Identification of a TonB-Dependent Receptor Involved in Lanthanide Switch by the Characterization of Laboratory-Adapted Methylosinus trichosporium OB3b.

Two methanol dehydrogenases (MDHs), MxaFI and XoxF, have been characterized in methylotrophic and methanotrophic bacteria. MxaFI contains a calcium ion in its active site, whereas XoxF contains a lanthanide ion. Importantly, the expression of MxaFI and XoxF is inversely regulated by lanthanide bioavailability, i.e., the "lanthanide switch." To reveal the genetic and environmental factors affecting the lanthanide switch, we focused on two Methylosinus trichosporium OB3b mutants isolated during routine cultivation. In these mutants, MxaF was constitutively expressed, but lanthanide-dependent XoxF1 was not, even in the presence of 25 µM cerium ions, which is sufficient for XoxF expression in the wild type. Genotyping showed that both mutants harbored a loss-of-function mutation in the CQW49_RS02145 gene, which encodes a TonB-dependent receptor. Gene disruption and complementation experiments demonstrated that CQW49_RS02145 was required for XoxF1 expression in the presence of 25 µM cerium ions. Phylogenetic analysis indicated that CQW49_RS02145 was homologous to the Methylorubrum extorquens AM1 lanthanide transporter gene (lutH). These findings suggest that CQW49_RS02145 is involved in lanthanide uptake across the outer membrane. Furthermore, we demonstrated that supplementation with cerium and glycerol caused severe growth arrest in the wild type. CQW49_RS02145 underwent adaptive laboratory evolution in the presence of cerium and glycerol ions, resulting in a mutation that partially mitigated the growth arrest. This finding implies that loss-of-function mutations in CQW49_RS02145 can be attributed to residual glycerol from the frozen stock. IMPORTANCE Lanthanides are widely used in many industrial applications, including catalysts, magnets, and polishing. Recently, lanthanide-dependent metabolism was characterized in methane-utilizing bacteria. Despite the global demand for lanthanides, few studies have investigated the mechanism of lanthanide uptake by these bacteria. In this study, we identify a lanthanide transporter in Methylosinus trichosporium OB3b and indicate the potential interaction between intracellular lanthanide and glycerol. Understanding the genetic and environmental factors affecting lanthanide uptake should not only help improve the use of lanthanides for the bioconversion of methane into valuable products like methanol but also be of value for developing biomining to extract lanthanides under neutral conditions.

The Proteobacterial Methanotroph Methylosinus trichosporium OB3b Remodels Membrane Lipids in Response to Phosphate Limitation.

Methane is a potent greenhouse gas in the atmosphere, and its concentration has continued to increase in recent decades. Aerobic methanotrophs, bacteria that use methane as the sole carbon source, are an important biological sink for methane, and they are widely distributed in the natural environment. However, relatively little is known on how methanotroph activity is regulated by nutrients, particularly phosphorus (P). P is the principal nutrient constraining plant and microbial productivity in many ecosystems, ranging from agricultural land to the open ocean. Using a model methanotrophic bacterium, Methylosinus trichosporium OB3b, we demonstrate here that this bacterium can produce P-free glycolipids to replace membrane phospholipids in response to P limitation. The formation of the glycolipid monoglucuronic acid diacylglycerol requires plcP-agt genes since the plcP-agt mutant is unable to produce this glycolipid. This plcP-agt-mediated lipid remodeling pathway appears to be important for M. trichosporium OB3b to cope with P stress, and the mutant grew significantly slower under P limitation. Interestingly, comparative genomics analysis shows that the ability to perform lipid remodeling appears to be a conserved trait in proteobacterial methanotrophs; indeed, plcP is found in all proteobacterial methanotroph genomes, and plcP transcripts from methanotrophs are readily detectable in metatranscriptomics data sets. Together, our study provides new insights into the adaptation to P limitation in this ecologically important group of bacteria. IMPORTANCE Methane is a potent greenhouse gas in the atmosphere, and its concentration has continued to increase steadily in recent decades. In the natural environment, bacteria known as methanotrophs help mitigate methane emissions at no cost to human beings. However, relatively little is known regarding how methane oxidation activity in methanotrophs is regulated by soil nutrients, particularly phosphorus. Here, we show that methanotrophs can modify their membrane in response to phosphorus limitation and that the ability to change membrane lipids is important for methanotroph activity. Genome and metatranscriptome analyses suggest that such an adaptation strategy appears to be strictly conserved in all proteobacterial methanotrophs and is used by these bacteria in the natural environment. Together, our study provides a plausible molecular mechanism for better understanding the role of phosphorus on methane oxidation in the natural environment.