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The function of acyl-CoA dehydrogenase gene acdA in Mesorhizobium huakuii

ABSTRACT: Fatty acids β-oxidation (FAO) is the main pathway of fatty acid degradation in organisms. Acyl-CoA dehydrogenase as the first major enzyme in FAO pathways, catalyzes the dehydrogenation reaction of α and β carbon atoms at the site under the action of FAD cofactor. Bioinformatics analysis showed that acdA gene in Mesorhizobium huakuii 7653R encodes the acyl-CoA dehydrogenase. M. huakuii 7653R acdA gene mutant strain and complementary strain were constructed by homologous recombination. mutant HKacdA exhibited delay in growth in AMS minimal medium with NH4Cl as nitrogen source and glucose as carbon source, and showed more sensitivity to H2O2. The function of M. huakuii acdA gene in symbiotic nitrogen fixation was studied through pot experiment of Astragalus sinicus. Mutant HKacdA was severely impaired in its ability to colonize the rhizosphere of A. sinicus. Compared with the plants inoculated with wild-type 7653R, the leaves of HKacdA-inoculated plants appeared were abnormally sparse and yellow, and exhibited obvious symptoms of nitrogen deficiency. Observation of root nodules and bacteroid morphology revealed that the nodules inoculated with HKacdA contained an abnormally thick cortex and bacteroid-containing cells, and some bacteroids accumulated poly-β-hydroxybutyrate (PHB) granules and appeared dissociation and senescent. The mass spectrometry combined with label-free technique was applied to study the differential expression of proteins in bacteroids. A total of 198 differentially expressed proteins were identifified were identifified including 120 up-regulated and 78 down-regulated proteins. Among the differentially expressed proteins, 38 proteins were related to redox，31 proteins were related to amino acid transport, and 11 proteins were connected with nitrogen fixation. Results showed that acdA gene plays a role in rhizobial bacteroid differentiation and nitrogen fixation by affecting amino acid transport, electron transport in nitrogen fixation and nitrogenase activity in nodule bacteroids.

ORGANISM(S): Mesorhizobium Huakuii 7653r

SUBMITTER: Guojun Cheng

PROVIDER: PXD046434 | iProX | Thu Oct 26 00:00:00 BST 2023

REPOSITORIES: iProX

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Project description:Resendis-Antonio2007 - Genome-scale metabolic network of Rhizobium etli (iOR363) This model is described in the article: Metabolic reconstruction and modeling of nitrogen fixation in Rhizobium etli. Resendis-Antonio O, Reed JL, Encarnación S, Collado-Vides J, Palsson BØ. PLoS Comput. Biol. 2007 Oct; 3(10): 1887-1895 Abstract: Rhizobiaceas are bacteria that fix nitrogen during symbiosis with plants. This symbiotic relationship is crucial for the nitrogen cycle, and understanding symbiotic mechanisms is a scientific challenge with direct applications in agronomy and plant development. Rhizobium etli is a bacteria which provides legumes with ammonia (among other chemical compounds), thereby stimulating plant growth. A genome-scale approach, integrating the biochemical information available for R. etli, constitutes an important step toward understanding the symbiotic relationship and its possible improvement. In this work we present a genome-scale metabolic reconstruction (iOR363) for R. etli CFN42, which includes 387 metabolic and transport reactions across 26 metabolic pathways. This model was used to analyze the physiological capabilities of R. etli during stages of nitrogen fixation. To study the physiological capacities in silico, an objective function was formulated to simulate symbiotic nitrogen fixation. Flux balance analysis (FBA) was performed, and the predicted active metabolic pathways agreed qualitatively with experimental observations. In addition, predictions for the effects of gene deletions during nitrogen fixation in Rhizobia in silico also agreed with reported experimental data. Overall, we present some evidence supporting that FBA of the reconstructed metabolic network for R. etli provides results that are in agreement with physiological observations. Thus, as for other organisms, the reconstructed genome-scale metabolic network provides an important framework which allows us to compare model predictions with experimental measurements and eventually generate hypotheses on ways to improve nitrogen fixation. This model is hosted on BioModels Database and identified by: MODEL1507180006. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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.

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The function of acyl-CoA dehydrogenase gene acdA in Mesorhizobium huakuii

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