Diversity and distribution of the lanthanome in aerobic methane-oxidising bacteria

Diversity and distribution of the lanthanome in aerobic methane-oxidising bacteria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Diversity and distribution of the lanthanome in aerobic methane-oxidising bacteria Shamsudeen Umar Dandare, Alexander Allenby, Eleonora Silvano, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6350491/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Sep, 2025 Read the published version in Environmental Microbiome → Version 1 posted 10 You are reading this latest preprint version Abstract Background Lanthanides (Ln) play essential roles in the metabolism of methanotrophs, catalysing key reactions in the methane oxidation pathway. However, the diversity, distribution, and ecological roles of Ln-dependent proteins (the lanthanome) in aerobic methane-oxidising bacteria (MOB) remain underexplored. This study investigates the lanthanome using genome, plasmid, and proteome data, as well as metatranscriptome data from methane-rich lake sediments. Results Comparative genomic analysis of a custom database of 179 MOB genomes revealed the presence of various methanol dehydrogenase (MDH) isoforms, including xoxF variants, distributed across Proteobacteria and Verrucomicrobia phyla. Using Methylosinus trichosporium OB3b as a model, we measured methane oxidation rates in response to CeCl₃ and ore containing a mixture of lanthanides. Using proteomics, we uncovered differential protein expression in response to Ln. Despite differences in adaptation times, methane oxidation rates were consistent across treatments, indicating similar overall metabolic efficiencies after Ln acclimatisation. The genomic analysis uncovered several Ln-binding proteins, including the TonB-dependent receptors (LanA and LutH-like), as well as Lanmodulin and LanPepsy, with unique phylogenetic patterns. Metatranscriptomic analysis confirmed the expression of lanthanome, particularly in Proteobacteria, with xoxF5 as the dominant MDH variant in MOB genomes. The discovery of Ln-binding proteins in plasmids suggests horizontal gene transfer, highlighting the adaptive mechanisms of MOB to Ln availability and their ecological role in methane cycling. Conclusion This work expands our understanding of Ln-dependent methane oxidation in MOB, highlighting their metabolic flexibility and ecological significance in methane cycling. The findings suggest potential applications for Ln-dependent processes in biotechnology, particularly in methane capture and bio-utilization, as well as lanthanide recovery from geological sources. Lanthanides Methane-oxidising bacteria Lanthanome Proteomics Metatranscriptomes Figures Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 Introduction The discovery in 2011 of bacteria capable of accumulating and utilising lanthanides (Ln) established the biological relevance of these elements. The first evidence of a biological role for Ln came from the identification of a lanthanoenzyme—an Ln-dependent methanol dehydrogenase (MDH) found in several methylotrophs[ 1 – 3 ] and methanotrophs [ 4 ]. This enzyme, which catalyses the oxidation of methanol to formaldehyde, contains a pyrroloquinoline quinone (PQQ) redox cofactor that coordinates the Ln ion [ 5 ]. The discovery and subsequent characterisation of lanthanoenzymes led to the identification of additional proteins involved in Ln sensing, acquisition, and transport, collectively referred to as the “lanthanome”. Cotruvo and colleagues identified lanmodulin (LanM), a highly selective Ln-binding protein structurally similar to the calcium-binding protein calmodulin [ 6 ]. Two distinct TonB-dependent receptors—LanA and LutH—were identified as critical for the uptake of lanthanum and the lanthanide switch in gammaproteobacterial[ 7 ] and alphaproteobacterial methanotrophs [ 8 ], respectively. The lutH gene is part of a recently characterised 10-gene lut cluster in Methylorubrum extorquens AM1 and the closely related phyllosphere bacterium, PA1 [ 9 , 10 ]. Recent research on the lanthanome in the obligate model methylotroph Methylobacillus flagellatus identified a 19 kDa periplasmic protein comprising two characteristic PepSY domains, referred to as lanpepsy (LanP) [ 11 ]. Research on the lanthanome has predominantly focused on methanol-oxidising methylotrophs, despite the widespread occurrence of Ln-utilising methane-oxidising bacteria (MOB)—a key player in global methane cycling and a promising microbial chassis for driving one-carbon biotechnology. Yet, there is a knowledge gap on the distribution and diversity of lanthanome in MOB. Here, we conducted a comprehensive survey of the genomes of aerobic MOB, plasmids, and metatranscriptomes from methane-rich lake sediments to investigate the distribution and diversity of Ln-dependent enzymes and transporters in these bacteria. In addition, we performed growth assays and proteomic analyses to compare the impact of cerium chloride (CeCl₃), widely used in laboratory experiments and mixed lanthanide ore on the activity and physiology of an alphaproteobacterial MOB Methylosinus trichosporium OB3b. Methods Acquisition of MOB genomes, plasmids, and environmental metatranscripts A total of 179 known aerobic MOB genomes were downloaded from the National Centre for Biotechnology Information (NCBI) database (accessed in October 2023) and concatenated to make a custom MOB database. The representative MOB database comprises 175 characterised genomes from the RefSeq database, three metagenome-assembled genomes (MAGs) representing atmospheric methane oxidisers [ 12 – 14 ], and a recently reported MOB belonging to the genus Mycobacterium reported to harbour only the soluble methane monooxygenase enzyme [ 15 ]. All information regarding the custom database, including genome accession numbers, metadata (source environment), and genome completeness determined using the CheckM tool (v1.0.12), is detailed in Table S1 . A phylogenomic tree of MOB genomes was generated using the GToTree workflow (v1.7.00). The GToTree pipeline predicts genes using Prodigal (v2.6.3), searches for a panel of 74 bacterial single copy marker genes (SCGs) from genomes using HMMER3 (v3.3.2), aligns the retrieved genes with Muscle (v5.1.linux64), trims those alignments with TrimAl (v1.4.rev15), concatenates the trimmed alignments and then performs phylogenetic reconstruction with FastTree (v2.1.11) [ 16 – 21 ]. To identify the presence of Ln-related genes in plasmids to understand their distribution within MOB taxa, we used the IMG/PR database ( https://img.jgi.doe.gov ) [ 22 ]. In order to assess the expression of MOB-specific genes in the environment, we used the assembled metatranscriptomes of Lake Washington microbial communities (Study name: Freshwater Sediment Methanotrophic Microbial Communities from Lake Washington under Simulated Oxygen Tension; [ 23 ] available via the IMG portal. Sequence similarity search, curation, and phylogenetic tree construction In order to retrieve homologues for specific genes, we carried out searches (E-value cut off of 1e-30) with specific hidden Markov model (HMM) profiles in the custom MOB genomes database using the HMMER tool (v3.4) [ 24 ]. HMM profiles from KOFAM were used for MxaF and XoxF, and custom HMMs were developed for the remaining genes using Refseq gene sequences [ 25 ]. Homologues flagged by the HMM searches were retrieved from the MOB protein database by seqkit (v2.9.0; [ 26 ]). The recovered sequences were then concatenated with curated reference sequences and were subsequently aligned using Clustal Omega (v1.2.4) with default parameters [ 27 ]. Conserved regions in the alignments were identified and trimmed using trimAl (v1.5. rev0) with the option –automated1 [ 17 ], and finally, a phylogenetic tree of the resulting aligned and trimmed sequences was constructed using FastTree (v2.1.11) with 1,000 replicates for bootstrap [ 18 ]. The phylogenetic trees were then used for manual curation of the retrieved homologues. Homologues of specific Ln-dependent enzymes and transporters in plasmids and lake sediment metatranscriptomes databases were retrieved using the BlastP (E-value cut off 1e-30; as described previously in [ 28 ]) within the IMG portal (Details on specific query sequences are included in Table S2 ). To remove any redundancy and for ease of analysis, candidate hits were clustered at 100% using CD-HIT [ 29 ]. Hits were curated for sequence length using CD-HIT, and sequences shorter than the specified length were discarded. Following clustering, downstream processing was carried out, as mentioned above, to build representative phylogenetic trees for each gene. Phylogenetic trees were visualised and annotated with metadata using the interactive tree of life (iTOL v5) tool [ 30 ]. For the genes with biochemically characterised motifs and domains, we analysed all the potential hits from the MOB genomes, plasmids, and metatranscriptomes to identify motifs and ascertain bona fide sequences. The motif and domain analyses were done using seqkit and the NCBI Conserved Domain Database (CDD), respectively. Growth of Methylosinus trichosporium (OB3b) with cerium and Ore Methylosinus trichosporium (OB3b) was grown in Nitrate Mineral Salts (NMS) [ 31 ] or dilute (DNMS) media and 20% (v/v) methane (CH 4 ) until the late exponential phase. Methane was added to the headspace by injection through the septum. All media were prepared using ultrapure water (18 MΩ cm) obtained from an Elga Purelab Classic Life Science water purification system (Veolia Water Technologies, High Wycombe, UK). The cells were harvested and washed twice in the growth medium, then used as the parent culture for the growth experiments. All experiments were conducted in 120 mL acid-washed glass serum vials, containing 20 mL of media, with a 100 mL headspace for the gases. The parent culture was grown in 20% (v/v) CH 4 , and four different treatments were set up: without added Ln, with CeCl 3 (25 µM), with mixed Ln ore (250 mg), and a no-cells treatment with ore (250 mg). Each treatment was performed in five biological replicates, incubated at 30°C with shaking at 150 rpm, and with an initial headspace methane concentration of 20% (v/v). The pre-concentrated ore contains a mixture of lanthanide oxides, including scandium and yttrium, with cerium dominating the composition (Table S3 ). The optical density (OD) of the cultures and CH 4 in the headspace was measured daily to monitor growth and CH 4 oxidation using a spectrophotometer (Clariostar microplate reader) at 540 nm and gas chromatography (Agilent 7890B equipped with a flame ionisation detector), respectively. For the gas chromatography, a FuSED-silica column TG-5MS (5% phenyl Methylpolysiloxane) was used. Both the front inlet and detector temperatures were set at 300 o C, with a front inlet pressure of 4.5 psi. Helium (greater than 99.9% purity) was used at a constant flow rate of 1.2 mL/min as the carrier gas. Proteomics analysis Cells were harvested at the end of the experiment through centrifugation, then resuspended in 1 mL of ddH 2 O and mixed with 200 µL 2X Laemmli buffer. Only three biological replicates were selected for each condition. Samples were boiled at 98°C for 15 min, after which 30 µL was loaded onto a precast NuPAGE Bis-Tris gel (Invitrogen) and electrophoresed for 5 min at 200 V. The gel was stained with Coomassie Brilliant Blue and destained overnight in ddH 2 O. Each band was excised, cut into small pieces, and subjected to in-gel digestion using trypsin (Roche). Peptides were extracted using a formic acid-acetonitrile solution (5%:25%, v/v), dried in a speed vacuum concentrator, and resuspended in acetonitrile-trifluoroacetate (2%:0.1%, v/v) for the nanoLC-ESI-MS/MS run. Peptide separation was performed using an Ultimate 3000 RSLCnano (Dionex-LC Packings) equipped with two C18 columns: an Acclaim PepMap µ-precolumn cartridge (300 µm i.d. x 5 mm, 5 µm, 100 Å; Thermo Fisher Scientific) and a Bruker nanoElute Forty analytical column (75 µm x 40 cm, 1.9 µm). Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The gradient program was as follows: 4–25% B over 36 min, 25–35% B over 10 min, 35–90% B over 3 min, followed by a 10 min re-equilibration at 4% B. The flow rate was maintained at 350 nL min-1. The Ultimate 3000 RSLCnano was coupled online to a hybrid timsTOF Pro mass spectrometer (Bruker Daltonics, Germany) via a CaptiveSpray nano-electrospray ion source [ 32 ]. The instrument operated in Data-Dependent Parallel Accumulation-Serial Fragmentation (PASEF) mode. Peptides were separated by ion mobility according to their collisional cross sections and charge states. Acquisition settings were as follows: mass range 100–1700 m/z, ion mobility range 1/K 0 (Start: 0.6 Vs/cm 2 ; End: 1.6 Vs/cm 2 ), ramp rate 9.42 Hz, and 100% duty cycle. Raw MS/MS files were searched against the M. trichosporium OB3b protein database using the MaxQuant software package. The label-free quantification (LFQ) was performed according to the framework described by Cox and Mann [ 33 ], with default parameters and the ‘match between runs' function enabled. Comparative proteomics analysis was performed using Perseus software (v1.6.5.0; MPI of Biochemistry). Statistical significance was assessed using a two-sample t-test, applying a false discovery rate (FDR) threshold of 0.01 and 0.05, and requiring a minimal log2 fold change of 2. Only proteins present in all replicates of at least one condition were considered to be considered valid. The proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE[ 34 ] partner repository with the dataset identifier PXD063434. The dataset was manually interrogated for changes in known lanthanide- and methanotrophy-related gene products. Functional assignment of gene products was performed using the KEGG Orthology (KO) database, based on the functional ortholog (K number) assignments of individual proteins in the IMG database. Results and Discussion We performed a comprehensive survey of the “lanthanome” in aerobic MOBs using a custom database of 179 MOB genomes that included 101 Gammaproteobacteria, 60 Alphaproteobacteria, 17 Verrucomicrobia and 1 Actinomycete (Figure 1a, Table S4). At the family level, the Methylococcaceae dominate with 97 representatives, while at the genus level, Methylomonas (39) show the highest representation, followed by Methylocystis (28). The key genes we surveyed encode the methanol dehydrogenases and known lanthanide transporter proteins (Lanmodulin, LanPepSy, LanA, and LutH), which together constitute the major known components of the lanthanome. We curated the hits to identify bona fide sequences that could be attributed to each functional class through phylogenetic trees, as well as domain and motif analyses. Distribution of methanol dehydrogenases in MOB genomes and sediment metatranscriptomes Our genome-wide survey using key biomarker genes revealed the distribution pattern of variants of lanthanide-dependent (XoxF1, XoxF2, XoxF3, and XoxF5) and calcium-dependent methanol dehydrogenases (MxaF ) (Table S4). A total of 175 out of the 179 MOB genomes in our database harboured at least one copy of the MDH, with several genomes possessing multiple copies of specific MDH variants, especially within the members of the Proteobacteria MOBs (Figure 1). The four MOB genomes that do not have any identifiable MDH include the recently characterised Actinobacterial MOB ( Candidatus M. methanotrophicum ) — the only Mycobacterium shown to oxidise methane and 3 Alphaproteobacterial genomes ( Methylovirgula sp. 4M-Z18, Methylocapsa sp. S129, and Methylocella sp. CPCC 101449) (Figure 1a). Candidatus M. methanotrophicum, though, does not possess any MDH; however, it contains a full suite of enzymes required for aerobic growth on CH 4 , including the soluble methane monooxygenase (sMMO) that oxidises CH 4 to methanol and alcohol dehydrogenase D, which may fulfil the role of MDH in other methanotrophs [15]. The gammaproteobacterial Methylogaea oryzae JCM 16910 is the only organism in our database with two copies of mxaF but none of the xoxF variants. A total of 154 mxaF and 288 xoxF hits, including multiple copies in single MOB genomes, were recovered, with the highest proportion of hits (71.9%) affiliated with xoxF5 and the lowest (3.8%) affiliated with xoxF1 (Figure 1b). No xoxF 4 variant was detected in MOB genomes, and it is well established that this clade occurs only in members within the family Methylophilaceae [35]. Most of the proteobacterial MOBs possess both mxaF and xoxF5 , consistent with the findings of Huang and colleagues [36]. This further confirms the metabolic potential of methanotrophs to switch between using lanthanides or calcium depending on the availability of either cofactor in their environment, commonly referred to as the “lanthanide switch”. The xoxF5 variant, the most prevalent and widely distributed variant among the xoxF genes in our survey, is harboured only by members of Proteobacterial MOBs (Figure S1). Most of the Alphaproteobacterial MOB genomes (56 out of 60 genomes) harboured the xoxF5 variant, with several genomes possessing more than one copy of the xoxF5 variant, e.g. Methylosinus sp. Ce-a6 harbours four xoxF5 variants, and 13 other MOB genomes, each possessing three copies of xoxF5 . These 13 genomes belong to the genera Methylocystis (7), Methylosinus (4), Methyloferulla (1) and Methylovirgula (1). In gammaproteobacterial MOBs, xoxF5 was detected in all genomes except in Methylogaea oryzae JCM 16910. Only three gammaproteobacterial genomes ( Methylospira mobilis Shm1, Methylospira mobilis SPMX and Methylococcus sp. EFPC2) harboured multiple copies of the xoxF5 gene, with each genome possessing two copies only (Table S4). The multiple copies of xoxF5 detected in the MOB genomes exhibit a patchy phylogenetic distribution pattern, with some copies clustering within the same clade and others grouping into distinct clades. This observed variation in the phylogenetic tree could provide insights into the potential evolutionary dynamics and divergent functional roles of the xoxF5 . In genomes, such as Methyloferula stellata AR4 and Methylovirgula sp. HY1 that harbour three copies of xoxF5, all clustered within the same clade, suggesting recent duplication events and potential for functional redundancy [37]. On the contrary, in other genomes, such as Methylosinus sp. Ce-a6 and Methylosinus sp. PW2, not all copies of xoxF5 were clustered together (Figure S1), potentially suggesting more ancient duplication events and subsequent divergence. This divergence may reflect adaptation to varied ecological niches, cofactor preferences, or substrate specificities [38,39]. The xoxF 2 variant was only detected in verrucomicrobial MOBs (Figure S2). In fact, all the Verrucomicrobiota harbour only XoxF2 as their MDH, except for four Verrucomicrobiota incertae sedis genomes (all belonging to the genus Methylacidimicrobium ), which possess both XoxF1 and XoxF2 variants. This suggests functional specialisation of XoxF to these organisms, enabling them to metabolise methanol under distinct environmental conditions or with different cofactors. XoxF1 was detected in five Alphaproteobacteria (three within the genus Methylocella, one Methylovirgula and one Methylocystis ) isolated from acidic soils, and the only four Verrucomicrobiota incertae sedis genomes isolated from acidophilic/thermophilic environments [40]. The distribution of xoxF1 , particularly in organisms present in acidic and high-temperature environments, suggests that the gene may confer ecological advantages to the organisms under such extreme conditions, playing a significant role in adaptation to pH and cofactor changes [4,35]. Only the Methylovirgula ligni BW863 has multiple copies of xoxF1 , possessing three copies, with two copies clustering together, while the other copy is grouped into a separate clade (Figure S3). Forty-five MOB genomes harboured the xoxF3 variant that includes 23 Alphaproteobacteria and 22 Gammaproteobacteria MOB. Six genomes possessed multiple copies: one alphaproteobacterial MOB, Methylocystis echinoides LMG27198, and five gammaproteobacterial MOBs belonging to the genera Methylocaldum (3) and Methylomonas (2). Although the multiple copies in each genome cluster distinctly into separate clades, the genes from members of the same genus cluster together (Figure S4). The lack of clustering of copies from the same organism suggests divergent evolution and possibly distinct functional roles of the different xoxF3 copies within individual genomes. Moreover, the gene copies that cluster closely with those from other organisms of the same genus indicate that horizontal gene transfer (HGT) may have occurred. This is common in MOB as they often exchange genetic material to adapt to various environmental conditions [41]. To understand the distribution of xoxF s and mxaF in the natural environment, assembled metatranscriptomes from Lake Washington sediment microbial communities [23] were analysed for the presence of MDH transcripts. A BlastP (1e-40) sequence similarity search on the IMG platform resulted in over 6800 hits. However, most sequences were very short, so a length cutoff of 500 was applied to exclude fragmented sequences. A total of 535 transcripts were retained for the final phylogenetic analysis. All xoxF variants were detected in the metatranscriptomes except xoxF2, highlighting that MOB XoxF-MDHs are expressed and actively involved in the methane oxidation pathway, i.e. catalysing the oxidation of methanol to formaldehyde. However, more than 50% of the xoxFs identified in the metatranscriptomes were affiliated with the xoxF4 clade, a group of MDH mainly found in non-MOBs ( Methylophilaceae ) (Figure 2a). This is unsurprising as Methylophilaceae are ubiquitous in the environment, including in freshwater ecosystems [42]. Studies in Lake Washington have also provided evidence of a cooperative metabolic relationship between Methylophilaceae and MOB [43,44]. The majority of xoxF metatranscripts are phylogenetically linked to MOB, particularly Methylobacter , a dominant MOB in freshwater environments. Notably, among the 94 xoxF5 metatranscripts, only two (belonging to Burkholderiales ) are not associated with MOB, accounting for just 2% of the total metatranscripts of this gene. Overall, our genome- and metatranscriptome-wide survey highlights the diverse distribution patterns and evolutionary dynamics of methanol dehydrogenases in MOBs, with clear lineage-specific preferences for specific MDH variants. The widespread occurrence of XoxF5 in proteobacterial MOBs, the specialisation of XoxF2 in Verrucomicrobiota, and the presence of XoxF1 in organisms that thrive in acidophilic and thermophilic environments underscore the ecological and functional adaptability of these enzymes. Distribution of lanthanide transporters in MOB genomes, plasmids, and metatranscriptomes The lanthanide-binding and transport proteins also show interesting patterns. While the lanthanide-binding protein LanM is only present in Alphaproteobacteria, the TonB-dependent receptor LanA and the Ln-binding protein LanPepSY were primarily found in the gammaproteobacterial MOB genomes. The LutH-like protein (also a TonB-dependent receptor) is more widely distributed and abundant than any other lanthanome candidate investigated, as it is found in all the MOB phyla (Figure 1b). LanA was identified in Methylovibrium buryatense [7] and CQW49_RS02145, a protein recently found in Methylosinus trichosporium OB3b homologous to the LutH of Methylorubrum extorquens AM1 [8] are the only lanthanide transport proteins identified in the MOBs characterised to date. Lanmodulin (LanM) was the first lanthanide-selective chelator to be discovered in M. extorquens , and it provided valuable insights into how Ln are selectively recognised and transported in methylotrophs [6]. Although it is similar to the calcium-binding protein calmodulin, as it possesses metal-binding EF hands, LanM was shown to uniquely respond to picomolar concentrations of all Ln 3+ while responding to Ca 2+ at millimolar concentrations – a remarkable 100-million-fold selectivity for Ln 3+ over Ca 2+ [6]. Our genome-wide survey detected the presence of LanM only in the MOB genomes belonging to the families Methylocystaceae and Beijerinkiaceae, which is consistent with the findings of Mattocks and colleagues [45]. We also identified LanM sequences in the plasmids most belonging to Bradyrhizobium and Methylobacterium (Figure 3a). The presence of this gene in plasmids suggests its potential role in the survival and adaptation of these organisms to specific environments, as well as the potential for horizontal transfer of the gene to other bacteria. Due to the close sequence relatedness of LanM and calmodulin, we performed a motif analysis to distinguish bona fide sequences from potential false positives. We used the identified motifs of the two biochemically characterised LanM from M. extorquens ( Mex ) and Hansschlegelia quercus ( Hans ) (Figure 3b) and created a single regular expression (regex) pattern (Figure 3b) for identifying LanM sequences. The motif analysis revealed that 17 MOB genome sequences (2 Methylocella and 15 Methylocystis ) contained motifs matching Mex- LanM, suggesting them as bona fide LanM. At the same time, only one metatranscript was identified to encode a bona fide LanM (Figure 3a). No sequences matched the Hans- LanM. Sequences lacking known motifs potentially represent LanM with novel or distinct motifs that diverge from those characterised in Mex-LanM. Lanpepsy (LanP) , a lanthanide-binding protein, was recently discovered in the obligate methylotroph Methylobaccillus flagellatus . Characterised by two PepSY domains, LanP represents the first member of the PepSY family shown to bind lanthanides [11]. Our survey (Figure 4a) revealed that LanP was predominantly detected within Methylococcales genomes belonging to the family Methylococcaceae , with a single hit identified in Methylothermaceae ( Methylohalobius crimeensis ). Additionally, five plasmid hits associated with the Methylococcaceae ( Methylomonas sp., Methylomicrobium sp., Methylobacter sp., and Methylomonas methanica ) were found to encode LanP. In sediment metatranscriptomes, we recovered 406 sequences, confirming their active expression in lake sediment. To verify the authenticity of the retrieved LanP sequences, we searched for the PepSY domain in all the curated 249 sequences (from genomes, metatranscriptomes, and plasmids) using the Conserved Domain Database (CDD). Notably, all the sequences had the two characteristic PepSY domains, each approximately 60 amino acids in length (Figure 4b). This finding suggests that while LanP is restricted to only a few MOB families, the gene is widespread and actively expressed in the environment, underscoring its ecological significance. We also analysed the TonB-dependent receptors LanA and LutH, first identified in Methylotuvimicrobium buryatense 5GB1C and M. extorquens AM1, respectively. Both genes were found to be crucial in controlling the lanthanide switch in these organisms, particularly in the lanthanide uptake system [7,10]. LanA from M. buryatense 5GB1C was the first lanthanum receptor identified in a methanotroph. Recently, Shiina and colleagues [8] Identified a lutH -like gene in Methylosinus trichosporium OB3b. Our work revealed that LanA is widely distributed in MOBs, predominantly within the Methylococcaceae family. We also detected two LanA genes from the family Crenotrichaceae and one each from Methylothermaceae , Methylocystaceae , and Beijerinckiaceae . Interestingly, this gene was also identified in metatranscriptomes related to Methylobacter , Methylomonas , and Methylotenera ; however, no LanA related to MOB was found in plasmids (Figure 5a). Similarly, the lutH -like gene was found in all the MOB families affiliated with LanA, including the Methylacidiphilaceae and unclassified Chromatiales (Figure 5b) . However, in both lanA and lutH -like genes retrieved, there is a pervasive phylogenetic incongruence, most likely due to horizontal gene transfer, as these genes are also found in the plasmids of other organisms. Impact of Cerium and Mixed Lanthanides on Methane Oxidation and Protein Expression in M. trichosporium OB3b. Rare earth elements (REE) are naturally present in the environment, where they predominantly occur as insoluble oxides. To explore how an MOB responds to this environmentally relevant form compared to the commonly used soluble laboratory form (CeCl 3 ), we assessed the growth of M. trichosporium OB3b on 20% CH 4 under three conditions: no added lanthanides (No Ln), with cerium (Ce), and with a lanthanide-rich ore. These treatments revealed distinct differences in bacterial response and growth in the presence and absence of these critical cofactors. Notably, we observed the longest lag phase in the ore treatment culture, followed by the Ce treatment, with the shortest lag phase in the No Ln control (Figure 6a). The duration of the lag phase provides insight into how the bacteria adapt to the presence and different Ln sources. This suggests that the bacteria require more time to express the genes necessary for the selection and mobilisation of specific metals from a mixture of similar elements, in which those elements may be in a less bioavailable form. Conceivably, this also enables them to overcome toxicity associated with the ore. In contrast, the cerium-only treatment shows a shorter lag phase, most likely because the bacteria are provided with a single cofactor and no additional, potentially inhibitory, components. The energy and resources required to mobilise cerium (Ce) are minimal, leading to a quicker adaptation. The shortest lag phase was observed in the No Ln control, likely because M. trichosporium OB3b is routinely cultured and maintained in our laboratory without the addition of lanthanides. Under this condition, the bacteria constitutively express and utilise the calcium-dependent MDH (MxaF) for methanol oxidation, making this condition the natural growth environment for the organism. Consequently, no additional energy is required for regulatory adjustments or enzyme switch to enable methane oxidation and growth in this treatment. Interestingly, despite the differences in lag phases across the treatments, the amount of methane oxidised over 98 hours is similar, with all treatments showing an average methane oxidation rate of approximately 0.08% CH 4 hr -1 . This indicates that in the presence of lanthanides, once the bacteria have acclimatised to the new conditions, their growth rate and methane oxidation capabilities become comparable or even exceed those observed in the No Ln treatment. Although the lanthanide-treated cultures exhibit a slower start, they achieve the same level of methane consumption within the same timeframe (approximately 98 h), indicating a higher effective oxidation rate once growth begins. This effect is particularly evident in the mixed lanthanide ore treatment, suggesting that when environmentally or geologically available lanthanides are present in sufficient quantities, the growth and methane oxidation of M. trichosporium OB3b proceed more rapidly than in their absence. Proteomic analysis revealed significant changes in protein expression in both the Ce and ore treatments compared to the control. A total of 1,414 proteins were detected, with 60 and 724 proteins significantly expressed in the Ce and ore treatments, respectively (Table S5). The 12-fold increase in significantly expressed proteins in the ore treatment compared to the Ce-only treatment is likely attributable to the complex nature and composition of the ore. The response of M. trichosporium OB3b to the ore is likely not only a response to the provision of lanthanides in a more complex and less bioavailable form but also a response to other metals in the ore, resulting in the induction of various proteins, including those associated with stress response and chemotaxis. This response is necessary for the organism to adapt and thrive in its environment. In the Ce-only treatment, among the 60 significantly expressed proteins, 33 were upregulated, and 27 were downregulated. In the ore treatment, 144 proteins were upregulated, while 580 were downregulated. Our genome analysis of M. trichosporium OB3b revealed the presence of genes such as mxaF, xoxF3 , xoxF5, lanM , and lutH -like genes. Except for LanM, all these proteins were expressed in both the Ce and ore treatments. Furthermore, the lanthanide-dependent enzymes XoxF3 and XoxF5 were upregulated, while the calcium-dependent MxaF and the LutH-like protein were downregulated under both conditions (Figure 6b). The gene encoding MxaF is in a 12-gene cluster implicated in calcium-dependent MDH activity, eight of which (in addition to mxaF ) were downregulated under both conditions. The protein MxaJ, which was upregulated under both conditions, is not part of this cluster but encoded by a gene adjacent to xoxF3 . The inverse expression/regulation of MxaF and XoxF and their associated genes in response to lanthanide bioavailability strongly suggests that XoxF lanthanide-binding proteins play a critical role in the utilisation of lanthanides in M. trichosporium OB3b. The inverse regulation of MxaF and XoxF due to lanthanide availability has been previously demonstrated [46]. Interestingly, LanM, which is known to respond to picomolar concentrations of lanthanides in obligate methylotrophs, was not expressed in the presence of Ce or ore treatments. This suggests that while M. trichosporium OB3b possesses the lanM gene, it may not be the primary gene responsible for lanthanide acquisition and trafficking in this bacterium under the growth conditions we have tested. Recent studies by Shiina et al. (2023) demonstrated that the LutH-like TonB-dependent receptor (CQW49_RS02145) is required for the expression of XoxF in the presence of cerium in a laboratory-adapted strain of M. trichosporium OB3b [8]. These findings imply a crucial role for this gene in lanthanide uptake across the outer membrane. However, in our study, the LutH-like gene (MettrDRAFT_0198) was downregulated in both the Ce and ore treatments. This observation aligns with findings by Gu and Semrau [47], who reported downregulation of the same gene (referred to as ADVE02_v2_10208) in the presence of cerium and the absence of copper. Such downregulation of the LutH-like gene may be due to the high concentration of Ce, as metal ion uptake systems are typically downregulated when intracellular metal levels reach sufficient concentrations to maintain homeostasis [48,49]. Our findings are consistent with MettrDRAFT_0198 LutH-like as a component of a primary route for lanthanide uptake and trafficking in M. trichosporium OB3b. Its downregulation under elevated Ce conditions supports a model in which the bacterium modulates uptake systems to avoid excess metal accumulation, thus maintaining intracellular metal homeostasis. The large number of proteins that are differently affected by the two lanthanide sources, including some 48 transport-associated proteins and 19 putative molecular chaperones, may indicate proteins that are necessary for liberating lanthanides from geological sources and growing in the presence of the stresses, lanthanide-related and others, that such materials present. Among the proteins significantly upregulated in both Ce and ore treatments, we identified a TonB-dependent siderophore receptor (MettrDRAFT_4452) and an adjacent cyclic peptide export ABC transporter (MettrDRAFT_4451). This co-upregulation suggests a coordinated role in lanthanide acquisition, potentially through a lanthanophore-mediated mechanism similar to bacterial siderophore-iron uptake systems. Recent work by Juma et al. (2022) demonstrated that Methylobacterium aquaticum strain 22A utilises a siderophore to solubilise lanthanides, facilitating their uptake through a TonB-dependent receptor. This raises the possibility that M. trichosporium OB3b employs a similar strategy, where the cyclic peptide export ABC transporter secretes metal-chelating peptides that enhance lanthanide solubilisation, while the TonB-dependent receptor mediates uptake of the resulting lanthanide-peptide complex [50]. This system may represent an important alternative lanthanide-scavenging strategy in M. trichosporium OB3b, distinct from the previously characterised LutH-like transport pathway. Conclusion The lanthanome is distributed across the MOB phyla, with the lanthanoenzyme (MDH) found across all phyla. However, the distribution of the MDH variants (XoxFs) is skewed, favouring XoxF5, where most proteobacterial MOBs have multiple copies. The lanthanide transporters are also not evenly distributed in MOBs. While LanM was only detected in alphaproteobacterial methanotrophs, LanA and LanPepSY were primarily found in the gammaproteobacterial MOB, mainly within the family Methylococcaceae . The lutH -like gene showed a wide distribution in all MOBs. The difference in the presence and distribution of the lanthanome, especially lanthanide transporters, strongly indicates that the Ln transport system in MOBs is not universal; different methanotrophs employ different proteins for acquiring, trafficking, and utilising Ln from their environment. The existence of a specific lanthanome that is identifiable in metatranscriptome data confirms MOB-lanthanides interaction and activities in the environment. We also found some lanthanide transporters in plasmids, suggesting their importance in the organisms’ evolution, adaptability, and survival within specific environmental niches. In M. trichosporium OB3b, the presence of cerium (Ce) and mixed lanthanides led to distinct methane oxidation and proteomic responses. Although the lag phase differed across treatments, the methane oxidation rate was similar (0.08% CH 4 hr − 1 ) once the bacteria acclimatised. Proteomic analysis revealed a greater number of significantly expressed proteins in the ore treatment compared to cerium alone, suggesting that the ore’s complex composition induced broader stress and adaptation responses. Key lanthanide-binding enzymes, including XoxF3 and XoxF5, were upregulated, while MxaF was downregulated, reflecting the “lanthanide switch” phenomenon. Interestingly, LanM, despite being present in the genome, was not detected in either treatment, suggesting that it is not the primary protein for lanthanide acquisition in M. trichosporium OB3b. The downregulation of the lutH -like gene (MettrDRAFT_0198) under both treatments may indicate that it plays a key role in lanthanide uptake that requires its downregulation to maintain metal homeostasis when intracellular lanthanide concentrations are sufficient. Overall, understanding the distribution of the lanthanome within MOBs is crucial for comprehending methane capture and utilisation in various environments. This knowledge also serves as a valuable resource for exploring lanthanome genes within MOB genomes or the environment, facilitating their potential application in lanthanide recovery from geological sources and other aspects of biotechnology. Abbreviations Ln Lanthanides MDH Methanol dehydrogenase MOB Methane oxidising bacteria LanM Lanmodulin LanP LanPepsy MAGs Metagenome assembled genomes HGT Horizontal gene transfer Declarations Author Contribution D.K. conceptualised the study; D.K. and Y.C. designed the experiments; P.N. provided material; S.U.D. and E.S. performed the experiments; S.U.D., A.A., E.S. and T.J.S. performed the analyses; S.U.D. wrote the manuscript with help from all co-authors. Acknowledgements We are grateful to the Natural Environmental Research Council for funding (NE/X005062; NE/X005119) Data Availability All data supporting the findings of this study are available within the paper and its Supplementary Information. 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Supplementary Files Supportinginformation.docx TableS1Customdatabase.xlsx TableS4Lanthanomecounttable.xlsx Cite Share Download PDF Status: Published Journal Publication published 29 Sep, 2025 Read the published version in Environmental Microbiome → Version 1 posted Editorial decision: Revision requested 03 Jul, 2025 Reviews received at journal 11 Jun, 2025 Reviewers agreed at journal 30 May, 2025 Reviews received at journal 29 May, 2025 Reviewers agreed at journal 27 May, 2025 Reviewers agreed at journal 25 May, 2025 Reviewers invited by journal 20 May, 2025 Editor assigned by journal 07 May, 2025 Submission checks completed at journal 03 May, 2025 First submitted to journal 29 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6350491","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":459566115,"identity":"393e4bbf-46e1-41a4-a0ab-58e16ad63878","order_by":0,"name":"Shamsudeen Umar Dandare","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYHACAwYGNiDF3sDADBFgI1YLzwGStUgkEKlFvoF54+eCMrt8fsnH26QLGOzkGSTSEvBbcYCtWHrGuWTLmbPTyqRnMCQbNkikHSDgKh4Dad42ZgOD2zlm0jwMzAkMEukNBBzGY/ybt63ewP7mGZCWesJaGA7wmAFtOWxgIMED0nIYqIWQww6zlVnznDtuIHEmrdh6hsFxwzaeZwn4HdbevPk2T1m1AX/74Y23Cyqq5fnZ0wzwO4wZyUZ4HBENCBg+CkbBKBgFIxYAAB54NpHnPvuwAAAAAElFTkSuQmCC","orcid":"","institution":"Queen’s University Belfast","correspondingAuthor":true,"prefix":"","firstName":"Shamsudeen","middleName":"Umar","lastName":"Dandare","suffix":""},{"id":459566116,"identity":"57179c61-d69c-4c0e-b5f1-517dbc9ab6a9","order_by":1,"name":"Alexander Allenby","email":"","orcid":"","institution":"Queen’s University Belfast","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Allenby","suffix":""},{"id":459566117,"identity":"bc24a946-cf23-4b72-a23a-2f549aca4e02","order_by":2,"name":"Eleonora Silvano","email":"","orcid":"","institution":"University of Warwick","correspondingAuthor":false,"prefix":"","firstName":"Eleonora","middleName":"","lastName":"Silvano","suffix":""},{"id":459566118,"identity":"1ad3b909-3601-4e7b-8c14-f3be2dd553a9","order_by":3,"name":"Peter Nockemann","email":"","orcid":"","institution":"Queen’s University Belfast","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Nockemann","suffix":""},{"id":459566119,"identity":"20691ab9-28ea-43d9-9490-449a255c5906","order_by":4,"name":"Yin Chen","email":"","orcid":"","institution":"University of Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Yin","middleName":"","lastName":"Chen","suffix":""},{"id":459566120,"identity":"120bb088-593a-4022-91ab-9f0215e386f2","order_by":5,"name":"Thomas J Smith","email":"","orcid":"","institution":"Sheffield Hallam University","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"J","lastName":"Smith","suffix":""},{"id":459566121,"identity":"b0062061-0c18-4643-bd92-01a2cfc2b0e2","order_by":6,"name":"Deepak Kumaresan","email":"","orcid":"","institution":"Queen’s University Belfast","correspondingAuthor":false,"prefix":"","firstName":"Deepak","middleName":"","lastName":"Kumaresan","suffix":""}],"badges":[],"createdAt":"2025-04-01 07:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6350491/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6350491/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40793-025-00776-5","type":"published","date":"2025-09-29T15:57:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83298295,"identity":"4fa35cd1-9f3a-49ac-bf89-23974944354c","added_by":"auto","created_at":"2025-05-22 14:28:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":283576,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenomic construction of MOB genes and distribution of methanol dehydrogenases and lanthanide-dependent genes in MOB.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003e Phylogenomics tree constructed using 1 MOB genomes with heatmap showing the presence or absence (empty space) of genes encoding for proteins of the lanthanome. The tree was annotated using ITOL, and the colored ranges are according to the bacteria family. After completing the HMM search against genomes, a presence/absence table was created, and a heatmap was added to the tree for each gene. \u003cstrong\u003eB) \u003c/strong\u003eStacked bar chart showing hits of each gene found in MOB. The hits are classified based on class level. The numbers in the squares show the number of genomes found to have multiple copies of the gene.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6350491/v1/541d75bc35109de48615ebab.png"},{"id":83297348,"identity":"77690628-9984-4732-87b9-6ace828d6bd4","added_by":"auto","created_at":"2025-05-22 14:20:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":316755,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of MxaF and XoxF in MOB genomes and metatranscriptomes. A) \u003c/strong\u003eCircular phylogenetic tree constructed showing all MDH XoxF variants from MOB genomes (grey strips), and lake sediment metatranscriptomes (MTR, red strips). Hits for the MxaF sequences, related alcohol dehydrogenases, and XoxF4 are collapsed. \u003cstrong\u003eB)\u003c/strong\u003e The rectangular phylogenetic tree shows all clades collapsed, with the number of sequences in each clade indicated in brackets; G and M denote the number of genome and metatranscriptome hits, respectively. The tree was annotated using ITOL, and the coloured ranges are according to the \u003cem\u003eXoxF\u003c/em\u003e variants.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6350491/v1/24785197a210cc327c9570c2.png"},{"id":83300156,"identity":"65d02800-34d9-4872-8e45-936a0f4aa1b5","added_by":"auto","created_at":"2025-05-22 14:52:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":342943,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLanmodulin in MOB genomes, plasmids, and metatranscriptomes. A) \u003c/strong\u003eCircular phylogenetic tree constructed showing all LanM hits from MOB genomes (coloured ranges), plasmids (blue strips), lake sediment metatranscriptomes (red strips), and reference sequences (grey strips). Red circles indicate bona fide LanM sequences matching the \u003cem\u003eMex\u003c/em\u003e-LanM motif. \u003cstrong\u003eB)\u003c/strong\u003e The Mex- and Hans-LanM motifs from the biochemically characterised proteins [45]. The tree was annotated using ITOL, and the coloured ranges are according to the family of the MOBs.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6350491/v1/34e3ff5c9bff71a5a9e3d95f.png"},{"id":83297358,"identity":"5822c0fc-3fcd-4966-bcd9-664f9402b481","added_by":"auto","created_at":"2025-05-22 14:20:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":397522,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of LanA and LutH in MOB genomes, plasmids, and metatranscriptomes. \u003c/strong\u003eCircular phylogenetic tree constructed showing all retrieved \u003cstrong\u003eA)\u003c/strong\u003e LanA sequences and \u003cstrong\u003eB) \u003c/strong\u003e\u003cem\u003elutH\u003c/em\u003e-like sequences from MOB genomes (coloured ranges), plasmids (blue strips), lake sediment metatranscriptomes (red strips), and reference sequences (grey strips). The trees were annotated using ITOL, and the coloured ranges are according to the family of the MOBs.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6350491/v1/117fde43f67b721f5be69c30.png"},{"id":83298730,"identity":"dcf91372-7ca7-4678-88f4-a5d9e80d43e8","added_by":"auto","created_at":"2025-05-22 14:36:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":67132,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of Ln on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. trichosporium\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e OB3b.\u003c/strong\u003e The methane oxidation rate of \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b with and without lanthanides (cerium and mixed lanthanides). \u003cstrong\u003eB) \u003c/strong\u003eHeatmap showing differential expression of lanthanide-binding proteins in cerium and ore-treated \u003cem\u003eM. trichosporium \u003c/em\u003eOB3b\u003cem\u003e. \u003c/em\u003eThe red color shows upregulated proteins, blue indicates downregulated proteins, while white means no expression. The numbers represent the Log\u003csub\u003e2 \u003c/sub\u003efold change.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6350491/v1/774fb6e0f7671b4b7d8ff446.png"},{"id":92883794,"identity":"c58c744c-0d17-4ede-9b01-3945f75ca172","added_by":"auto","created_at":"2025-10-06 16:09:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2282571,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6350491/v1/8e55016c-5001-43f5-908c-d291e4637cb7.pdf"},{"id":83298299,"identity":"85c5978d-8a1e-40c5-aac9-9134580c6aef","added_by":"auto","created_at":"2025-05-22 14:28:34","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4245859,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6350491/v1/b9954b094c499f944a3ea243.docx"},{"id":83297353,"identity":"f7d2a40b-30ed-4ed5-bbd3-39b258da50c9","added_by":"auto","created_at":"2025-05-22 14:20:34","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":113501,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1Customdatabase.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6350491/v1/36043982ff1d50e88e7f50cf.xlsx"},{"id":83298296,"identity":"dada1727-23c5-430f-a0dc-21dfe0901fe5","added_by":"auto","created_at":"2025-05-22 14:28:34","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":70797,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4Lanthanomecounttable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6350491/v1/4b382f225447c969d4b065c1.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Diversity and distribution of the lanthanome in aerobic methane-oxidising bacteria","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe discovery in 2011 of bacteria capable of accumulating and utilising lanthanides (Ln) established the biological relevance of these elements. The first evidence of a biological role for Ln came from the identification of a lanthanoenzyme\u0026mdash;an Ln-dependent methanol dehydrogenase (MDH) found in several methylotrophs[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and methanotrophs [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This enzyme, which catalyses the oxidation of methanol to formaldehyde, contains a pyrroloquinoline quinone (PQQ) redox cofactor that coordinates the Ln ion [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The discovery and subsequent characterisation of lanthanoenzymes led to the identification of additional proteins involved in Ln sensing, acquisition, and transport, collectively referred to as the \u0026ldquo;lanthanome\u0026rdquo;. Cotruvo and colleagues identified lanmodulin (LanM), a highly selective Ln-binding protein structurally similar to the calcium-binding protein calmodulin [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Two distinct TonB-dependent receptors\u0026mdash;LanA and LutH\u0026mdash;were identified as critical for the uptake of lanthanum and the lanthanide switch in gammaproteobacterial[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and alphaproteobacterial methanotrophs [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], respectively. The \u003cem\u003elutH\u003c/em\u003e gene is part of a recently characterised 10-gene \u003cem\u003elut\u003c/em\u003e cluster in \u003cem\u003eMethylorubrum extorquens\u003c/em\u003e AM1 and the closely related phyllosphere bacterium, PA1 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Recent research on the lanthanome in the obligate model methylotroph \u003cem\u003eMethylobacillus flagellatus\u003c/em\u003e identified a 19 kDa periplasmic protein comprising two characteristic PepSY domains, referred to as lanpepsy (LanP) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResearch on the lanthanome has predominantly focused on methanol-oxidising methylotrophs, despite the widespread occurrence of Ln-utilising methane-oxidising bacteria (MOB)\u0026mdash;a key player in global methane cycling and a promising microbial chassis for driving one-carbon biotechnology. Yet, there is a knowledge gap on the distribution and diversity of lanthanome in MOB.\u003c/p\u003e \u003cp\u003eHere, we conducted a comprehensive survey of the genomes of aerobic MOB, plasmids, and metatranscriptomes from methane-rich lake sediments to investigate the distribution and diversity of Ln-dependent enzymes and transporters in these bacteria. In addition, we performed growth assays and proteomic analyses to compare the impact of cerium chloride (CeCl₃), widely used in laboratory experiments and mixed lanthanide ore on the activity and physiology of an alphaproteobacterial MOB \u003cem\u003eMethylosinus trichosporium\u003c/em\u003e OB3b.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAcquisition of MOB genomes, plasmids, and environmental metatranscripts\u003c/h2\u003e \u003cp\u003eA total of 179 known aerobic MOB genomes were downloaded from the National Centre for Biotechnology Information (NCBI) database (accessed in October 2023) and concatenated to make a custom MOB database. The representative MOB database comprises 175 characterised genomes from the RefSeq database, three metagenome-assembled genomes (MAGs) representing atmospheric methane oxidisers [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and a recently reported MOB belonging to the genus \u003cem\u003eMycobacterium\u003c/em\u003e reported to harbour only the soluble methane monooxygenase enzyme [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. All information regarding the custom database, including genome accession numbers, metadata (source environment), and genome completeness determined using the CheckM tool (v1.0.12), is detailed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. A phylogenomic tree of MOB genomes was generated using the GToTree workflow (v1.7.00). The GToTree pipeline predicts genes using Prodigal (v2.6.3), searches for a panel of 74 bacterial single copy marker genes (SCGs) from genomes using HMMER3 (v3.3.2), aligns the retrieved genes with Muscle (v5.1.linux64), trims those alignments with TrimAl (v1.4.rev15), concatenates the trimmed alignments and then performs phylogenetic reconstruction with FastTree (v2.1.11) [\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo identify the presence of Ln-related genes in plasmids to understand their distribution within MOB taxa, we used the IMG/PR database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://img.jgi.doe.gov\u003c/span\u003e\u003cspan address=\"https://img.jgi.doe.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In order to assess the expression of MOB-specific genes in the environment, we used the assembled metatranscriptomes of Lake Washington microbial communities (Study name: Freshwater Sediment Methanotrophic Microbial Communities from Lake Washington under Simulated Oxygen Tension; [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] available via the IMG portal.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSequence similarity search, curation, and phylogenetic tree construction\u003c/h3\u003e\n\u003cp\u003eIn order to retrieve homologues for specific genes, we carried out searches (E-value cut off of 1e-30) with specific hidden Markov model (HMM) profiles in the custom MOB genomes database using the HMMER tool (v3.4) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. HMM profiles from KOFAM were used for MxaF and XoxF, and custom HMMs were developed for the remaining genes using Refseq gene sequences [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Homologues flagged by the HMM searches were retrieved from the MOB protein database by seqkit (v2.9.0; [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]). The recovered sequences were then concatenated with curated reference sequences and were subsequently aligned using Clustal Omega (v1.2.4) with default parameters [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Conserved regions in the alignments were identified and trimmed using trimAl (v1.5. rev0) with the option \u0026ndash;automated1 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and finally, a phylogenetic tree of the resulting aligned and trimmed sequences was constructed using FastTree (v2.1.11) with 1,000 replicates for bootstrap [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The phylogenetic trees were then used for manual curation of the retrieved homologues.\u003c/p\u003e \u003cp\u003eHomologues of specific Ln-dependent enzymes and transporters in plasmids and lake sediment metatranscriptomes databases were retrieved using the BlastP (E-value cut off 1e-30; as described previously in [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]) within the IMG portal (Details on specific query sequences are included in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). To remove any redundancy and for ease of analysis, candidate hits were clustered at 100% using CD-HIT [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Hits were curated for sequence length using CD-HIT, and sequences shorter than the specified length were discarded. Following clustering, downstream processing was carried out, as mentioned above, to build representative phylogenetic trees for each gene. Phylogenetic trees were visualised and annotated with metadata using the interactive tree of life (iTOL v5) tool [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor the genes with biochemically characterised motifs and domains, we analysed all the potential hits from the MOB genomes, plasmids, and metatranscriptomes to identify motifs and ascertain \u003cem\u003ebona fide\u003c/em\u003e sequences. The motif and domain analyses were done using seqkit and the NCBI Conserved Domain Database (CDD), respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth of\u003c/b\u003e \u003cb\u003eMethylosinus trichosporium\u003c/b\u003e \u003cb\u003e(OB3b) with cerium and Ore\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eMethylosinus trichosporium\u003c/em\u003e (OB3b) was grown in Nitrate Mineral Salts (NMS) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] or dilute (DNMS) media and 20% (v/v) methane (CH\u003csub\u003e4\u003c/sub\u003e) until the late exponential phase. Methane was added to the headspace by injection through the septum. All media were prepared using ultrapure water (18 MΩ cm) obtained from an Elga Purelab Classic Life Science water purification system (Veolia Water Technologies, High Wycombe, UK). The cells were harvested and washed twice in the growth medium, then used as the parent culture for the growth experiments. All experiments were conducted in 120 mL acid-washed glass serum vials, containing 20 mL of media, with a 100 mL headspace for the gases. The parent culture was grown in 20% (v/v) CH\u003csub\u003e4\u003c/sub\u003e, and four different treatments were set up: without added Ln, with CeCl\u003csub\u003e3\u003c/sub\u003e (25 \u0026micro;M), with mixed Ln ore (250 mg), and a no-cells treatment with ore (250 mg). Each treatment was performed in five biological replicates, incubated at 30\u0026deg;C with shaking at 150 rpm, and with an initial headspace methane concentration of 20% (v/v). The pre-concentrated ore contains a mixture of lanthanide oxides, including scandium and yttrium, with cerium dominating the composition (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The optical density (OD) of the cultures and CH\u003csub\u003e4\u003c/sub\u003e in the headspace was measured daily to monitor growth and CH\u003csub\u003e4\u003c/sub\u003e oxidation using a spectrophotometer (Clariostar microplate reader) at 540 nm and gas chromatography (Agilent 7890B equipped with a flame ionisation detector), respectively. For the gas chromatography, a FuSED-silica column TG-5MS (5% phenyl Methylpolysiloxane) was used. Both the front inlet and detector temperatures were set at 300 \u003csup\u003eo\u003c/sup\u003eC, with a front inlet pressure of 4.5 psi. Helium (greater than 99.9% purity) was used at a constant flow rate of 1.2 mL/min as the carrier gas.\u003c/p\u003e\n\u003ch3\u003eProteomics analysis\u003c/h3\u003e\n\u003cp\u003eCells were harvested at the end of the experiment through centrifugation, then resuspended in 1 mL of ddH\u003csub\u003e2\u003c/sub\u003eO and mixed with 200 \u0026micro;L 2X Laemmli buffer. Only three biological replicates were selected for each condition. Samples were boiled at 98\u0026deg;C for 15 min, after which 30 \u0026micro;L was loaded onto a precast NuPAGE Bis-Tris gel (Invitrogen) and electrophoresed for 5 min at 200 V. The gel was stained with Coomassie Brilliant Blue and destained overnight in ddH\u003csub\u003e2\u003c/sub\u003eO. Each band was excised, cut into small pieces, and subjected to in-gel digestion using trypsin (Roche).\u003c/p\u003e \u003cp\u003ePeptides were extracted using a formic acid-acetonitrile solution (5%:25%, v/v), dried in a speed vacuum concentrator, and resuspended in acetonitrile-trifluoroacetate (2%:0.1%, v/v) for the nanoLC-ESI-MS/MS run. Peptide separation was performed using an Ultimate 3000 RSLCnano (Dionex-LC Packings) equipped with two C18 columns: an Acclaim PepMap \u0026micro;-precolumn cartridge (300 \u0026micro;m i.d. x 5 mm, 5 \u0026micro;m, 100 \u0026Aring;; Thermo Fisher Scientific) and a Bruker nanoElute Forty analytical column (75 \u0026micro;m x 40 cm, 1.9 \u0026micro;m). Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The gradient program was as follows: 4\u0026ndash;25% B over 36 min, 25\u0026ndash;35% B over 10 min, 35\u0026ndash;90% B over 3 min, followed by a 10 min re-equilibration at 4% B. The flow rate was maintained at 350 nL min-1. The Ultimate 3000 RSLCnano was coupled online to a hybrid timsTOF Pro mass spectrometer (Bruker Daltonics, Germany) via a CaptiveSpray nano-electrospray ion source [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The instrument operated in Data-Dependent Parallel Accumulation-Serial Fragmentation (PASEF) mode. Peptides were separated by ion mobility according to their collisional cross sections and charge states. Acquisition settings were as follows: mass range 100\u0026ndash;1700 m/z, ion mobility range 1/K\u003csub\u003e0\u003c/sub\u003e (Start: 0.6 Vs/cm\u003csup\u003e2\u003c/sup\u003e; End: 1.6 Vs/cm\u003csup\u003e2\u003c/sup\u003e), ramp rate 9.42 Hz, and 100% duty cycle.\u003c/p\u003e \u003cp\u003eRaw MS/MS files were searched against the \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b protein database using the MaxQuant software package. The label-free quantification (LFQ) was performed according to the framework described by Cox and Mann [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], with default parameters and the \u0026lsquo;match between runs' function enabled. Comparative proteomics analysis was performed using Perseus software (v1.6.5.0; MPI of Biochemistry). Statistical significance was assessed using a two-sample t-test, applying a false discovery rate (FDR) threshold of 0.01 and 0.05, and requiring a minimal log2 fold change of 2. Only proteins present in all replicates of at least one condition were considered to be considered valid. The proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] partner repository with the dataset identifier PXD063434.\u003c/p\u003e \u003cp\u003eThe dataset was manually interrogated for changes in known lanthanide- and methanotrophy-related gene products. Functional assignment of gene products was performed using the KEGG Orthology (KO) database, based on the functional ortholog (K number) assignments of individual proteins in the IMG database.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eWe performed a comprehensive survey of the \u0026ldquo;lanthanome\u0026rdquo; in aerobic MOBs using a custom database of 179 MOB genomes that included 101 Gammaproteobacteria, 60 Alphaproteobacteria, 17 Verrucomicrobia and 1 Actinomycete (Figure 1a, Table S4). At the family level, the Methylococcaceae dominate with 97 representatives, while at the genus level, \u003cem\u003eMethylomonas\u0026nbsp;\u003c/em\u003e(39) show the highest representation, followed by \u003cem\u003eMethylocystis\u003c/em\u003e (28). The key genes we surveyed encode the methanol dehydrogenases and known lanthanide transporter proteins (Lanmodulin, LanPepSy, LanA, and LutH), which together constitute the major known components of the lanthanome. We curated the hits to identify \u003cem\u003ebona fide\u003c/em\u003e sequences that could be attributed to each functional class through phylogenetic trees, as well as domain and motif analyses.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eDistribution of methanol dehydrogenases in MOB genomes and sediment metatranscriptomes\u003c/h2\u003e\n\u003cp\u003eOur genome-wide survey using key biomarker genes revealed the distribution pattern of variants of lanthanide-dependent (XoxF1, XoxF2, XoxF3, and XoxF5) and calcium-dependent methanol dehydrogenases (MxaF\u003cem\u003e)\u003c/em\u003e (Table S4). A total of 175 out of the 179 MOB genomes in our database harboured at least one copy of the MDH, with several genomes possessing multiple copies of specific MDH variants, especially within the members of the \u003cem\u003eProteobacteria\u0026nbsp;\u003c/em\u003eMOBs (Figure 1). The four MOB genomes that do not have any identifiable MDH include the recently characterised Actinobacterial MOB (\u003cem\u003eCandidatus M. methanotrophicum\u003c/em\u003e)\u0026nbsp;\u0026mdash;\u0026nbsp;the only \u003cem\u003eMycobacterium\u003c/em\u003e shown to oxidise methane and 3 Alphaproteobacterial genomes (\u003cem\u003eMethylovirgula\u003c/em\u003e sp. 4M-Z18, \u003cem\u003eMethylocapsa\u003c/em\u003e sp. S129, and \u003cem\u003eMethylocella\u003c/em\u003e sp. CPCC 101449) (Figure 1a). \u003cem\u003eCandidatus\u003c/em\u003e M. methanotrophicum, though, does not possess any MDH; however, it contains a full suite of enzymes required for aerobic growth on CH\u003csub\u003e4\u003c/sub\u003e, including the soluble methane monooxygenase (sMMO) that oxidises CH\u003csub\u003e4\u0026nbsp;\u003c/sub\u003eto methanol and alcohol dehydrogenase D, which may fulfil the role of MDH in other methanotrophs [15]. The gammaproteobacterial \u003cem\u003eMethylogaea oryzae\u003c/em\u003e JCM 16910 is the only organism in our database with two copies of \u003cem\u003emxaF\u003c/em\u003e but none of the \u003cem\u003exoxF\u003c/em\u003e variants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA total of 154 \u003cem\u003emxaF\u0026nbsp;\u003c/em\u003eand 288 \u003cem\u003exoxF\u003c/em\u003e hits, including multiple copies in single MOB genomes, were recovered, with the highest proportion of hits (71.9%) affiliated with \u003cem\u003exoxF5\u003c/em\u003e and the lowest (3.8%) affiliated with \u003cem\u003exoxF1\u0026nbsp;\u003c/em\u003e(Figure 1b). No \u003cem\u003exoxF\u003c/em\u003e4 variant was detected in MOB genomes, and it is well established that this clade occurs only in members within the family Methylophilaceae [35]. Most of the proteobacterial MOBs possess both \u003cem\u003emxaF\u003c/em\u003e and \u003cem\u003exoxF5\u003c/em\u003e, consistent with the findings of Huang and colleagues [36]. This further confirms the metabolic potential of methanotrophs to switch between using lanthanides or calcium depending on the availability of either cofactor in their environment, commonly referred to as the \u0026ldquo;lanthanide switch\u0026rdquo;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003exoxF5\u003c/em\u003e variant, the most prevalent and widely distributed variant among the \u003cem\u003exoxF\u003c/em\u003e genes in our survey, is harboured only by members of Proteobacterial\u003cem\u003e\u0026nbsp;\u003c/em\u003eMOBs (Figure S1). Most of the Alphaproteobacterial MOB genomes (56 out of 60 genomes) harboured the \u003cem\u003exoxF5\u003c/em\u003e variant, with several genomes possessing more than one copy of the \u003cem\u003exoxF5\u003c/em\u003e variant, e.g. \u003cem\u003eMethylosinus\u0026nbsp;\u003c/em\u003esp. Ce-a6 harbours four \u003cem\u003exoxF5\u003c/em\u003e variants, and 13 other MOB genomes, each possessing three copies of \u003cem\u003exoxF5\u003c/em\u003e. These 13 genomes belong to the genera \u003cem\u003eMethylocystis\u003c/em\u003e (7), \u003cem\u003eMethylosinus\u003c/em\u003e (4), \u003cem\u003eMethyloferulla\u003c/em\u003e (1) and \u003cem\u003eMethylovirgula\u003c/em\u003e (1). In gammaproteobacterial MOBs, \u003cem\u003exoxF5\u0026nbsp;\u003c/em\u003ewas detected in all genomes except in \u003cem\u003eMethylogaea oryzae\u003c/em\u003e JCM 16910. Only three gammaproteobacterial genomes (\u003cem\u003eMethylospira mobilis\u003c/em\u003e Shm1, \u003cem\u003eMethylospira mobilis\u003c/em\u003e SPMX and \u003cem\u003eMethylococcus\u0026nbsp;\u003c/em\u003esp. EFPC2) harboured multiple copies of the \u003cem\u003exoxF5\u003c/em\u003e gene, with each genome possessing two copies only (Table S4). The multiple copies of \u003cem\u003exoxF5\u003c/em\u003e detected in the MOB genomes exhibit a patchy phylogenetic distribution pattern, with some copies clustering within the same clade and others grouping into distinct clades. This observed variation in the phylogenetic tree could provide insights into the potential evolutionary dynamics and divergent functional roles of the \u003cem\u003exoxF5\u003c/em\u003e. In genomes, such as \u003cem\u003eMethyloferula stellata\u0026nbsp;\u003c/em\u003eAR4 and \u003cem\u003eMethylovirgula\u0026nbsp;\u003c/em\u003esp. HY1\u003cem\u003e\u0026nbsp;\u003c/em\u003ethat harbour three copies of \u003cem\u003exoxF5,\u003c/em\u003e all clustered within the same clade, suggesting recent duplication events and potential for functional redundancy [37]. On the contrary, in other genomes, such as \u003cem\u003eMethylosinus\u0026nbsp;\u003c/em\u003esp. Ce-a6\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eMethylosinus\u0026nbsp;\u003c/em\u003esp. PW2,\u003cem\u003e\u0026nbsp;\u003c/em\u003enot all copies of \u003cem\u003exoxF5\u003c/em\u003e were clustered together (Figure S1), potentially suggesting more ancient duplication events and subsequent divergence. This divergence may reflect adaptation to varied ecological niches, cofactor preferences, or substrate specificities [38,39].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The \u003cem\u003exoxF\u003c/em\u003e2 variant was only detected in verrucomicrobial MOBs (Figure S2). In fact, all the Verrucomicrobiota harbour only XoxF2 as their MDH, except for four \u003cem\u003eVerrucomicrobiota incertae sedis\u003c/em\u003e genomes (all belonging to the genus \u003cem\u003eMethylacidimicrobium\u003c/em\u003e), which possess both XoxF1 and XoxF2 variants. This suggests functional specialisation of XoxF to these organisms, enabling them to metabolise methanol under distinct environmental conditions or with different cofactors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eXoxF1 was detected in five \u003cem\u003eAlphaproteobacteria\u0026nbsp;\u003c/em\u003e(three within the genus \u003cem\u003eMethylocella,\u0026nbsp;\u003c/em\u003eone \u003cem\u003eMethylovirgula\u003c/em\u003e and one \u003cem\u003eMethylocystis\u003c/em\u003e) isolated from acidic soils, and\u003cem\u003e\u0026nbsp;\u003c/em\u003ethe only\u003cem\u003e\u0026nbsp;\u003c/em\u003efour\u003cem\u003e\u0026nbsp;Verrucomicrobiota incertae sedis\u003c/em\u003e genomes isolated from acidophilic/thermophilic environments [40]. The distribution of \u003cem\u003exoxF1\u003c/em\u003e, particularly in organisms present in acidic and high-temperature environments, suggests that the gene may confer ecological advantages to the organisms under such extreme conditions, playing a significant role in adaptation to pH and cofactor changes [4,35]. Only the \u003cem\u003eMethylovirgula ligni\u003c/em\u003e BW863 has multiple copies of \u003cem\u003exoxF1\u003c/em\u003e, possessing three copies, with two copies clustering together, while the other copy is grouped into a separate clade (Figure S3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Forty-five MOB genomes harboured the \u003cem\u003exoxF3\u003c/em\u003e variant that includes 23 Alphaproteobacteria and 22 Gammaproteobacteria MOB. Six genomes possessed multiple copies: one alphaproteobacterial MOB,\u0026nbsp;\u003cem\u003eMethylocystis echinoides\u003c/em\u003e LMG27198, and five gammaproteobacterial MOBs belonging to the genera\u0026nbsp;\u003cem\u003eMethylocaldum\u003c/em\u003e (3) and\u0026nbsp;\u003cem\u003eMethylomonas\u003c/em\u003e (2). Although the multiple copies in each genome cluster distinctly into separate clades, the genes from members of the same genus cluster together (Figure S4). The lack of clustering of copies from the same organism suggests divergent evolution and possibly distinct functional roles of the different \u003cem\u003exoxF3\u003c/em\u003e copies within individual genomes. Moreover, the gene copies that cluster closely with those from other organisms of the same genus indicate that horizontal gene transfer (HGT) may have occurred. This is common in MOB as they often exchange genetic material to adapt to various environmental conditions [41].\u003c/p\u003e\n\u003cp\u003eTo understand the distribution of \u003cem\u003exoxF\u003c/em\u003es and \u003cem\u003emxaF\u003c/em\u003e in the natural environment, assembled metatranscriptomes from Lake Washington sediment microbial communities [23] were analysed for the presence of MDH transcripts. A BlastP (1e-40) sequence similarity search on the IMG platform resulted in over 6800 hits. However, most sequences were very short, so a length cutoff of 500 was applied to exclude fragmented sequences. A total of 535 transcripts were retained for the final phylogenetic analysis. All \u003cem\u003exoxF\u003c/em\u003e variants were detected in the metatranscriptomes except \u003cem\u003exoxF2,\u0026nbsp;\u003c/em\u003ehighlighting that MOB XoxF-MDHs are expressed and actively involved in the methane oxidation pathway, i.e. catalysing the oxidation of methanol to formaldehyde.\u003cem\u003e\u0026nbsp;\u003c/em\u003eHowever, more than 50% of the \u003cem\u003exoxFs\u003c/em\u003e identified in the metatranscriptomes were affiliated with the \u003cem\u003exoxF4\u003c/em\u003e clade, a group of MDH mainly found in non-MOBs (\u003cem\u003eMethylophilaceae\u003c/em\u003e) (Figure 2a). This is unsurprising as \u003cem\u003eMethylophilaceae\u003c/em\u003e are ubiquitous in the environment, including in freshwater ecosystems [42]. Studies in Lake Washington have also provided evidence of a cooperative metabolic relationship between \u003cem\u003eMethylophilaceae\u0026nbsp;\u003c/em\u003eand MOB [43,44]. The majority of \u003cem\u003exoxF\u003c/em\u003e metatranscripts are phylogenetically linked to MOB, particularly \u003cem\u003eMethylobacter\u003c/em\u003e, a dominant MOB in freshwater environments. Notably, among the 94 \u003cem\u003exoxF5\u003c/em\u003e metatranscripts, only two (belonging to \u003cem\u003eBurkholderiales\u003c/em\u003e) are not associated with MOB, accounting for just 2% of the total metatranscripts of this gene. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOverall, our genome- and metatranscriptome-wide survey highlights the diverse distribution patterns and evolutionary dynamics of methanol dehydrogenases in MOBs, with clear lineage-specific preferences for specific MDH variants. The widespread occurrence of XoxF5 in proteobacterial MOBs, the specialisation of XoxF2 in Verrucomicrobiota, and the presence of XoxF1 in organisms that thrive in acidophilic and thermophilic environments underscore the ecological and functional adaptability of these enzymes. \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eDistribution of lanthanide transporters in MOB genomes, plasmids, and metatranscriptomes\u003c/h2\u003e\n\u003cp\u003eThe lanthanide-binding and transport proteins also show interesting patterns. While the lanthanide-binding protein LanM is only present in Alphaproteobacteria, the TonB-dependent receptor LanA and the Ln-binding protein LanPepSY were primarily found in the \u003cem\u003egammaproteobacterial\u003c/em\u003e MOB genomes. The LutH-like protein (also a TonB-dependent receptor) is more widely distributed and abundant than any other lanthanome candidate investigated, as it is found in all the MOB phyla (Figure 1b). \u0026nbsp; LanA was identified in \u003cem\u003eMethylovibrium buryatense\u003c/em\u003e [7] and CQW49_RS02145, a protein recently found in \u003cem\u003eMethylosinus trichosporium\u003c/em\u003e OB3b homologous to the LutH\u0026nbsp;of \u003cem\u003eMethylorubrum extorquens\u003c/em\u003e AM1 [8]\u0026nbsp;are the only lanthanide transport proteins identified in the MOBs characterised to date.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLanmodulin (LanM)\u003c/strong\u003e was the first lanthanide-selective chelator to be discovered in \u003cem\u003eM. extorquens\u003c/em\u003e, and it provided valuable insights into how Ln are selectively recognised and transported in methylotrophs [6]. Although it is similar to the calcium-binding protein calmodulin, as it possesses metal-binding EF hands, LanM was shown to uniquely respond to picomolar concentrations of all Ln\u003csup\u003e3+\u003c/sup\u003e while responding to Ca\u003csup\u003e2+\u003c/sup\u003e at millimolar concentrations \u0026ndash; a remarkable 100-million-fold selectivity for Ln\u003csup\u003e3+\u003c/sup\u003e over Ca\u003csup\u003e2+\u003c/sup\u003e [6]. Our genome-wide survey detected the presence of LanM only in the MOB genomes belonging to the families Methylocystaceae and Beijerinkiaceae, which is consistent with the findings of Mattocks and colleagues [45]. We also identified LanM sequences in the plasmids most belonging to \u003cem\u003eBradyrhizobium\u003c/em\u003e and \u003cem\u003eMethylobacterium\u003c/em\u003e (Figure 3a). The presence of this gene in plasmids suggests its potential role in the survival and adaptation of these organisms to specific environments, as well as the potential for horizontal transfer of the gene to other bacteria.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDue to the close sequence relatedness of LanM and calmodulin, we performed a motif analysis to distinguish \u003cem\u003ebona fide\u003c/em\u003e sequences from potential false positives. We used the identified motifs of the two biochemically characterised LanM from \u003cem\u003eM. extorquens\u0026nbsp;\u003c/em\u003e(\u003cem\u003eMex\u003c/em\u003e) and \u003cem\u003eHansschlegelia quercus\u0026nbsp;\u003c/em\u003e(\u003cem\u003eHans\u003c/em\u003e)\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Figure 3b) and created a single regular expression (regex) pattern (Figure 3b) for identifying LanM sequences. The motif analysis revealed that 17 MOB genome sequences (2 \u003cem\u003eMethylocella\u003c/em\u003e and 15 \u003cem\u003eMethylocystis\u003c/em\u003e) contained motifs matching \u003cem\u003eMex-\u003c/em\u003eLanM, suggesting them as \u003cem\u003ebona fide\u003c/em\u003e LanM. At the same time, only one metatranscript was identified to encode a \u003cem\u003ebona fide\u003c/em\u003e LanM (Figure 3a). No sequences matched the \u003cem\u003eHans-\u003c/em\u003eLanM. Sequences lacking known motifs potentially represent LanM with novel or distinct motifs that diverge from those characterised in Mex-LanM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLanpepsy (LanP)\u003c/strong\u003e, a lanthanide-binding protein, was recently discovered in the obligate methylotroph \u003cem\u003eMethylobaccillus flagellatus\u003c/em\u003e. Characterised by two PepSY domains, LanP represents the first member of the PepSY family shown to bind lanthanides [11]. Our survey (Figure 4a) revealed that LanP was predominantly detected within Methylococcales genomes belonging to the family \u003cem\u003eMethylococcaceae\u003c/em\u003e, with a single hit identified in \u003cem\u003eMethylothermaceae\u0026nbsp;\u003c/em\u003e(\u003cem\u003eMethylohalobius crimeensis\u003c/em\u003e). Additionally, five plasmid hits associated with the \u003cem\u003eMethylococcaceae\u003c/em\u003e (\u003cem\u003eMethylomonas\u0026nbsp;\u003c/em\u003esp., \u003cem\u003eMethylomicrobium\u0026nbsp;\u003c/em\u003esp., \u003cem\u003eMethylobacter\u0026nbsp;\u003c/em\u003esp., and \u003cem\u003eMethylomonas methanica\u003c/em\u003e) were found to encode LanP. In sediment metatranscriptomes, we recovered 406 sequences, confirming their active expression in lake sediment.\u003c/p\u003e\n\u003cp\u003eTo verify the authenticity of the retrieved LanP sequences, we searched for the PepSY domain in all the curated 249 sequences (from genomes, metatranscriptomes, and plasmids) using the Conserved Domain Database (CDD). Notably, all the sequences had the two characteristic PepSY domains, each approximately 60 amino acids in length (Figure 4b). This finding suggests that while LanP is restricted to only a few MOB families, the gene is widespread and actively expressed in the environment, underscoring its ecological significance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe also analysed the TonB-dependent receptors \u003cstrong\u003eLanA\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;LutH,\u003c/strong\u003e first identified in \u003cem\u003eMethylotuvimicrobium buryatense\u003c/em\u003e 5GB1C and \u003cem\u003eM. extorquens\u003c/em\u003e AM1, respectively. Both genes were found to be crucial in controlling the lanthanide switch in these organisms, particularly in the lanthanide uptake system [7,10]. LanA from \u003cem\u003eM. buryatense\u003c/em\u003e 5GB1C was the first lanthanum receptor identified in a methanotroph. Recently, Shiina and colleagues [8] Identified a \u003cem\u003elutH\u003c/em\u003e-like gene in \u003cem\u003eMethylosinus trichosporium\u003c/em\u003e OB3b. Our work revealed that LanA is widely distributed in MOBs, predominantly within the \u003cem\u003eMethylococcaceae\u003c/em\u003e family. We also detected two LanA genes from the family \u003cem\u003eCrenotrichaceae\u003c/em\u003e and one each from \u003cem\u003eMethylothermaceae\u003c/em\u003e, \u003cem\u003eMethylocystaceae\u003c/em\u003e, and \u003cem\u003eBeijerinckiaceae\u003c/em\u003e. Interestingly, this gene was also identified in metatranscriptomes related to \u003cem\u003eMethylobacter\u003c/em\u003e, \u003cem\u003eMethylomonas\u003c/em\u003e, and \u003cem\u003eMethylotenera\u003c/em\u003e; however, no LanA related to MOB was found in plasmids (Figure 5a). Similarly, the \u003cem\u003elutH\u003c/em\u003e-like gene was found in all the MOB families affiliated with LanA, including the \u003cem\u003eMethylacidiphilaceae\u003c/em\u003e and unclassified \u003cem\u003eChromatiales\u0026nbsp;\u003c/em\u003e(Figure 5b)\u003cem\u003e.\u003c/em\u003e However, in both \u003cem\u003elanA\u003c/em\u003e and \u003cem\u003elutH\u003c/em\u003e-like genes retrieved, there is a pervasive phylogenetic incongruence, most likely due to horizontal gene transfer, as these genes are also found in the plasmids of other organisms.\u003c/p\u003e\n\u003ch2\u003eImpact of Cerium and Mixed Lanthanides on Methane Oxidation and Protein Expression in \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b.\u003c/h2\u003e\n\u003cp\u003eRare earth elements (REE) are naturally present in the environment, where they predominantly occur as insoluble oxides. To explore how an MOB responds to this environmentally relevant form compared to the commonly used soluble laboratory form (CeCl\u003csub\u003e3\u003c/sub\u003e), we assessed the growth of \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b on 20% CH\u003csub\u003e4\u003c/sub\u003e under three conditions: no added lanthanides (No Ln), with cerium (Ce), and with a lanthanide-rich ore. These treatments revealed distinct differences in bacterial response and growth in the presence and absence of these critical cofactors. Notably, we observed the longest lag phase in the ore treatment culture, followed by the Ce treatment, with the shortest lag phase in the No Ln control (Figure 6a). The duration of the lag phase provides insight into how the bacteria adapt to the presence and different Ln sources.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis suggests that the bacteria require more time to express the genes necessary for the selection and mobilisation of specific metals from a mixture of similar elements, in which those elements may be in a less bioavailable form. Conceivably, this also enables them to overcome toxicity associated with the ore. In contrast, the cerium-only treatment shows a shorter lag phase, most likely because the bacteria are provided with a single cofactor and no additional, potentially inhibitory, components. The energy and resources required to mobilise cerium (Ce) are minimal, leading to a quicker adaptation. The shortest lag phase was observed in the No Ln control, likely because \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b is routinely cultured and maintained in our laboratory without the addition of lanthanides. Under this condition, the bacteria constitutively express and utilise the calcium-dependent MDH (MxaF) for methanol oxidation, making this condition the natural growth environment for the organism. Consequently, no additional energy is required for regulatory adjustments or enzyme switch to enable methane oxidation and growth in this treatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInterestingly, despite the differences in lag phases across the treatments, the amount of methane oxidised over 98 hours is similar, with all treatments showing an average methane oxidation rate of approximately 0.08% CH\u003csub\u003e4\u0026nbsp;\u003c/sub\u003ehr\u003csup\u003e-1\u003c/sup\u003e. This indicates that in the presence of lanthanides, once the bacteria have acclimatised to the new conditions, their growth rate and methane oxidation capabilities become comparable or even exceed those observed in the No Ln treatment. Although the lanthanide-treated cultures exhibit a slower start, they achieve the same level of methane consumption within the same timeframe (approximately 98 h), indicating a higher effective oxidation rate once growth begins. This effect is particularly evident in the mixed lanthanide ore treatment, suggesting that when environmentally or geologically available lanthanides are present in sufficient quantities, the growth and methane oxidation of \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b proceed more rapidly than in their absence.\u003c/p\u003e\n\u003cp\u003eProteomic analysis revealed significant changes in protein expression in both the Ce and ore treatments compared to the control. A total of 1,414 proteins were detected, with 60 and 724 proteins significantly expressed in the Ce and ore treatments, respectively (Table S5). The 12-fold increase in significantly expressed proteins in the ore treatment compared to the Ce-only treatment is likely attributable to the complex nature and composition of the ore. \u0026nbsp;The response of \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b to the ore is likely not only a response to the provision of lanthanides in a more complex and less bioavailable form but also a response to other metals in the ore, resulting in the induction of various proteins, including those associated with stress response and chemotaxis. This response is necessary for the organism to adapt and thrive in its environment. In the Ce-only treatment, among the 60 significantly expressed proteins, 33 were upregulated, and 27 were downregulated. In the ore treatment, 144 proteins were upregulated, while 580 were downregulated.\u003c/p\u003e\n\u003cp\u003eOur genome analysis of \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b revealed the presence of genes such as \u003cem\u003emxaF, xoxF3\u003c/em\u003e, \u003cem\u003exoxF5, lanM\u003c/em\u003e, and \u003cem\u003elutH\u003c/em\u003e-like genes. Except for LanM, all these proteins were expressed in both the Ce and ore treatments. Furthermore, the lanthanide-dependent enzymes XoxF3 and XoxF5 were upregulated, while the calcium-dependent MxaF and the LutH-like protein were downregulated under both conditions (Figure 6b). The gene encoding MxaF is in a 12-gene cluster implicated in calcium-dependent MDH activity, eight of which (in addition to \u003cem\u003emxaF\u003c/em\u003e) were downregulated under both conditions. The protein MxaJ, which was upregulated under both conditions, is not part of this cluster but encoded by a gene adjacent to \u003cem\u003exoxF3\u003c/em\u003e. \u0026nbsp;The inverse expression/regulation of MxaF and XoxF and their associated genes in response to lanthanide bioavailability strongly suggests that XoxF lanthanide-binding proteins play a critical role in the utilisation of lanthanides in \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b. The inverse regulation of MxaF and XoxF due to lanthanide availability has been previously demonstrated [46].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInterestingly, LanM, which is known to respond to picomolar concentrations of lanthanides in obligate methylotrophs, was not expressed in the presence of Ce or ore treatments. This suggests that while \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b possesses the \u003cem\u003elanM\u003c/em\u003e gene, it may not be the primary gene responsible for lanthanide acquisition and trafficking in this bacterium under the growth conditions we have tested. Recent studies by Shiina et al. (2023) demonstrated that the LutH-like TonB-dependent receptor (CQW49_RS02145) is required for the expression of XoxF in the presence of cerium in a laboratory-adapted strain of \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b [8]. These findings imply a crucial role for this gene in lanthanide uptake across the outer membrane. \u0026nbsp;However, in our study, the LutH-like gene (MettrDRAFT_0198) was downregulated in both the Ce and ore treatments. This observation aligns with findings by Gu and Semrau [47], who reported downregulation of the same gene (referred to as ADVE02_v2_10208) in the presence of cerium and the absence of copper. Such downregulation of the LutH-like gene may be due to the high concentration of Ce, as metal ion uptake systems are typically downregulated when intracellular metal levels reach sufficient concentrations to maintain homeostasis [48,49]. Our findings are consistent with MettrDRAFT_0198 LutH-like as a component of a primary route for lanthanide uptake and trafficking in \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b. Its downregulation under elevated Ce conditions supports a model in which the bacterium modulates uptake systems to avoid excess metal accumulation, thus maintaining intracellular metal homeostasis. \u0026nbsp;The large number of proteins that are differently affected by the two lanthanide sources, including some 48 transport-associated proteins and 19 putative molecular chaperones, may indicate proteins that are necessary for liberating lanthanides from geological sources and growing in the presence of the stresses, lanthanide-related and others, that such materials present.\u003c/p\u003e\n\u003cp\u003eAmong the proteins significantly upregulated in both Ce and ore treatments, we identified a TonB-dependent siderophore receptor (MettrDRAFT_4452) and an adjacent cyclic peptide export ABC transporter (MettrDRAFT_4451). This co-upregulation suggests a coordinated role in lanthanide acquisition, potentially through a lanthanophore-mediated mechanism similar to bacterial siderophore-iron uptake systems. Recent work by Juma et al. (2022) demonstrated that \u003cem\u003eMethylobacterium aquaticum\u003c/em\u003e strain 22A utilises a siderophore to solubilise lanthanides, facilitating their uptake through a TonB-dependent receptor. This raises the possibility that \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b employs a similar strategy, where the cyclic peptide export ABC transporter secretes metal-chelating peptides that enhance lanthanide solubilisation, while the TonB-dependent receptor mediates uptake of the resulting lanthanide-peptide complex [50]. This system may represent an important alternative lanthanide-scavenging strategy in \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b, distinct from the previously characterised LutH-like transport pathway.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe lanthanome is distributed across the MOB phyla, with the lanthanoenzyme (MDH) found across all phyla. However, the distribution of the MDH variants (XoxFs) is skewed, favouring XoxF5, where most proteobacterial MOBs have multiple copies. The lanthanide transporters are also not evenly distributed in MOBs. While LanM was only detected in alphaproteobacterial methanotrophs, LanA and LanPepSY were primarily found in the gammaproteobacterial MOB, mainly within the family \u003cem\u003eMethylococcaceae\u003c/em\u003e. The \u003cem\u003elutH\u003c/em\u003e-like gene showed a wide distribution in all MOBs. The difference in the presence and distribution of the lanthanome, especially lanthanide transporters, strongly indicates that the Ln transport system in MOBs is not universal; different methanotrophs employ different proteins for acquiring, trafficking, and utilising Ln from their environment. The existence of a specific lanthanome that is identifiable in metatranscriptome data confirms MOB-lanthanides interaction and activities in the environment. We also found some lanthanide transporters in plasmids, suggesting their importance in the organisms\u0026rsquo; evolution, adaptability, and survival within specific environmental niches.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b, the presence of cerium (Ce) and mixed lanthanides led to distinct methane oxidation and proteomic responses. Although the lag phase differed across treatments, the methane oxidation rate was similar (0.08% CH\u003csub\u003e4\u003c/sub\u003e hr\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) once the bacteria acclimatised. Proteomic analysis revealed a greater number of significantly expressed proteins in the ore treatment compared to cerium alone, suggesting that the ore\u0026rsquo;s complex composition induced broader stress and adaptation responses. Key lanthanide-binding enzymes, including XoxF3 and XoxF5, were upregulated, while MxaF was downregulated, reflecting the \u0026ldquo;lanthanide switch\u0026rdquo; phenomenon. Interestingly, LanM, despite being present in the genome, was not detected in either treatment, suggesting that it is not the primary protein for lanthanide acquisition in \u003cem\u003eM. trichosporium\u003c/em\u003e OB3b. The downregulation of the \u003cem\u003elutH\u003c/em\u003e-like gene (MettrDRAFT_0198) under both treatments may indicate that it plays a key role in lanthanide uptake that requires its downregulation to maintain metal homeostasis when intracellular lanthanide concentrations are sufficient.\u003c/p\u003e \u003cp\u003eOverall, understanding the distribution of the lanthanome within MOBs is crucial for comprehending methane capture and utilisation in various environments. This knowledge also serves as a valuable resource for exploring lanthanome genes within MOB genomes or the environment, facilitating their potential application in lanthanide recovery from geological sources and other aspects of biotechnology.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eLn Lanthanides\u003c/p\u003e\n\u003cp\u003eMDH Methanol dehydrogenase\u003c/p\u003e\n\u003cp\u003eMOB Methane oxidising bacteria\u003c/p\u003e\n\u003cp\u003eLanM Lanmodulin\u003c/p\u003e\n\u003cp\u003eLanP LanPepsy\u003c/p\u003e\n\u003cp\u003eMAGs Metagenome assembled genomes\u003c/p\u003e\n\u003cp\u003eHGT Horizontal gene transfer\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eD.K. conceptualised the study; D.K. and Y.C. designed the experiments; P.N. provided material; S.U.D. and E.S. performed the experiments; S.U.D., A.A., E.S. and T.J.S. performed the analyses; S.U.D. wrote the manuscript with help from all co-authors.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe are grateful to the Natural Environmental Research Council for funding (NE/X005062; NE/X005119)\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information. The proteomics data is available in the ProteomeXchange via the PRIDE partner repository with the dataset identifier PXD063434. https://www.ebi.ac.uk/pride/archive/projects/PXD063434\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHibi Y, Asai K, Arafuka H, Hamajima M, Iwama T, Kawai K. Molecular structure of La3+-induced methanol dehydrogenase-like protein in Methylobacterium radiotolerans. J Biosci Bioeng. 2011;111:547\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eFitriyanto NA, Fushimi M, Matsunaga M, Pertiwiningrum A, Iwama T, Kawai K. Molecular structure and gene analysis of Ce3+ -induced methanol dehydrogenase of Bradyrhizobium sp. MAFF211645. J Biosci Bioeng. 2011;111:613\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eNakagawa T, Mitsui R, Tani A, Sasa K, Tashiro S, Iwama T, et al. A Catalytic Role of XoxF1 as La3+-Dependent Methanol Dehydrogenase in Methylobacterium extorquens Strain AM1. 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Front Microbiol. 2022;13. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sigs","sideBox":"Learn more about [Environmental Microbiome](https://environmentalmicrobiome.biomedcentral.com)","snPcode":"40793","submissionUrl":"https://submission.nature.com/new-submission/40793/3","title":"Environmental Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lanthanides, Methane-oxidising bacteria, Lanthanome, Proteomics, Metatranscriptomes","lastPublishedDoi":"10.21203/rs.3.rs-6350491/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6350491/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eLanthanides (Ln) play essential roles in the metabolism of methanotrophs, catalysing key reactions in the methane oxidation pathway. However, the diversity, distribution, and ecological roles of Ln-dependent proteins (the lanthanome) in aerobic methane-oxidising bacteria (MOB) remain underexplored. This study investigates the lanthanome using genome, plasmid, and proteome data, as well as metatranscriptome data from methane-rich lake sediments.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eComparative genomic analysis of a custom database of 179 MOB genomes revealed the presence of various methanol dehydrogenase (MDH) isoforms, including xoxF variants, distributed across Proteobacteria and Verrucomicrobia phyla. Using \u003cem\u003eMethylosinus trichosporium\u003c/em\u003e OB3b as a model, we measured methane oxidation rates in response to CeCl₃ and ore containing a mixture of lanthanides. Using proteomics, we uncovered differential protein expression in response to Ln. Despite differences in adaptation times, methane oxidation rates were consistent across treatments, indicating similar overall metabolic efficiencies after Ln acclimatisation. The genomic analysis uncovered several Ln-binding proteins, including the TonB-dependent receptors (LanA and LutH-like), as well as Lanmodulin and LanPepsy, with unique phylogenetic patterns. Metatranscriptomic analysis confirmed the expression of lanthanome, particularly in Proteobacteria, with xoxF5 as the dominant MDH variant in MOB genomes. The discovery of Ln-binding proteins in plasmids suggests horizontal gene transfer, highlighting the adaptive mechanisms of MOB to Ln availability and their ecological role in methane cycling.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis work expands our understanding of Ln-dependent methane oxidation in MOB, highlighting their metabolic flexibility and ecological significance in methane cycling. The findings suggest potential applications for Ln-dependent processes in biotechnology, particularly in methane capture and bio-utilization, as well as lanthanide recovery from geological sources.\u003c/p\u003e","manuscriptTitle":"Diversity and distribution of the lanthanome in aerobic methane-oxidising bacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-22 14:20:29","doi":"10.21203/rs.3.rs-6350491/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-03T11:23:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-12T03:33:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119725890964213447762920137270289473442","date":"2025-05-30T23:11:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-29T16:51:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41993594821020040836743713784047666922","date":"2025-05-27T15:09:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156742520515502217382421023938120875200","date":"2025-05-25T19:20:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-20T09:06:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-07T10:59:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-03T15:24:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Microbiome","date":"2025-04-29T18:06:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sigs","sideBox":"Learn more about [Environmental Microbiome](https://environmentalmicrobiome.biomedcentral.com)","snPcode":"40793","submissionUrl":"https://submission.nature.com/new-submission/40793/3","title":"Environmental Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dadce69a-e621-4b6b-967e-cd0cb41c6555","owner":[],"postedDate":"May 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:03:25+00:00","versionOfRecord":{"articleIdentity":"rs-6350491","link":"https://doi.org/10.1186/s40793-025-00776-5","journal":{"identity":"environmental-microbiome","isVorOnly":false,"title":"Environmental Microbiome"},"publishedOn":"2025-09-29 15:57:54","publishedOnDateReadable":"September 29th, 2025"},"versionCreatedAt":"2025-05-22 14:20:29","video":"","vorDoi":"10.1186/s40793-025-00776-5","vorDoiUrl":"https://doi.org/10.1186/s40793-025-00776-5","workflowStages":[]},"version":"v1","identity":"rs-6350491","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6350491","identity":"rs-6350491","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}