Dataset Information

Regulation of hierarchical carbon substrate utilization, nitrogen fixation, and root colonization by the Hfq/Crc/CrcZY genes in Pseudomonas stutzeri.

ABSTRACT: Bacteria of the genus Pseudomonas consume preferred carbon substrates in nearly reverse order to that of enterobacteria, and this process is controlled by RNA-binding translational repressors and regulatory ncRNA antagonists. However, their roles in microbe-plant interactions and the underlying mechanisms remain uncertain. Here we show that root-associated diazotrophic Pseudomonas stutzeri A1501 preferentially catabolizes succinate, followed by the less favorable substrate citrate, and ultimately glucose. Furthermore, the Hfq/Crc/CrcZY regulatory system orchestrates this preference and contributes to optimal nitrogenase activity and efficient root colonization. Hfq has a central role in this regulatory network through different mechanisms of action, including repressing the translation of substrate-specific catabolic genes, activating the nitrogenase gene nifH posttranscriptionally, and exerting a positive effect on the transcription of an exopolysaccharide gene cluster. Our results illustrate an Hfq-mediated mechanism linking carbon metabolism to nitrogen fixation and root colonization, which may confer rhizobacteria competitive advantages in rhizosphere environments.

SUBMITTER: Lv F

PROVIDER: S-EPMC9730152 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Publications

Regulation of hierarchical carbon substrate utilization, nitrogen fixation, and root colonization by the Hfq/Crc/CrcZY genes in Pseudomonas stutzeri.

Lv Fanyang F Zhan Yuhua Y Lu Wei W Ke Xiubin X Shao Yahui Y Ma Yiyuan Y Zheng Juan J Yang Zhimin Z Jiang Shanshan S Shang Liguo L Ma Yao Y Cheng Lei L Elmerich Claudine C Yan Yongliang Y Lin Min M

iScience 20221123 12

Bacteria of the genus Pseudomonas consume preferred carbon substrates in nearly reverse order to that of enterobacteria, and this process is controlled by RNA-binding translational repressors and regulatory ncRNA antagonists. However, their roles in microbe-plant interactions and the underlying mechanisms remain uncertain. Here we show that root-associated diazotrophic Pseudomonas stutzeri A1501 preferentially catabolizes succinate, followed by the less favorable substrate citrate, ...[more]

PMID: 36505936

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Project description:The RNA chaperone Hfq acts as a global regulator of numerous biological processes, such as carbon/nitrogen metabolism and environmental adaptation in plant-associated diazotrophs; however, its target RNAs and the mechanisms underlying nitrogen fixation remain largely unknown. Here, we used enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing to identify hundreds of Hfq-binding RNAs probably involved in nitrogen fixation, carbon substrate utilization, biofilm formation, and other functions. Collectively, these processes endow strain A1501 with the requisite capabilities to thrive in the highly competitive rhizosphere. Our findings revealed a previously uncharted landscape of Hfq target genes. Notable among these is nifM, encoding an isomerase necessary for nitrogenase reductase solubility; amtB, encoding an ammonium transporter; oprB, encoding a carbohydrate porin; and cheZ, encoding a chemotaxis protein. Furthermore, we identified more than 100 genes of unknown function, which expands the potential direct regulatory targets of Hfq in diazotrophs. Our data showed that Hfq directly interacts with the mRNA of regulatory proteins (RsmA, AlgU, and NifA), regulatory ncRNA RsmY, and other potential targets, thus revealing the mechanistic links in nitrogen fixation and other metabolic pathways.ImportanceNumerous experimental approaches often face challenges in distinguishing between direct and indirect effects of Hfq-mediated regulation. New technologies based on high-throughput sequencing are increasingly providing insight into the global regulation of Hfq in gene expression. Here, enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing was employed to identify the Hfq-binding sites and potential targets in the root-associated Pseudomonas stutzeri A1501 and identify hundreds of novel Hfq-binding RNAs that are predicted to be involved in metabolism, environmental adaptation, and nitrogen fixation. In particular, we have shown Hfq interactions with various regulatory proteins' mRNA and their potential targets at the posttranscriptional level. This study not only enhances our understanding of Hfq regulation but, importantly, also provides a framework for addressing integrated regulatory network underlying root-associated nitrogen fixation.

Project description:BACKGROUND: Biological nitrogen fixation is highly controlled at the transcriptional level by regulatory networks that respond to the availability of fixed nitrogen. In many diazotrophs, addition of excess ammonium in the growth medium results in immediate repression of nif gene transcription. Although the regulatory cascades that control the transcription of the nif genes in proteobacteria have been well investigated, there are limited data on the kinetics of ammonium-dependent repression of nitrogen fixation. RESULTS: Here we report a global transcriptional profiling analysis of nitrogen fixation and ammonium repression in Pseudomonas stutzeri A1501, a root-associated and nitrogen-fixing bacterium. A total of 166 genes, including those coding for the global nitrogen regulation (Ntr) and Nif-specific regulatory proteins, were upregulated under nitrogen fixation conditions but rapidly downregulated as early as 10 min after ammonium shock. Among these nitrogen fixation-inducible genes, 95 have orthologs in each of Azoarcus sp. BH72 and Azotobacter vinelandii AvoP. In particular, a 49-kb expression island containing nif and other associated genes was markedly downregulated by ammonium shock. Further functional characterization of pnfA, a new NifA-sigma54-dependent gene chromosomally linked to nifHDK, is reported. This gene encodes a protein product with an amino acid sequence similar to that of five hypothetical proteins found only in diazotrophic strains. No noticeable differences in the transcription of nifHDK were detected between the wild type strain and pnfA mutant. However, the mutant strain exhibited a significant decrease in nitrogenase activity under microaerobic conditions and lost its ability to use nitrate as a terminal electron acceptor for the support of nitrogen fixation under anaerobic conditions. CONCLUSIONS: Based on our results, we conclude that transcriptional regulation of nif gene expression in A1501 is mediated by the nif-specific and ntr gene regulatory systems. Furthermore, microarray and mutational analyses revealed that many genes of unknown function may play some essential roles in controlling the expression or activity of nitrogenase. The findings presented here establish the foundation for further studies on the physiological function of nitrogen fixation-inducible genes.

Project description:Diazotrophs can produce bioavailable nitrogen from inert N2 gas by bioelectrochemical nitrogen fixation (e-BNF), which is emerging as an energy-saving and highly selective strategy for agriculture and industry. However, current e-BNF technology is impeded by requirements for NH4+ assimilation inhibitors to facilitate intracellular ammonia secretion and precious metal catalysts to generate H2 as the energy-carrying intermediate. Here, we initially demonstrate inhibitor- and catalystless extracellular NH4+ production by the diazotroph Pseudomonas stutzeri A1501 using an electrode as the sole electron donor. Multiple lines of evidence revealed that P. stutzeri produced 2.32 ± 0.25 mg/liter extracellular NH4+ at a poised potential of -0.3 V (versus standard hydrogen electrode [SHE]) without the addition of inhibitors or expensive catalysts. The electron uptake mechanism was attributed to the endogenous electron shuttle phenazine-1-carboxylic acid, which was excreted by P. stutzeri and mediated electron transfer from electrodes into cells to directly drive N2 fixation. The faradaic efficiency was 20% ± 3%, which was 2 to 4 times that of previous e-BNF attempts using the H2-mediated pathway. This study reports a diazotroph capable of producing secretable NH4+ via extracellular electron uptake, which has important implications for optimizing the performance of e-BNF systems and exploring the novel nitrogen-fixing mode of syntrophic microbial communities in the natural environment. IMPORTANCE Ammonia greatly affects global ecology, agriculture, and the food industry. Diazotrophs with an enhanced capacity of extracellular NH4+ excretion have been proven to be more beneficial to the growth of microalgae and plants, whereas most previously reported diazotrophs produce intracellular organic nitrogen in the absence of chemical suppression and genetic manipulation. Here, we demonstrate that Pseudomonas stutzeri A1501 is capable of extracellular NH4+ production without chemical suppression or genetic manipulation when the extracellular electrode is used as the sole electron donor. We also reveal the electron uptake pathway from the extracellular electron-donating partner to P. stutzeri A1501 via redox electron shuttle phenazines. Since both P. stutzeri A1501 and potential electron-donating partners (such as electroactive microbes and natural semiconductor minerals) are abundant in diverse soils and sediments, P. stutzeri A1501 has broader implications on the improvement of nitrogen fertilization in the natural environment.

Project description:In Mexico, the center of maize origin (Zea mays ssp. mays), there are landraces from the highlands that develop extensive aerial root systems which secrete a carbohydrate-rich mucilage. This mucilage produces a favorable environment for nitrogenase activity by diazotrophs. This plant-microbial interaction enables the fixation of nitrogen (N) from the atmosphere, reducing the required N that otherwise must come from the soil and/or fertilizers. The objective of this research was to investigate the degree to which other landraces of maize and nutrient management affect aerial root growth and the ability of maize to perform and benefit from N2 fixation. In two replicated field experiments in Columbus, Ohio, USA in 2019 and 2020, we planted 21 maize landraces and three improved varieties with and without fertilizer to measure their growth, production of aerial roots, and rate of atmospheric N2 fixation using the 15N natural abundance method. Maize accessions varied in the growth rate and number of nodes with aerial roots. Up to 36% of plant N was derived from the atmosphere, with values varying by accession, the reference plant used, and the fertilizer level. Moreover, there was a positive relationship between early growth parameters and numbers of nodes with aerial roots, which, in turn, predicted the amount of N derived from the atmosphere. Thus, larger seedlings may experience enhanced root growth and thereby benefit more from N fixation. By phenotyping a diverse set of maize accessions with and without fertilizer, this study explores both environmental and quantitative genetic variation in the traits involved in N fixation capacity, clarifying that N fixation found in the Sierra Mixe landrace is more broadly distributed than previously thought. In sum, farmers stewarding genetic diversity in a crop center of origin have preserved traits essential for biological symbioses that contribute to maize's nutrient requirements. These traits may enable maize crops grown by Mexican farmers, and farmers globally, to benefit from N fixation from the atmosphere.

Dataset Information

Regulation of hierarchical carbon substrate utilization, nitrogen fixation, and root colonization by the Hfq/Crc/CrcZY genes in Pseudomonas stutzeri.

Publications

Regulation of hierarchical carbon substrate utilization, nitrogen fixation, and root colonization by the Hfq/Crc/CrcZY genes in <i>Pseudomonas stutzeri</i>.

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