Lanthanide transport, storage, and beyond: gene products and processes contributing to lanthanide and methanol metabolism inMethylorubrum extorquensAM1
Authors: Roszczenko-Jasińska Paula , Vu Huong N. , Subuyuj Gabriel A. , Crisostomo Ralph Valentine , Ayala Elena M. , Cai James , Lien Nicholas F. , Clippard Erik J. , Ngo Richard T. , Yarza Fauna , Wingett Justin P. , Raghuraman Charumathi , Hoeber Caitlin A. , Martinez-Gomez Norma C. , Skovran Elizabeth 
Published: May 2019

ABSTRACTLanthanide elements have been recently recognized as “new life metals” for diverse environmental microorganisms including Gram-negative methylotrophic bacteria and strains ofPseudomonasandBradyrhizobium. Yet much remains unknown regarding lanthanide acquisition and homeostasis. InMethylorubrum extorquensAM1, the periplasmic lanthanide-dependent methanol dehydrogenase XoxF1 produces formaldehyde, which is lethal if allowed to accumulate. This property enabled a transposon mutagenesis study to expand knowledge of the metabolic network required for methanol oxidation when lanthanides are available. Growth studies were conducted to detail the involvement of novel gene products that impact the ability of XoxF-type enzymes to oxidize methanol to formaldehyde. The identified genes encode an MxaD homolog, an ABC-type transporter, an aminopeptidase, a putative homospermidine synthase, and two genes of unknown function annotated asorf6andorf7. Lanthanide transport and trafficking genes were also identified. Growth and lanthanide uptake were measured using strains lacking individual lanthanide transport cluster genes and transmission electron microscopy was used to visualize lanthanide localization. We corroborated previous reports that a TonB-ABC transport system is required for lanthanide incorporation to the cytoplasm. However, cells are able to acclimate overtime and bypass the requirement for the TonB outer membrane transporter to allow expression ofxoxF1and growth. Transcriptional reporter fusions show that excess lanthanides repress the gene encoding the TonB-receptor. Using growth studies along with energy dispersive X-ray spectroscopy and transmission electron microscopy, we demonstrate that lanthanides are stored as cytoplasmic inclusions that resemble polyphosphate granules.IMPORTANCEThe increasing genetic and biochemical evidence that lanthanide-dependent enzymes are widespread among numerous environmental microbes leads to the parallel questions of how these insoluble metals are scavenged, transported, and used by bacteria. Results herein describe the contribution of the different gene products that constitute the lanthanide utilization and transport machinery in the methylotrophM. extorquensAM1 and highlight possible redundancies by periplasmic components. The discovery and characterization of intracellular lanthanide storage in mineral form by these microbes opens the possibility of using methylotrophic platforms for concentration and recovery of these critical energy metals from diverse sources. In addition, methylotrophs are effective biotechnological platforms for the production of biofuels and bioplastics from pollutants such as methane, and inexpensive carbon feedstocks like methanol. Defining the lanthanide acquisition, transport, and storage machinery is a step forward in designing a sustainable platform to recover lanthanides efficiently.