be used in future remediation strategies. The biodegradation capability of the isolated strain as well as endosulfan's impact on some of the strain's biological functions was studied. Endosulfan and its metabolites were analyzed using TLC and GC-MS. The isolated strain, capable of growing in a liquid culture treated with this insecticide as the sole sulfur source rather than a carbon source, was selected for further study. The isolated bacterium is Gram-negative, having the morphological and biochemical characteristics of Azotobacter sp. The remaining concentrations after 6 days, using 2 and 10 mg l(-1) of endosulfan, were 57.6 and 72.3% respectively, and the degradation constants were 0.12 d(-1) and 0.26 d(-1). Four metabolites were detected, one of which was identified as endosulfan ether. Endosulfan reduced nitrogenase activity but had no impact on indole 3-acetic acid production. Thus, these results suggest that this strain has the potential to act as a biocatalyst in endosulfan degradation.

Topics: Azotobacter; Biodegradation, Environmental; Chromatography, Thin Layer; Coffee; Colombia; Endosulfan; Gas Chromatography-Mass Spectrometry; Indoleacetic Acids; Insecticides; Nitrogenase; Plant Growth Regulators; Soil Microbiology

Biological systems are getting more relevance than chemical control of plant pathogens as they are not only eco-friendly and economic in approach but are also involved in improving the soil consistency and maintenance of natural soil flora. Plant growth promoting rhizosphere microorganisms were isolated from three different tree rhizospheres using selective culture media. Five microorganisms were selected from each rhizosphere soil based on their efficiency and screened for their ability to promote plant growth as a consortium. Each of the developed consortium has a phosphate solubilizer, nitrogen fixer, growth hormone producer, heterotrophic member and an antagonist. The plant growth promoting ability of the microbial members present in the consortium was observed by estimating the IAA production level and also by the nitrogenase activity of the nitrogen fixers. The biocontrol potentiality of the consortium and the antagonist present in the consortium were checked by both dual plate assay and cross-streaking technique. Consortial treatments effected very good growth promotion in Lycopersicon esculentum Mill and the treated plants also developed resistance against wilt pathogen, Fusarium oxysporum f. sp. lycopersici though the effect was well pronounced with consortium developed from Santalum album.

Topics: Antibiosis; Bacteria; Indoleacetic Acids; Nitrogenase; Plant Development; Plant Diseases; Plant Roots; Plants; Soil Microbiology; Symbiosis; Trees

Nine diazotrophic bacteria were isolated from surface-sterilized roots and culms of wheat variety Malviya-234, which is grown with very low or no inputs of nitrogen fertilizer. Out of the nine bacteria, four showed indole acetic acid (IAA) production, and five were positive for P solubilization. One isolate, WM234C-3, showed appreciable level of nitrogenase activity, IAA production, and P solubilization ability, and was further characterized with a view to exploiting its plant growth promoting activity. Based on 16S rDNA sequence analysis, this isolate was identified as Achromobacter xylosoxidans. Diazotrophic nature of this particular isolate was confirmed by Western blot analysis of dinitrogenase reductase and amplification of nifH. Analysis of the nifH sequence showed close homology with typical diazotrophic bacteria. Endophytic nature and cross-infection ability of WM234C-3 were tested by molecular tagging with gusA fused to a constitutive promoter followed by inoculation onto rice seedlings in axenic conditions. At 21 days after inoculation, the roots showed blue staining, the most intense color being at the emergence of lateral roots and root tips. Microscopic observation confirmed colonization of gus-tagged WM234C-3 in the intercellular spaces of cortical as well as vascular zones of roots. Inoculation of gus-tagged WM234C-3 to rice plants resulted in significant increase in root/shoot length, fresh weight, and chlorophyll a content. Plant growth promoting features coupled with cross-infection ability suggest that this endophytic bacterium may be exploited as agricultural agent for various crops after a thorough and critical pathogenicity test.

Topics: Achromobacter denitrificans; Colony Count, Microbial; DNA, Bacterial; Indoleacetic Acids; Nitrogenase; Phosphorus; Plant Roots; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Triticum

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.

Topics: Azospirillum lipoferum; Drug Resistance; Ethanol; Fatty Acids; Gluconacetobacter; Indoleacetic Acids; Nitrogen Fixation; Nitrogenase; Pseudomonas putida; RNA, Ribosomal, 16S; Zea mays

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.

Topics: Coffee; Crops, Agricultural; Gluconacetobacter; India; Indoleacetic Acids; Nitrogenase; Phosphorus; Plant Leaves; Plant Roots; Polymerase Chain Reaction; Tropical Climate; Vegetables; Zinc

This study aims at evaluating the ability of Beijerinckia derxii, a free-living nitrogen (N)-fixing bacterium frequently isolated from tropical soils, to release certain plant growth regulators [indoleacetic acid (IAA), ethylene, polyamines] and amino acids into the growth medium.. The production of those substances was compared using both cultures in which nitrogenase was active (N-free medium) and cultures in which nitrogenase was repressed (combined-N cultures). Those cultures were grown under agitation and in absence of agitation. Total IAA production was higher in agitated, N-free cultures but specific production was greater in combined-N cultures under agitation. Putrescine and spermidine were detected under all conditions tested. Ethylene was produced in both N-free and combined-N cultures. A greatest diversity of amino acids was released in N-free cultures.. There was no inhibition of the production of the analysed substances under conditions where nitrogenase was inactive.. Beijerinckia derxii is potentially a producer of plant-active substances; its presence in the natural environment suggests that this bacterium may contribute to the development of other living organisms.

Topics: Amino Acids; Beijerinckiaceae; Culture Media; Ethylenes; Indoleacetic Acids; Nitrogenase; Plant Growth Regulators; Polyamines; Putrescine; Soil Microbiology; Spermidine

A study of the diversity of endophytic bacteria present in seeds of a deepwater rice variety revealed the presence of seven types of BOX-PCR fingerprints. In order to evaluate the plant growth promoting potential the presence of nitrogenase, indole acetic acid production and mineral phosphate solubilization were estimated in the representative BOX-PCR types. The seven representatives of BOX-PCR types produced indole acetic acid, reduced acetylene and showed specific immunological cross-reaction with anti-dinitrogenase reductase antibody. Only four types showed mineral phosphate solubilizing ability. Comparison of cellulase and pectinase activities showed differences among different BOX-PCR types. PCR fingerprinting data showed that one strain isolated from the surface sterilized seeds as well as the aerial parts of the seedlings of rice variety showed low cellulase and pectinase but relatively high ARA. On the basis of 16S rDNA nucleotide sequence and BIOLOG system of bacterial identification, this strain was identified as Pantoea agglomerans. For studying the endophytic colonization this strain was genetically tagged with the reporter gene, gusA. Histochemical analysis of the seedling grown in hydroponics showed that the tagged strain colonized the root surface, root hairs, root cap, points of lateral root emergence, root cortex and the stelar region. Treatment of the roots with 2,4-D produced short thickened lateral roots which showed better colonization by P. agglomerans.

Topics: 2,4-Dichlorophenoxyacetic Acid; Agriculture; Cellulase; DNA, Ribosomal; Enterobacter; Fertilizers; Herbicides; Hydroponics; Indoleacetic Acids; Nitrogen Fixation; Nitrogenase; Oryza; Pantoea; Phosphates; Plant Roots; Polygalacturonase; Polymerase Chain Reaction; Seeds

Fourteen Azospirillum brasilense strains growing at a sub-optimum temperature were selected based on their ability to grow and carry out plant growth promoting activities at 22 degrees C. The strains were tested for their response to inoculation in wheat (two popular cultivars, HD2285 and WH547, under sterile conditions) crop using sterile and nonsterile rooting medium. Significant increase in plant growth parameters was observed; the overall response to inoculation was better in cultivar HD2285. Based on their performance under sterile conditions, 4 strains were selected and compared under nonsterile conditions with strain sensitive to a sub-optimum temperature in pots using wheat variety HD2285. The strains capable of growing at the sub-optimum temperature can colonize the wheat endorhizosphere efficiently and improve the plant growth and yield as compared to sensitive strain; a 25-27% increase in grain yield was found on inoculating two selected strains compared to NO3- control.

Topics: Azospirillum brasilense; Culture Media; Indoleacetic Acids; Nitrogenase; Plant Growth Regulators; Plant Roots; Siderophores; Soil Microbiology; Temperature; Triticum

The phenotypic characteristics of four Burkholderia cepacia strains isolated from the rhizosphere and the clinical environment were compared. Tests included optimum growth temperature, utilization of carbon sources, production of HCN, indole-3-acetic acid (IAA) and siderophores, proteolytic activity, nitrogen fixation, inhibition of some phytopathogenic fungi, adherence to human mucosal and plant root epithelia, and greenhouse-based plant-growth promotion experiments using cucumber (Cucumis sativus). Results indicated that the strains of B. cepacia isolated from the rhizosphere differ markedly from their clinical counterparts. Strains isolated from the rhizosphere grew over a wider temperature range, fixed nitrogen and produced IAA, did not produce proteases, displayed a wider antibiosis against the phytopathogenic fungi studied, did not adhere to human uroepithelial cells, promoted growth of C. sativus and only produced a hydroxamate-like siderophore. In contrast, clinical isolates could not fix nitrogen or produce IAA, produced proteases, adhered to human uroepithelial cells, did not promote the growth of C. sativus and, in addition to a hydroxamate-like siderophore, produced pyochelin and salicylate siderophores. All four isolates exhibited the ability to adhere to the root tissue of C. sativus and were unable to produce HCN.

Topics: Antibiosis; Bacterial Adhesion; Burkholderia cepacia; Cell Division; Cyanides; Endopeptidases; Epithelium; Female; Humans; Indoleacetic Acids; Nitrogen Fixation; Nitrogenase; Siderophores; Vegetables; Virulence

A recombinant plasmid carrying the trp operon from Escherichia coli, which synthesizes tryptophan from chorismate, was constructed by using a broad host range plasmid vector pRK290; a mutant trp plasmid for tryptophan overproduction was then selected. The physiological, biochemical, and genetic properties of the Azospirillum lipoferum KY6, a potential nitrogen fixer of rice, harbouring the recombinant trp plasmid pMJC1 and its mutant pMJC101, were compared with those of the wild-type bacteria. Anthranilate synthetase is known to be the trpE gene product which plays a key role in the regulatory step in the feedback control of tryptophan biosynthesis. The enzyme activity of the Azospirillum lipoferum KY6 carrying pMJC1 or pMJC101 was respectively 7- and 30-fold higher than that of the wild type in the presence of 10(-4)M tryptophan. As expected, the amount of tryptophan biosynthesis in A. lipoferum KY6 (pMJC101) was increased approximately 100-fold as compared with the wild type, which led to overproduction of indole acetic acid even without addition of exogenous tryptophan. Moreover, the recombinant trp plasmid was fairly stable in A. lipoferum KY6 host, showing only 25% loss of the plasmid itself or the trp insert after 40 generations.

Topics: Azospirillum; Cloning, Molecular; Conjugation, Genetic; Escherichia coli; Indoleacetic Acids; Mutagenesis; Nitrogenase; Operon; Plasmids; Tryptophan