XoxF and the Calvin-Benson cycle mediate lanthanide-dependent growth on methanol in <i>Bradyrhizobium</i> and <i>Sinorhizobium</i>

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-fixing <i>Rhizobiales</i> are examined. Prior work has demonstrated that select <i>Bradyrhizobium</i> strains can assimilate methanol only in the presence of lanthanide metals; however, the pathway enabling assimilation remains unknown. In this study, we characterize <i>Bradyrhizobium diazoefficiens</i> USDA 110, <i>Bradyrhizobium</i> sp. USDA 3456, and <i>Sinorhizobium meliloti</i> 2011 to determine the pathways involved in methanol metabolism in previously characterized strains, other clades of <i>Bradyrhizobium</i>, and the more distantly related <i>Sinorhizobium</i>. 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) cycle. Transcriptomics revealed upregulation of the aforementioned pathways in <i>Bradyrhizobium</i> sp. USDA 3456 during growth with methanol. Enzymatic assays demonstrated increased activity of the glutathione-linked oxidation pathway and formate dehydrogenases in all strains during growth with methanol compared to succinate. ¹³C-labeling studies confirmed the presence of CBB intermediates and label incorporation during growth with 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 <i>Bradyrhizobium</i> and <i>Sinorhizobium</i> species.IMPORTANCENitrogen-fixing soil bacteria such as <i>Bradyrhizobium</i> and <i>Sinorhizobium</i> promote plant growth while reducing dependence on artificial, energy-intensive fertilizers. Numerous studies have attempted to increase bacterial nitrogen fixation and colonization of plant tissues by identifying the micronutrients and plant exudates that promote successful symbiotic relationships. Among the compounds encountered by rhizobacteria, lanthanides have received little attention, despite reports that plant growth is affected by the presence of lanthanides. We characterized three agriculturally relevant <i>Bradyrhizobium</i> and <i>Sinorhizobium</i> strains, demonstrated that they gain the capacity to utilize methanol when lanthanides are present, and experimentally determined the pathway by which this metabolism occurs. This study provides a foundation for understanding the lanthanide-dependent metabolism of <i>Bradyrhizobium</i> and <i>Sinorhizobium,</i> which may influence their physiology and abundance in the environment.