XoxF and the Calvin-Benson Cycle Mediate Lanthanide-Dependent Growth on Methanol inBradyrhizobiumandSinorhizobium

Nodule-forming bacteria play crucial roles in plant health and nutrition by providing fixed nitrogen to leguminous plants. Despite the importance of this relationship, how nodule-forming bacteria are affected by plant exudates and soil minerals is not fully characterized. Here, the effects of plant-derived methanol and lanthanide metals on the growth of nitrogen-fixingRhizobialesare examined. Prior work has demonstrated that selectBradyrhizobiumstrains can assimilate methanol only in the presence of lanthanide metals; however, the pathway enabling assimilation remains unknown. In this study, we characterizeBradyrhizobium diazoefficiensUSDA 110,Bradyrhizobium sp. USDA 3456, andSinorhizobium meliloti2011 to determine the pathways involved in methanol metabolism in previously characterized strains, other clades ofBradyrhizobium, and the more distantly relatedSinorhizobium. 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 CO₂via the Calvin-Benson-Bassham (CBB) cycle. Transcriptomics revealed upregulation of the aforementioned pathways inBradyrhizobium sp.USDA 3456 during growth on methanol. Enzymatic assays demonstrated increased activity of the glutathione-linked oxidation pathway and formate dehydrogenases in all strains during growth on methanol compared to succinate.¹³C-labeling studies confirmed 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 amongBradyrhizobiumandSinorhizobiumspecies.