Project description:Symbiotic nitroegn fixation in functional (Fix+) and non-functional (Fix-) nodules of Vicia faba infected with Rhizobium leguminosarum was investigated using label-free shotgun tandem MS. Proteins involved in symbiotic nitrogen fixation and maintenance of the symbiosis were identified.
Project description:Hairy vetch (Vicia villosa Roth) is recognized as a beneficial winter cover crop in the Midwestern U.S. DNA microarrays are used for assessing gene expression significance. The objective of the study was to identify a set of genes expressed in hairy vetch, that could be further analyzed for their potential in improving the crop. The RNA of four targets (soybean (Glycine max), hairy vetch, Vicia pannonica PI 170008, and Vicia pannonica PI 515988) were purified, labeled, and hybridized to 142 cDNA clones of biotic stress genes and gene sequences of soybean that were robotically spotted onto aminosilicated slides with duplicate spots and three arrays per slide. The microarray experiments were completed in a reference design experiment incorporating a two-dye system. The data were analyzed using the individual fluorescence intensities to fit two statistical models in a mixed model analysis of variance. In this analysis, systematic error effects were observed to account for 24% of the total variation. The use of a Bonferroni adjusted significance threshold allowed for adequate control over the number of falsely identified significant genes showing expression in the different target comparisons. We observed that 64 of the 142 gene sequences (45%) were differentially expressed in at least one of the target comparisons. Two of these expressed genes in hairy vetch encoded for a cold tolerance indicator, proline, and two other gene sequences encode for stress tolerance indicators, myo-inositol and calmodulin. A soybean cDNA microarray was used effectively to differentiate gene expression in hairy vetch. Keywords: comparative genomic hybridization (cross-species)
Project description:To investigate the effect that biological nitrogen fixation will have on plant responses to nitrogen dose at elevated CO2, alfalfa (Medicago sativa) lines were grown at three nitrogen doses and ambient or elevated CO2. Four lines were used in the study, two lines that can form nodules capable of fixing nitrogen (effective lines) and two lines that can not form nodules capable of nitrogen fixation (ineffective lines). The ineffective lines are the result of a complementary mutation in the same gene.
Project description:Our study revealed a synergistic effect between biological nitrogen fixation and current generation by G. sulfurreducens, providing a green nitrogen fixation alternative through shifting the nitrogen fixation field from energy consumption to energy production and having implications for N-deficient wastewater treatment.
Project description:The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (nifL disrupted) and wild-type strain, and to what was reported for the wild-type strain under exponential growth related to key processes involved in driving the process of nitrogen fixation in A. vinelandii. These results further illuminate a number of additional genes associated with siderophore synthesis, molybdate transfer and electron transfer that are likely associated with biological nitrogen fixation.
Project description:A1501 NFI is a genomic island derived from Pseudomonas stutzeri A1501. To study the molecular interactions of the P. stutzeri nif genes with the E. coli genome during nitrogen fixation, the NIF of A1501 was transferred into E. coli and comparative transcriptomics analyses were performed between nitrogen fixation conditions and nitrogen excess conditions.
Project description:Model endophyte Azoarcus sp. BH72 is known to contribute fixed nitrogen to its host Kallar grass by nitrogen fixation and also expresses nitrogenase genes endophytically in rice seedlings in gnotobiotic culture. Availability of fixed nitrogen is one of the important signals regulating the transcription of nitrogenase genes and hence nitrogen fixing activity. Therefore, we analysed global transcription in response to differences in the nitrogen source. Transcription profiles of cells grown microaerobically (0.6% oxygen) on minimal medium with nitrogen (N2-fixing) versus ammonium (combined nitrogen) were compared using a genome-wide microarray approach and differences in the gene expression profile were monitored.
Project description:Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCE Azotobacter vinelandii is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered A. vinelandii strains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.
Project description:Model endophyte Azoarcus sp. BH72 is known to contribute fixed nitrogen to its host Kallar grass by nitrogen fixation and also expresses nitrogenase genes endophytically in rice seedlings in gnotobiotic culture. Availability of fixed nitrogen is one of the important signals regulating the transcription of nitrogenase genes and hence nitrogen fixing activity. Therefore, we analysed global transcription in response to differences in the nitrogen source. Transcription profiles of cells grown microaerobically (0.6% oxygen) on minimal medium with nitrogen (N2-fixing) versus ammonium (combined nitrogen) were compared using a genome-wide microarray approach and differences in the gene expression profile were monitored. RNA from cells grown on nitrogen-free synthetic medium under nitrogen fixation (experiment) and combined nitrogen source as ammonium chloride (control) was used for two-color whole-genome microarray approach.