Direct electrochemistry of two genetically distinct flavodoxins isolated from Azotobacter chroococcum grown under nitrogen-fixing conditions.
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ABSTRACT:
SUBMITTER: Bagby S
PROVIDER: S-EPMC1151234 | biostudies-other | 1991 Jul
REPOSITORIES: biostudies-other
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Project description:Melanins, the ubiquitous hetero-polymer pigments found widely dispersed among various life forms, are usually dark brown/black in colour. Although melanins have variety of biological functions, including protection against ultraviolet radiation of sunlight and are used in medicine, cosmetics, extraction of melanin from the animal and plant kingdoms is not an easy task. Using complementary physicochemical techniques (i.e. MALDI-TOF, FTIR absorption and cross-polarization magic angle spinning solid-state (13)C NMR), we report here the characterization of melanins extracted from the nitrogen-fixing non-virulent bacterium Azotobacter chroococcum, a safe viable source. Moreover, considering dihydroxyindole moiety as the main constituent, an effort is made to propose the putative molecular structure of the melanin hetero-polymer extracted from the bacterium. Characterization of the melanin obtained from Azotobacter chroococcum would provide an inspiration in extending research activities on these hetero-polymers and their use as protective agent against UV radiation.
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Project description:Azotobacter chroococcum is a widespread free-living soil bacterium within the genus of Azotobacter known for assimilation of atmospheric nitrogen and subsequent conversion into nitrogenous compounds, which henceforth enrich the nitrogen content of soils. A. chroococcum SBUG 1484, isolated from composted earth, exhibits phenol oxidase (PO) activity when growing under nitrogen-fixing conditions. In the present study we provide incipient analysis of the crude PO activity expressed by A. chroococcum SBUG 1484 within comparative analysis to fungal crude PO from the white-rot fungus Pycnoporus cinnabarinus SBUG-M 1044 and tyrosinase (PPO) from the mushroom Agaricus bisporus in an attempt to reveal desirable properties for exploitation with future recombinant expression of this enzyme. Catalytic activity increased with pre-incubation at 35°C; however 70% of activity remained after pre-treatment at 50°C. Native A. chroococcum crude PO exhibited not only strong preference for 2,6-dimethoxyphenol, but also towards related methoxy-activated substrates as well as substituted ortho-benzenediols from over 40 substrates tested. Presence of CuSO4 enhanced crude phenol oxidase activity up to 30%, whereas NaN3 (0.1 mM) was identified as the most inhibiting substance of all inhibitors tested. Lowest inhibition of crude PO activity occurred after 60 minutes of incubation in presence of 15% methanol and ethanol with 63% and 77% remaining activities respectively, and presence of DMSO even led to increasing oxidizing activities. Substrate scope and inhibitor spectrum strongly differentiated A. chroococcum PO activity comprised in crude extracts from those of PPO and confirmed distinct similarities to fungal PO.
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Project description:With the purpose of isolating and characterizing free nitrogen fixing bacteria (FNFB) of the genus Azotobacter, soil samples were collected randomly from different vegetable organic cultures with neutral pH in different zones of Boyacá-Colombia. Isolations were done in selective free nitrogen Ashby-Sucrose agar obtaining a recovery of 40%. Twenty four isolates were evaluated for colony and cellular morphology, pigment production and metabolic activities. Molecular characterization was carried out using amplified ribosomal DNA restriction analysis (ARDRA). After digestion of 16S rDNA Y1-Y3 PCR products (1487pb) with AluI, HpaII and RsaI endonucleases, a polymorphism of 16% was obtained. Cluster analysis showed three main groups based on DNA fingerprints. Comparison between ribotypes generated by isolates and in silico restriction of 16S rDNA partial sequences with same restriction enzymes was done with Gen Workbench v.2.2.4 software. Nevertheless, Y1-Y2 PCR products were analysed using BLASTn. Isolate C5T from tomato (Lycopersicon esculentum) grown soils presented the same in silico restriction patterns with A. chroococcum (AY353708) and 99% of similarity with the same sequence. Isolate C5CO from cauliflower (Brassica oleracea var. botrytis) grown soils showed black pigmentation in Ashby-Benzoate agar and high similarity (91%) with A. nigricans (AB175651) sequence. In this work we demonstrated the utility of molecular techniques and bioinformatics tools as a support to conventional techniques in characterization of the genus Azotobacter from vegetable-grown soils.
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Project description:Nitrogen is a limiting nutrient for degraders function in hydrocarbon-contaminated environments. Biological nitrogen fixation by diazotrophs is a natural solution for supplying bioavailable nitrogen. Here, we determined whether the diazotroph Azotobacter chroococcum HN can provide nitrogen to the polycyclic aromatic hydrocarbon-degrading bacterium Paracoccus aminovorans HPD-2 and further explored the synergistic interactions that facilitate pyrene degradation in nitrogen-deprived environments. We found that A. chroococcum HN and P. aminovorans HPD-2 grew and degraded pyrene more quickly in co-culture than in monoculture. Surface-enhanced Raman spectroscopy combined with 15N stable isotope probing (SERS - 15N SIP) demonstrated that A. chroococcum HN provided nitrogen to P. aminovorans HPD-2. Metabolite analysis and feeding experiments confirmed that cross-feeding occurred between A. chroococcum HN and P. aminovorans HPD-2 during pyrene degradation. Transcriptomic and metabolomic analyses further revealed that co-culture significantly upregulated key pathways such as nitrogen fixation, aromatic compound degradation, protein export, and the TCA cycle in A. chroococcum HN and quorum sensing, aromatic compound degradation and ABC transporters in P. aminovorans HPD-2. Phenotypic and fluorescence in situ hybridization (FISH) assays demonstrated that A. chroococcum HN produced large amounts of biofilm and was located at the bottom of the biofilm in co-culture, whereas P. aminovorans HPD-2 attached to the surface layer and formed a bridge-like structure with A. chroococcum HN. This study demonstrates that distinct syntrophic interactions occur between A. chroococcum HN and P. aminovorans HPD-2 and provides support for their combined use in organic pollutant degradation in nitrogen-deprived environments.
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Project description:Background and objectivesAzotobacter is a diazotroph bacterium reported to possess various plant growth-promoting characteristics.The aim of this study was to isolate Azotobacter strains capable of fixing nitrogen and effectively hydrolyzing both organic and inorganic Pi compounds.Materials and methodsIn this study, soil samples collected from a diverse range of slightly alkaline soil types were screened for Azotobacter isolates. The inorganic and organic phosphate solubilization potentials of twenty competent phosphate solubilizing Azotobacter isolates were assessed.Variations were noted in the solubilization potentials.ResultThree isolates, identified as Azotobacter vinelandii strains O2, O4 and O6, were able to fix atmospheric N2 effectively. The nitrogenase activity of these isolates ranged between 158.6 and 326.4 C2H4h(-1)vial(-1) (ethylene). Bacterial growth rates and phosphate solubilization activities were measured quantitatively under various environmental conditions. A close association was evident between phosphate solubilizing ability and growth rate as an indicator of active metabolism. All three phosphate solubilizing bacteria (PSB) were able to withstand temperature as high as 45°C, high concentration of NaCl (upto 5%) and a wide range of initial pH from 5 to 10 while hydrolyzing phosphate compounds actively.ConclusionAzotobacter vinelandii strains O2, O4 and O6 are superior candidates for biofertilizers that may result in the reduction of chemical nitrogen and phosphate fertilizers leading to increase crop production.
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Project description:The genome of the soil-dwelling heterotrophic N2-fixing Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 (ATCC 4412) (Ac-8003) has been determined. It consists of 7 circular replicons totalling 5,192,291 bp comprising a circular chromosome of 4,591,803 bp and six plasmids pAcX50a, b, c, d, e, f of 10,435 bp, 13,852, 62,783, 69,713, 132,724, and 311,724 bp respectively. The chromosome has a G+C content of 66.27% and the six plasmids have G+C contents of 58.1, 55.3, 56.7, 59.2, 61.9, and 62.6% respectively. The methylome has also been determined and 5 methylation motifs have been identified. The genome also contains a very high number of transposase/inactivated transposase genes from at least 12 of the 17 recognised insertion sequence families. The Ac-8003 genome has been compared with that of Azotobacter vinelandii ATCC BAA-1303 (Av-DJ), a derivative of strain O, the only other member of the Azotobacteraceae determined so far which has a single chromosome of 5,365,318 bp and no plasmids. The chromosomes show significant stretches of synteny throughout but also reveal a history of many deletion/insertion events. The Ac-8003 genome encodes 4628 predicted protein-encoding genes of which 568 (12.2%) are plasmid borne. 3048 (65%) of these show > 85% identity to the 5050 protein-encoding genes identified in Av-DJ, and of these 99 are plasmid-borne. The core biosynthetic and metabolic pathways and macromolecular architectures and machineries of these organisms appear largely conserved including genes for CO-dehydrogenase, formate dehydrogenase and a soluble NiFe-hydrogenase. The genetic bases for many of the detailed phenotypic differences reported for these organisms have also been identified. Also many other potential phenotypic differences have been uncovered. Properties endowed by the plasmids are described including the presence of an entire aerobic corrin synthesis pathway in pAcX50f and the presence of genes for retro-conjugation in pAcX50c. All these findings are related to the potentially different environmental niches from which these organisms were isolated and to emerging theories about how microbes contribute to their communities.
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Project description:The enhancement of nitrogen fixation activity of diazotrophs is essential for safe crop production. Lysine succinylation (KSuc) is widely present in eukaryotes and prokaryotes and regulates various biological process. However, knowledge of the extent of KSuc in nitrogen fixation of Azotobacter chroococcum is scarce. In this study, we found that 250 mg/l of rhamnolipid (RL) significantly increased the nitrogen fixation activity of A. chroococcum by 39%, as compared with the control. Real-time quantitative reverse transcription PCR (qRT-PCR) confirmed that RL could remarkably increase the transcript levels of nifA and nifHDK genes. In addition, a global KSuc of A. chroococcum was profiled using a 4D label-free quantitative proteomic approach. In total, 5,008 KSuc sites were identified on 1,376 succinylated proteins. Bioinformatics analysis showed that the addition of RL influence on the KSuc level, and the succinylated proteins were involved in various metabolic processes, particularly enriched in oxidative phosphorylation, tricarboxylic acid cycle (TCA) cycle, and nitrogen metabolism. Meanwhile, multiple succinylation sites on MoFe protein (NifDK) may influence nitrogenase activity. These results would provide an experimental basis for the regulation of biological nitrogen fixation with KSuc and shed new light on the mechanistic study of nitrogen fixation.
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Project description:Nitrogen (N) and phosphorus (P) deficiencies are two of the most agronomic problems that cause significant decrease in crop yield and quality. N and P chemical fertilizers are widely used in current agriculture, causing environmental problems and increasing production costs. Therefore, the development of alternative strategies to reduce the use of chemical fertilizers while maintaining N and P inputs are being investigated. Although dinitrogen is an abundant gas in the atmosphere, it requires biological nitrogen fixation (BNF) to be transformed into ammonium, a nitrogen source assimilable by living organisms. This process is bioenergetically expensive and, therefore, highly regulated. Factors like availability of other essential elements, as phosphorus, strongly influence BNF. However, the molecular mechanisms of these interactions are unclear. In this work, a physiological characterization of BNF and phosphorus mobilization (PM) from an insoluble form (Ca3(PO4)2) in Azotobacter chroococcum NCIMB 8003 was carried out. These processes were analyzed by quantitative proteomics in order to detect their molecular requirements and interactions. BNF led to a metabolic change beyond the proteins strictly necessary to carry out the process, including the metabolism related to other elements, like phosphorus. Also, changes in cell mobility, heme group synthesis and oxidative stress responses were observed. This study also revealed two phosphatases that seem to have the main role in PM, an exopolyphosphatase and a non-specific alkaline phosphatase PhoX. When both BNF and PM processes take place simultaneously, the synthesis of nitrogenous bases and L-methionine were also affected. Thus, although the interdependence is still unknown, possible biotechnological applications of these processes should take into account the indicated factors.
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