Project description:Nodule-forming bacteria play crucial roles in plant health and nutrition by fixing atmospheric nitrogen. Despite the importance of this relationship, how nodule-forming bacteria are affected by plant exudates and soil minerals is not fully characterized. Of particular interest are the effects of plant-derived methanol and lanthanide metals on the growth of nitrogen-fixing Rhizobiales. Prior work has demonstrated that select Bradyrhizobium are able to assimilate methanol only in the presence of lanthanide metals; however, the pathway enabling assimilation remains unknown. Here we characterize Bradyrhizobium sp. USDA 3456 to determine the pathways involved in methanol metabolism. Based on genomic analyses, we hypothesized that methanol assimilation in these organisms occurs via the lanthanide-dependent methanol dehydrogenase XoxF, followed by oxidation of formaldehyde via the glutathione-linked oxidation pathway, subsequent oxidation of formate via formate dehydrogenases, and finally assimilation of CO2 via the Calvin Benson Bassham (CBB) pathway. Transcriptomics revealed upregulation of the aforementioned pathways in Bradyrhizobium sp. USDA 3456 during growth on methanol. Assays demonstrated increased activity of the glutathione-linked oxidation pathway and formate dehydrogenases during growth on methanol compared to succinate. 13C-labeling studies demonstrate the presence of CBB intermediates and label incorporation during growth on methanol. Our findings provide multiple lines of evidence supporting the proposed XoxF-CBB pathway and, combined with genomic analyses, suggest that this metabolism is widespread among Bradyrhizobium and Sinorhizobium species.
Project description:Bradyrhizobium diazoefficiens can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR encodes a transcription factor annotated as PHA (polyhydroxyalkanoate) accumulation regulator. We found that PhaR not only controls the PHA cycle but also acts as a global regulator of excess carbon allocation by controlling the expression of fixK2 and nifA genes, both encoding key transcription factors for microoxic and symbiotic metabolism in B. diazoefficiens. The function of PhaR was expanded by a multi-pronged approach that includes analysis of the effects of phaR mutation at transcriptional and protein levels of putative PhaR targets and direct control mediated by PhaR determined by EMSA assays. We also were able to identify PhaR, phasins and other proteins associated with PHA granules which confirmed a global function of PhaR in microoxia.
