A Mutagenic Screen Identifies a TonB-Dependent Receptor Required for the Lanthanide Metal Switch in the Type I Methanotroph “Methylotuvimicrobium buryatense” 5GB1C

Abstract

Several of the metabolic enzymes in methanotrophic bacteria rely on metals for both their expression and their catalysis. The MxaFI methanol dehydrogenase enzyme complex uses calcium as a cofactor to oxidize methanol, while the alternative methanol dehydrogenase XoxF uses lanthanide metals such as lanthanum and cerium for the same function. Lanthanide metals, abundant in the earth’s crust, strongly repress the transcription of mxaF yet activate the transcription of xoxF. This regulatory program, called the “lanthanide switch,” is central to methylotrophic metabolism, but only some of its components are known. To uncover additional components of the lanthanide switch, we developed a chemical mutagenesis system in the type I gammaproteobacterial methanotroph “Methylotuvimicrobium buryatense” 5GB1C and designed a selection system for mutants unable to repress the mxaF promoter in the presence of lanthanum. Whole-genome resequencing for multiple lanthanide switch mutants identified several unique point mutations in a single gene encoding a TonB-dependent receptor, which we have named LanA. The LanA TonB-dependent receptor is absolutely required for the lanthanide switch and controls the expression of a small set of genes. While mutation of the lanA gene does not affect the amount of cell-associated lanthanum, it is essential for growth in the absence of the MxaF methanol dehydrogenase, suggesting that LanA is involved in lanthanum uptake to supply the XoxF methanol dehydrogenase with its critical metal ion cofactor. The discovery of this novel component of the lanthanide regulatory system highlights the complexity of this circuit and suggests that further components are likely involved. IMPORTANCE Lanthanide metals, or rare earth elements, are abundant in nature and used heavily in technological devices. Biological interactions with lanthanides are just beginning to be unraveled. Until very recently, microbial mechanisms of lanthanide metal interaction and uptake were unknown. The TonB-dependent receptor LanA is the first lanthanum receptor identified in a methanotroph. Sequence homology searches with known metal transporters and regulators could not be used to identify LanA or other lanthanide metal switch components, and this method for mutagenesis and selection was required to identify the receptor. This work advances the knowledge of microbe-metal interactions in environmental niches that impact atmospheric methane levels and are thus relevant to climate change.

Gene products and processes contributing to lanthanide homeostasis and methanol metabolism in Methylorubrum extorquens AM1

“…Addition of exogenous La 3+ repressed expression fourfold (RFU/OD 600 : 210 ± 7 without La 3+ and 47 ± 5 with La 3+ ) showing that when Ln are in excess, transport is down-regulated. Similar repression has been reported for transcription of the TonB-dependent transporter of Methylotuvimicrobium buryatense 5GB1C 41 .…”

Biological Function of Lanthanide in Plant-Symbiotic Bacteria: Lanthanide-Dependent Methanol Oxidation System

“…As shown in Fig. 3, an Ln-uptake system has already been postulated in methylotrophic and methanotrophic bacteria (Cotruvo Jr, 2019;Featherston et al, 2019;Groom et al, 2019;Ochsner et al, 2019;Roszczenko-Jasińska et al, 2019;Cotruvo Jr et al, 2018). In this system, the cells are believed to uptake Ln species bonded by a lanthanophore, and the Ln species is transferred into the cell using a TonB-dependent system, which is suspected to be similar to siderophore-and chalcophore-mediated iron and copper uptake (Daumann, 2019).…”

Rare earth element (REE)-dependent growth of Pseudomonas putida KT2440 depends on the ABC-transporter PedA1A2BC and is influenced by iron availability

“…In M. extorquens PA1 and M. extorquens AM1, almost identical TonB-dependent receptor proteins (>99 % sequence identity) were found to be crucial for REE-dependent growth suggesting a specific Ln 3+ -binding chelator system in these organisms (Ochsner et al, 2019; Roszczenko-Jasinska et al, 2019). Interestingly, also in Methylotuvimicrobium buryatense 5GB 1C a TonB-dependent receptor was identified that is crucial for the REE-switch to occur (Groom et al, 2019). Interestingly, the latter receptor only shows < 20% sequence identity to those of the M. extorquens strains and could thus not have been identified by homology searches.…”

“…In addition to these findings, it has been found that a specific TonB-dependent receptor protein as well as a TonB-like transporter protein are highly conserved in bacteria that carry genes encoding for Ln 3+ -dependent MDHs (Keltjens et al, 2014; Wu et al, 2015). Only very recently, different studies indeed identified both an ABC-transporter and TonB-dependent receptor proteins that are needed for REE-dependent growth of methano- and methylotrophs, strongly suggesting the existence of an uptake system that specifically transports a Ln 3+ -chelator complex in these organisms (Groom et al, 2019; Ochsner et al, 2019; Roszczenko-Jasińska et al, 2019).…”