School of Environmental and Forest Sciences, College of the Environment, University of Washington, Seattle, WA, 98195-2100, United States.
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, 99354, United States.
ISME J. 2024 Jan 8;18(1). doi: 10.1093/ismejo/wrad012.
Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte-poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only ~11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through 15N2 enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant-endophyte interactions.
微生物固氮菌的生物固氮作用可以显著提高非结瘤植物物种的氮素供应。在这项关于与生物固氮相关的分子机制和基因表达的研究中,好氧固氮内生菌 Burkholderia vietnamiensis WPB 菌株被用作内生菌-杨树相互作用的模型。在无氮培养基上观察到固氮活性是动态的,一部分菌落生长形成健壮的、凸起的球状结构。二次离子质谱(NanoSIMS)证实了种群内氮固定的不均匀性。荧光转录报告基因(GFP)表明,固氮酶亚基 nifH 在 WPB 的遗传上相同的菌落中不是均匀表达的,只有约 11%的种群在积极表达 nifH 基因。通过监测单个细菌细胞的单分子荧光原位杂交,我们观察到在聚集的细胞中观察到更高的 nifH 基因表达。通过 15N2 富集,我们鉴定了 WPB 合成的关键含氮代谢物和蛋白质,并在活跃和不活跃的种群中采用了靶向代谢组学。我们在 RhizoChip 中共同培养 WPB Pnif-GFP 和杨树,这是一种合成土壤生境,使我们能够直接观察到根表皮细胞中微生物 nifH 表达。我们观察到 nifH 表达定位于根伸长区,菌株在该区域与根细胞形成独特的物理相互作用。这项工作采用了综合实验来确定调节生物固氮和有益植物-内生菌相互作用的新机制。