Lanthanide transport, storage, and beyond: genes and processes contributing to XoxF function in Methylorubrum extorquens AM1

Lanthanides are elements that have been recently recognized as “new life metals” in bacterial metabolism, yet much remains unknown regarding lanthanide acquisition, regulation, and use. The lanthanide-dependent methanol dehydrogenase XoxF produces formaldehyde, which is lethal to AM1 if allowed to accumulate. This property enabled a transposon mutagenesis study to expand knowledge of the lanthanide-dependent metabolic network. Mutant strains were reconstructed and growth studies were conducted for over 40 strains detailing the involvement of 8 novel genes in lanthanide-dependent and independent methanol growth, including a fused ABC-transporter, aminopeptidase, LysR-type transcriptional regulator, putative homospermidine synthase, homolog (), porin family protein, and genes of unknown function previously published as and . Using genetic and biochemical analyses, strains lacking individual genes in the lanthanide transport cluster were characterized and named for anthanide tilization and ransport ( to ). Consistent with previous reports, we corroborated that a TonB-ABC transport system is required for lanthanide transport to the cytoplasm. However, an additional outer membrane transport mechanism became apparent after longer growth incubations. Additionally, suppressor mutations that rescued growth of the ABC-transporter mutants were identified. Transcriptional reporter fusions were used to show that like iron transport, expression from the TonB-dependent receptor promoter, , is repressed when lanthanides are in excess. Energy dispersive X-ray spectroscopy analysis was used to visualize the localization of lanthanum in wild-type and TonB-ABC transport mutant strains and showed for the first time, that AM1 stores cytoplasmic lanthanides in mineral form.