TY - JOUR T1 - Lanthanide transport, storage, and beyond: genes and processes contributing to XoxF function in <em>Methylorubrum extorquens</em> AM1 JF - bioRxiv DO - 10.1101/647677 SP - 647677 AU - Paula Roszczenko-Jasińska AU - Huong N. Vu AU - Gabriel A. Subuyuj AU - Ralph Valentine Crisostomo AU - James Cai AU - Charumathi Raghuraman AU - Elena M. Ayala AU - Erik J. Clippard AU - Nicholas F. Lien AU - Richard T. Ngo AU - Fauna Yarza AU - Caitlin A. Hoeber AU - Norma C. Martinez-Gomez AU - Elizabeth Skovran Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/05/24/647677.abstract N2 - 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 Methylorubrum extorquens 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, mxaD homolog (xoxD), porin family protein, and genes of unknown function previously published as orf6 and orf7. Using genetic and biochemical analyses, strains lacking individual genes in the lanthanide transport cluster were characterized and named lut for lanthanide utilization and transport (META1_1778 to META1_1787). 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, lutH, 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 M. extorquens AM1 stores cytoplasmic lanthanides in mineral form.IMPORTANCE Understanding the role of lanthanides in bacterial systems is an emergent field of study. Results from this work define mechanisms by which the methylotrophic bacterium, M. extorquens AM1, acquires and accumulates lanthanides. Lanthanides are critical metals for modern technologies and medical applications, yet lanthanide mining is cost-prohibitive and harmful to the environment. Methylotrophs are an attractive platform for the recovery of lanthanides from discarded electronics, mining leachate, and other waste streams as these microorganisms have been proven to sense and transport these metals for use in alcohol oxidation. Further, methylotrophs are effective biotechnological platforms for chemical productions and can use pollutants such as methane, and inexpensive feedstocks such as methanol. Defining the lanthanide acquisition, transport, and accumulation machinery is a step forward in designing a sustainable, clean platform to recover lanthanides in an efficient and less environmentally destructive manner. ER -