Abstract
Lanthanide elements have been recently recognized as “new life metals” yet much remains unknown regarding lanthanide acquisition and homeostasis. In Methylorubrum extorquens AM1, the periplasmic lanthanide-dependent methanol dehydrogenase XoxF1 produces formaldehyde, which is lethal if allowed to accumulate. This property enabled a transposon mutagenesis study and growth studies to confirm novel gene products required for XoxF1 function. The identified genes encode an MxaD homolog, an ABC-type transporter, an aminopeptidase, a putative homospermidine synthase, and two genes of unknown function annotated as orf6 and orf7. 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 were able to acclimate over time and bypass the requirement for the TonB outer membrane transporter to allow expression of xoxF1 and 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.
Highlights
Lanthanide elements have been recently recognized as “new life metals” yet much remains unknown regarding lanthanide acquisition and homeostasis
A transposon mutagenesis study was designed to take advantage of the in vivo formaldehyde production capability of XoxF1 and XoxF2 to identify genes required for XoxF-dependent methanol oxidation
The strain used to conduct the mutant hunt contained a mutation in mxaF to make cells dependent on the exogenously provided L a3+ for formaldehyde production, and a second mutation in fae, which would result in formaldehyde accumulation and cell death when methanol is oxidized to formaldehyde by XoxF1 and XoxF2 (Fig. 1)
Summary
Lanthanide elements have been recently recognized as “new life metals” yet much remains unknown regarding lanthanide acquisition and homeostasis. In Methylorubrum extorquens AM1, the periplasmic lanthanide-dependent methanol dehydrogenase XoxF1 produces formaldehyde, which is lethal if allowed to accumulate. This property enabled a transposon mutagenesis study and growth studies to confirm novel gene products required for XoxF1 function. MxaFI is a two-subunit Ca2+-dependent ADH that was long considered to be the predominant methanol dehydrogenase (MeDH) in nature until Ln-dependent XoxF enzymes were first described in 2 0115. Since their role in methanol oxidation became apparent, XoxF enzymes have been classified into five phylogenetically distinct clades (Type 1 to 5)[14,15,16]. When Ln are available, the XoxF enzymes produce formaldehyde from methanol whereas ExaF oxidizes methanol further to formate[25]
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