Lanthanide-Dependent Methanol Metabolism of a Proteobacteria-Dominated Community in a Light Lanthanide-Rich Deep Environment.

This study investigated the occurrence and diversity of proteobacterial XoxF-type methanol dehydrogenases (MDHs) in the microbial community that inhabits a fossil organic matter- and sedimentary lanthanide (Ln3+)-rich underground mine environment using a metagenomic and metaproteomic approach. A total of 8 XoxF-encoding genes (XoxF-EGs) and 14 protein sequences matching XoxF were identified. XoxF-type MDHs were produced by Alpha-, Beta-, and Gammaproteobacteria represented by the four orders Methylococcales, Nitrosomonadales, Rhizobiales, and Xanthomonadales. The highest number of XoxF-EG- and XoxF-matching protein sequences were affiliated with Nitrosomonadales and Rhizobiales, respectively. Among the identified XoxF-EGs, two belonged to the XoxF1 clade, five to the XoxF4 clade, and one to the XoxF5 clade, while seven of the identified XoxF proteins belonged to the XoxF1 clade, four to the XoxF4 clade, and three to the XoxF5 clade. Moreover, the accumulation of light lanthanides and the presence of methanol in the microbial mat were confirmed. This study is the first to show the occurrence of XoxF in the metagenome and metaproteome of a deep microbial community colonizing a fossil organic matter- and light lanthanide-rich sedimentary environment. The presented results broaden our knowledge of the ecology of XoxF-producing bacteria as well as of the distribution and diversity of these enzymes in the natural environment.

Lanthanide-dependent alcohol dehydrogenases require an essential aspartate residue for metal coordination and enzymatic function.

The lanthanide elements (Ln3+), those with atomic numbers 57-63 (excluding promethium, Pm3+), form a cofactor complex with pyrroloquinoline quinone (PQQ) in bacterial XoxF methanol dehydrogenases (MDHs) and ExaF ethanol dehydrogenases (EDHs), expanding the range of biological elements and opening novel areas of metabolism and ecology. Other MDHs, known as MxaFIs, are related in sequence and structure to these proteins, yet they instead possess a Ca2+-PQQ cofactor. An important missing piece of the Ln3+ puzzle is defining what features distinguish enzymes that use Ln3+-PQQ cofactors from those that do not. Here, using XoxF1 MDH from the model methylotrophic bacterium Methylorubrum extorquens AM1, we investigated the functional importance of a proposed lanthanide-coordinating aspartate residue. We report two crystal structures of XoxF1, one with and another without PQQ, both with La3+ bound in the active-site region and coordinated by Asp320 Using constructs to produce either recombinant XoxF1 or its D320A variant, we show that Asp320 is needed for in vivo catalytic function, in vitro activity, and La3+ coordination. XoxF1 and XoxF1 D320A, when produced in the absence of La3+, coordinated Ca2+ but exhibited little or no catalytic activity. We also generated the parallel substitution in ExaF to produce ExaF D319S and found that this variant loses the capacity for efficient ethanol oxidation with La3+ These results provide evidence that a Ln3+-coordinating aspartate is essential for the enzymatic functions of XoxF MDHs and ExaF EDHs, supporting the notion that sequences of these enzymes, and the genes that encode them, are markers for Ln3+ metabolism.

Methylotenera oryzisoli sp. nov., a lanthanide-dependent methylotrophic bacteria isolated from rice field soil.

A new lanthanide (Ln3+)-dependent methanol-utilizing bacterial strain, La3113T, was isolated from rice field soil and its taxonomic position was investigated using polyphasic approaches. The strain was aerobic, Gram-stain-negative, strongly motile, catalase-positive and cytochrome oxidase-positive. It could neither catalyse the hydrolysis of urea nor reduce nitrate to nitrite. Growth was observed within a temperature range of 10-40 °C and a pH range of 6-8, with optimum growth at 28 °C and pH 7. Methylamine was utilized as the single source of energy, carbon and nitrogen, and it was oxidized by methylamine dehydrogenase. C16 : 1 ω7c, C16 : 1 ω6c and C16 : 0 were the dominant cellular fatty acids. Its draft genome (2.67 Mbp and 44.9 mol% G+C content) encodes genes including three Ln3+-dependent methanol dehydrogenase (XoxF-type MDH) genes, those for formaldehyde assimilation (ribulose monophosphate pathway), formate dehydrogenases and methylamine dehydrogenases, but not Ca2+-dependent MDH (MxaFI-MDH), which characterizes the species as a Ln3+-dependent methylotroph. The 16S rRNA gene sequence showed that strain La3113T belongs to the genus Methylotenera and is closely related to Methylotenera mobilis JLW8T (98.29 % identity). The digital DNA-DNA hybridization (dDDH) values (less than 30 %) and average nucleotide identity (ANI) values (less than 85 %) between genomes of strain La3113T and related type strains were lower than the thresholds for species delineation (70 % for dDDH and 95-96 % for ANI). On the basis of these polyphasic approaches, we propose a novel Methylotenera species, Methylotenera oryzisoli sp. nov. (type strain La3113T=NBRC 111954T=DSM 103219T).

Structure and function of the lanthanide-dependent methanol dehydrogenase XoxF from the methanotroph Methylomicrobium buryatense 5GB1C.

In methylotrophic bacteria, which use one-carbon (C1) compounds as a carbon source, methanol is oxidized by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) enzymes. Methylotrophic genomes generally encode two distinct MDHs, MxaF and XoxF. MxaF is a well-studied, calcium-dependent heterotetrameric enzyme whereas XoxF is a lanthanide-dependent homodimer. Recent studies suggest that XoxFs are likely the functional MDHs in many environments. In methanotrophs, methylotrophs that utilize methane, interactions between particulate methane monooxygenase (pMMO) and MxaF have been detected. To investigate the possibility of interactions between pMMO and XoxF, XoxF was isolated from the methanotroph Methylomicrobium buryatense 5GB1C (5G-XoxF). Purified 5G-XoxF exhibits a specific activity of 0.16 µmol DCPIP reduced min-1 mg-1. The 1.85 Å resolution crystal structure reveals a La(III) ion in the active site, in contrast to the calcium ion in MxaF. The overall fold is similar to other MDH structures, but 5G-XoxF is a monomer in solution. An interaction between 5G-XoxF and its cognate pMMO was detected by biolayer interferometry, with a KD value of 50 ± 17 µM. These results suggest an alternative model of MDH-pMMO association, in which a XoxF monomer may bind to pMMO, and underscore the potential importance of lanthanide-dependent MDHs in biological methane oxidation.

Review of the genus Methylobacterium and closely related organisms: a proposal that some Methylobacterium species be reclassified into a new genus, Methylorubrum gen. nov.

The genus Methylobacterium, when first proposed by Patt et al. in 1976, was a monospecific genus created to accommodate a single pink pigmented facultatively methylotrophic bacterium. The genus now has over 50 validly published species, however, the percentage 16S rRNA sequence divergence within Methylobacterium questions whether or not they can still be accommodated within one genus. Additionally, several strains are described as belonging to Methylobacterium, but nodulate legumes and in some cases are unable to utilize methanol as a sole carbon source. This study reviews and discusses the current taxonomic status of Methylobacterium. Based on 16S rRNA gene, multi-locus sequence analysis, genomic and phenotypic data, the 52 Methylobacterium species can no longer be retained in one genus. Consequently, a new genus, Methylorubrum gen. nov., is proposed to accommodate 11 species previously held in Methylobacterium. The reclassified species names are proposed as: Methylorubrum aminovorans comb. nov. (type strain TH-15T=NCIMB 13343T=DSM 8832T), Methylorubrum extorquens comb. nov. (type strain NCIMB 9399T=DSM 1337T), Methylorubrum podarium comb. nov. (type strain FM4T=NCIMB 14856T=DSM 15083T), Methylorubrum populi comb. nov. (type strain BJ001T=NCIMB 13946T=ATCC BAA-705T), Methylorubrum pseudosasae comb. nov. (type strain BL44T=ICMP 17622T=NBRC 105205T), Methylorubrum rhodesianum comb. nov. (type strain NCIMB 12249T=DSM 5687T), Methylorubrum rhodinum comb. nov. (type strain NCIMB 9421T=DSM 2163T), Methylorubrum salsuginis comb. nov. (type strain MRT=NCIMB 14847T=NCCB 100140T), Methylorubrum suomiense comb. nov. (type strain F20T=NCIMB 13778T=DSM 14458T), Methylorubrum thiocyanatum comb. nov. (type strain ALL/SCN-PT=NCIMB 13651T=DSM 11490T) and Methylorubrum zatmanii comb. nov. (type strain NCIMB 12243T=DSM 5688T). The taxonomic position of several remaining species is also discussed.

Lanthanides: New life metals?

Lanthanides (Ln(3+)) that are Rare Earth Elements, until recently thought to be biologically inert, have recently emerged as essential metals for activity and expression of a special type of methanol dehydrogenase, XoxF. As XoxF enzyme homologs are encoded in a wide variety of microbes, including microbes active in important environmental processes such as methane and methanol metabolism, Ln(3+) may represent some of the key biogeochemical drivers in cycling of carbon and other elements. However, significant gaps in understanding the role of Ln(3+) in biological systems remain as the functions of most of the proteins potentially dependent of Ln(3+) and their roles in specific metabolic networks/respective biogeochemical cycles remain unknown. Moreover, enzymes dependent on Ln(3+) but not related to XoxF enzymes may exist, and these so far have not been recognized. Through connecting the recently uncovered genetic divergence and phylogenetic distribution of XoxF-like enzymes and through elucidation of their activities, metal and substrate specificities, along with the biological contexts of respective biochemical pathways, most parsimonious scenarios for their evolution could be uncovered. Generation of such data will firmly establish the role of Ln(3+) in the biochemistry of Life inhabiting this planet.

Expression, purification and properties of the enzymes involved in lanthanide-dependent alcohol oxidation: XoxF4, XoxF5, ExaF/PedH, and XoxG4.

In this chapter we describe logistics, protocols and conditions for expression, purification and characterization of Ln3+-dependent alcohol dehydrogenases representing three distinct phylogenetic clades of these enzymes, classified as XoxF4, XoxF5 and ExaF/PedH. We present data on the biochemical properties of a dozen enzymes, all generated by our group, in a comparative fashion. These enzymes display a range of properties in terms of substrate and metal specificities, pH and ammonium requirement, as well as catalytic constants. In addition, we describe a single novel cytochrome, XoxG4, that likely serves as a natural electron acceptor from XoxF5 in methanotrophs of the Gammaproteobacteria class.

Occurrence of XoxF-type methanol dehydrogenases in bacteria inhabiting light lanthanide-rich shale rock.

This study analyzed the occurrence of lanthanide-dependent (XoxF type) methanol dehydrogenases in the bacterial community dominated by Proteobacteria inhabiting shale rock. In total, 22 sequence matches of XoxF were identified in the metaproteome of the community. This enzyme was produced by bacteria represented by orders such as Rhizobiales, Rhodobacterales, Rhodospiralles, Burkholderiales and Nitrosomonadales. Among the identified XoxF proteins, seven belonged to XoxF1 clade and 15 to XoxF5 clade. This study is the first to show the occurrence of XoxF proteins in the metaproteome of environmental lithobiontic bacterial community colonizing an underground rock rich in light lanthanides. The presented results broaden our understanding of the ecology of XoxF producing bacteria as well as the distribution and diversity of these enzymes in the natural environment.

Discovery of lanthanide-dependent methylotrophy and screening methods for lanthanide-dependent methylotrophs.

The lanthanide elements (Lns) affect the physiology and growth of certain microorganisms known as "Ln-responsive microorganisms." Among them, in 2011, it was first reported that strains of Methylobacterium exhibited high methanol dehydrogenase (MDH) activity when grown in the presence of Lns; the purified Ln-inducible MDH was identified as XoxF-type MDH, whose catalytic function had previously been unknown. XoxF was the first enzyme to be identified as Ln-dependent, and its function in methylotrophy is more fundamental and important than that of the corresponding Ca2+-dependent MDH MxaFI. XoxF is encoded in the genomes of methylotrophic as well as non-methylotrophic bacteria. Thus, Lns are among the most fascinating and important growth factors for bacteria that potentially utilize methanol. Bacteria that require Lns for methanol growth are called "Ln-dependent methylotrophs." Recent findings indicate that these microorganisms comprise an "Ln-dependent ecosystem" that we have not been able to reconstruct under laboratory conditions without Lns. In this chapter, we summarize methods for (1) screening of Ln-responsive microorganisms, (2) purification of native XoxFs from Ln-dependent methylotrophs, and (3) screening of Ln-dependent methylotrophs from natural environments, while providing a history of the discovery of the Ln-dependent methylotrophs.

Preference for particular lanthanide species and thermal stability of XoxFs in Methylorubrum extorquens strain AM1.

XoxF-type methanol dehydrogenase was recently found to be lanthanide-dependent, while its counterpart MxaF is Ca2+-dependent. The lanthanide (Ln) series consists of 15 different elements, all of which exist in nature, although at different relative abundances. XoxF from Methylorubrum extorquens strain AM1 has been shown to be induced by four light Ln species (La3+ to Nd3+). The preference of XoxFs for certain co-existing Ln species and the catalytic activity and stability of XoxF metallated with different Ln species have not been well investigated. In this study, we found that (i) strain AM1 cells preferentially utilize La3+ rather than Nd3+ for growth, (ii) XoxF purified from cells grown with a La3+ and Nd3+ mixture contained a larger proportion of La3+, and (iii) La3+-metallated XoxF has higher activity and thermal stability than Nd3+-metallated XoxF, although (iv) both enzymes showed unchanged surface charges. Thermal shift assay in particular revealed that metallation affects the temperature for subunit denaturation but not for subunit dissociation. We concluded that, although La3+ and Nd3+ have similar distributions in nature, XoxF could chose La3+ preferentially.

Lanthanide-Dependent Methanol and Formaldehyde Oxidation in Methylobacterium aquaticum Strain 22A.

Lanthanides (Ln) are an essential cofactor for XoxF-type methanol dehydrogenases (MDHs) in Gram-negative methylotrophs. The Ln3+ dependency of XoxF has expanded knowledge and raised new questions in methylotrophy, including the differences in characteristics of XoxF-type MDHs, their regulation, and the methylotrophic metabolism including formaldehyde oxidation. In this study, we genetically identified one set of Ln3+- and Ca2+-dependent MDHs (XoxF1 and MxaFI), that are involved in methylotrophy, and an ExaF-type Ln3+-dependent ethanol dehydrogenase, among six MDH-like genes in Methylobacterium aquaticum strain 22A. We also identified the causative mutations in MxbD, a sensor kinase necessary for mxaF expression and xoxF1 repression, for suppressive phenotypes in xoxF1 mutants defective in methanol growth even in the absence of Ln3+. Furthermore, we examined the phenotypes of a series of formaldehyde oxidation-pathway mutants (fae1, fae2, mch in the tetrahydromethanopterin (H4MPT) pathway and hgd in the glutathione-dependent formaldehyde dehydrogenase (GSH) pathway). We found that MxaF produces formaldehyde to a toxic level in the absence of the formaldehyde oxidation pathways and that either XoxF1 or ExaF can oxidize formaldehyde to alleviate formaldehyde toxicity in vivo. Furthermore, the GSH pathway has a supportive role for the net formaldehyde oxidation in addition to the H4MPT pathway that has primary importance. Studies on methylotrophy in Methylobacterium species have a long history, and this study provides further insights into genetic and physiological diversity and the differences in methylotrophy within the plant-colonizing methylotrophs.

Regulation of lanthanide-dependent methanol oxidation pathway in the legume symbiotic nitrogen-fixing bacterium Bradyrhizobium sp. strain Ce-3.

Lanthanide (Ln)-dependent XoxF-type methanol dehydrogenase (MDH) genes can be found in bacteria that are not believed to be methylotrophs, and studies on their methylotrophic pathways and their use of Ln are now emerging. Ln-dependent methanol utilization in Bradyrhizobium sp. strain Ce-3, which belongs to the Bradyrhizobium elkanii superclade (clade II), was investigated in this study. Strain Ce-3 was able to grow in a media containing methanol as a sole carbon source and light Ln (L-Ln, i.e., La3+, Ce3+, Pr3+, and Nd3+), whereas the strain did not show any growth with Ca2+ or the heavy Ln, Sm3+. We found that the uptake of L-Ln is enhanced mainly by methanol and L-Ln species, and the strain incorporates each L-Ln species evenly into the cell. The genome of strain Ce-3 encodes the xox cluster for Ln-dependent methanol dehydrogenase (xoxF) and the enzymes participating in the methanol oxidation pathway (xoxG, fldA, and gfaA) and regulation (xoxR), but the gene encoding formate dehydrogenase (FDH) was not found in the cluster. MDH, formaldehyde dehydrogenase, and FDH activities were induced by methanol/Ln. Moreover, expression of the genes on the xox cluster was upregulated by methanol/Ln. Based on these results, we concluded that strain Ce-3 possesses a complete L-Ln-dependent methanol oxidation pathway, which is dissimilar to plant phyllospheric bacteria, Methylobacterium species, with a transport system for L-Ln species.

Lanthanide-dependent methanol dehydrogenase from the legume symbiotic nitrogen-fixing bacterium Bradyrhizobium diazoefficiens strain USDA110.

The legume symbiotic nitrogen-fixing bacterium, B. diazoefficiens strain USDA110, utilizes methanol for growth in the presence of light lanthanides, such as La3+, Ce3+, Pr3+ or Nd3+, and its cells possess significant methanol dehydrogenase (MDH) activity. We purified MDH to homogeneity from B. diazoefficiens strain USDA110 grown in a methanol/Ce3+ medium; the protein was identified as XoxF5-type MDH (blr6213). The purified XoxF contained 0.58 cerium atoms per enzyme subunit. Moreover, the in-solution structure of XoxF was analyzed by small angle X-ray scattering (SAXS) analysis; the radius of gyration (Rg) and maximum particle dimension (Dmax) of XoxF were calculated to be 32.3 and 96.8 Å, respectively, suggesting that XoxF adopts a dimer structure in solution. These results show that B. diazoefficiens strain USDA110 has XoxF, a lanthanides-dependent MDH, which has methanol oxidation activity and is induced by methanol/lanthanaides, and that lanthanide is one of the important factors in methanol utilization by the strain.

Novel facultative Methylocella strains are active methane consumers at terrestrial natural gas seeps.

BACKGROUND: Natural gas seeps contribute to global climate change by releasing substantial amounts of the potent greenhouse gas methane and other climate-active gases including ethane and propane to the atmosphere. However, methanotrophs, bacteria capable of utilising methane as the sole source of carbon and energy, play a significant role in reducing the emissions of methane from many environments. Methylocella-like facultative methanotrophs are a unique group of bacteria that grow on other components of natural gas (i.e. ethane and propane) in addition to methane but a little is known about the distribution and activity of Methylocella in the environment. The purposes of this study were to identify bacteria involved in cycling methane emitted from natural gas seeps and, most importantly, to investigate if Methylocella-like facultative methanotrophs were active utilisers of natural gas at seep sites. RESULTS: The community structure of active methane-consuming bacteria in samples from natural gas seeps from Andreiasu Everlasting Fire (Romania) and Pipe Creek (NY, USA) was investigated by DNA stable isotope probing (DNA-SIP) using 13C-labelled methane. The 16S rRNA gene sequences retrieved from DNA-SIP experiments revealed that of various active methanotrophs, Methylocella was the only active methanotrophic genus common to both natural gas seep environments. We also isolated novel facultative methanotrophs, Methylocella sp. PC1 and PC4 from Pipe Creek, able to utilise methane, ethane, propane and various non-gaseous multicarbon compounds. Functional and comparative genomics of these new isolates revealed genomic and physiological divergence from already known methanotrophs, in particular, the absence of mxa genes encoding calcium-containing methanol dehydrogenase. Methylocella sp. PC1 and PC4 had only the soluble methane monooxygenase (sMMO) and lanthanide-dependent methanol dehydrogenase (XoxF). These are the first Alphaproteobacteria methanotrophs discovered with this reduced functional redundancy for C-1 metabolism (i.e. sMMO only and XoxF only). CONCLUSIONS: Here, we provide evidence, using culture-dependent and culture-independent methods, that Methylocella are abundant and active at terrestrial natural gas seeps, suggesting that they play a significant role in the biogeochemical cycling of these gaseous alkanes. This might also be significant for the design of biotechnological strategies for controlling natural gas emissions, which are increasing globally due to unconventional exploitation of oil and gas.

Lanthanide-Dependent Methanol Dehydrogenases of XoxF4 and XoxF5 Clades Are Differentially Distributed Among Methylotrophic Bacteria and They Reveal Different Biochemical Properties.

Lanthanide-dependent alcohol dehydrogenases have recently emerged as environmentally important enzymes, most prominently represented in methylotrophic bacteria. The diversity of these enzymes, their environmental distribution, and their biochemistry, as well as their evolutionary relationships with their calcium-dependent counterparts remain virtually untapped. Here, we make important advances toward understanding lanthanide-dependent methylotrophy by assessing the distribution of XoxF4 and XoxF5 clades of lanthanide methanol dehydrogenases among, respectively, Methylophilaceae and non-Methylophilaceae methylotrophs, and we carry out comparative biochemical characterization of XoxF4 and XoxF5 enzymes, demonstrating differences in their properties, including catalytic efficiencies. We conclude that one subtype of the XoxF4 enzyme, XoxF4-1 is the dominant type in nature while other XoxF4 subtypes appear to be auxiliary, representatives of this clade only found in the Methylophilaceae (Betaproteobacteria). In contrast, we demonstrate that XoxF5 enzymes are widespread among Alpha-, Beta-, and Gammaproteobacteria. We purified and biochemically characterized two XoxF4 enzymes (XoxF4-1 and XoxF4-2), both from Methylotenera mobilis, and one XoxF5 enzyme, from Methylomonas sp., after expressing their His-tagged versions in respective natural hosts. All three enzymes showed broad specificities toward alcohols and aldehydes and strict dependence on lighter lanthanides. However, they revealed differences in their properties in terms of optimal pH for in vitro activity, ammonia dependence, the range of lanthanides that could serve as cofactors, and in kinetic properties. Overall, our data advance the understanding of the biochemistry and environmental distribution of these recently discovered enzymes that appear to be key enzymes in lanthanide-dependent methylotrophy.

Lanthanide-Dependent Regulation of Methylotrophy in Methylobacteriumaquaticum Strain 22A.

Methylobacterium species are representative of methylotrophic bacteria. Their genomes usually encode two types of methanol dehydrogenases (MDHs): MxaF and XoxF. The former is a Ca2+-dependent enzyme, and the latter was recently determined to be a lanthanide-dependent enzyme that is necessary for the expression of mxaF. This finding revealed the unexpected and important roles of lanthanides in bacterial methylotrophy. In this study, we performed transcriptome sequencing (RNA-seq) analysis using M. aquaticum strain 22A grown in the presence of different lanthanides. Expression of mxaF and xoxF1 genes showed a clear inverse correlation in response to La3+. We observed downregulation of formaldehyde oxidation pathways, high formaldehyde dehydrogenase activity, and low accumulation of formaldehyde in the reaction with cells grown in the presence of La3+; this might be due to the direct oxidation of methanol to formate by XoxF1. Lanthanides induced the transcription of AT-rich genes, the function of most of which was unknown, and genes possibly related to cellular survival, as well as other MDH homologues. These results revealed not only the metabolic response toward altered primary methanol oxidation, but also the possible targets to be investigated further in order to better understand methylotrophy in the presence of lanthanides. IMPORTANCE Lanthanides have been considered unimportant for biological processes. In methylotrophic bacteria, however, a methanol dehydrogenase (MDH) encoded by xoxF was recently found to be lanthanide dependent, while the classic-type mxaFI is calcium dependent. XoxF-type MDHs are more widespread in diverse bacterial genera, suggesting their importance for methylotrophy. Methylobacterium species, representative methylotrophic and predominating alphaproteobacteria in the phyllosphere, contain both types and regulate their expression depending on the availability of lanthanides. RNA-seq analysis showed that the regulation takes place not only for MDH genes but also the subsequent formaldehyde oxidation pathways and respiratory chain, which might be due to the direct oxidation of methanol to formate by XoxF. In addition, a considerable number of genes of unknown function, including AT-rich genes, were found to be upregulated in the presence of lanthanides. This study provides first insights into the specific reaction of methylotrophic bacteria to the presence of lanthanides, emphasizing the biological relevance of this trace metal.

Physiological Effect of XoxG(4) on Lanthanide-Dependent Methanotrophy.

A recent surprising discovery of the activity of rare earth metals (lanthanides) as enzyme cofactors as well as transcriptional regulators has overturned the traditional assumption of biological inertia of these metals. However, so far, examples of such activities have been limited to alcohol dehydrogenases. Here we describe the physiological effects of a mutation in xoxG, a gene encoding a novel cytochrome, XoxG(4), and compare these to the effects of mutation in XoxF, a lanthanide-dependent methanol dehydrogenase, at the enzyme activity level and also at the community function level, using Methylomonas sp. strain LW13 as a model organism. Through comparative phenotypic characterization, we establish XoxG as the second protein directly involved in lanthanide-dependent metabolism, likely as a dedicated electron acceptor from XoxF. However, mutation in XoxG caused a phenotype that was dramatically different from the phenotype of the mutant in XoxF, suggesting a secondary function for this cytochrome, in metabolism of methane. We also purify XoxG(4) and demonstrate that this protein is a true cytochrome c, based on the typical absorption spectra, and we demonstrate that XoxG can be directly reduced by a purified XoxF, supporting one of its proposed physiological functions. Overall, our data continue to suggest the complex nature of the interplay between the calcium-dependent and lanthanide-dependent alcohol oxidation systems, while they also suggest that addressing the roles of these alternative systems is essential at the enzyme and community function level, in addition to the gene transcription level.IMPORTANCE The lanthanide-dependent biochemistry of living organisms remains a barely tapped area of knowledge. So far, only a handful of lanthanide-dependent alcohol dehydrogenases have been described, and their regulation by lanthanides has been demonstrated at the transcription level. Little information is available regarding the concentrations of lanthanides that could support sufficient enzymatic activities to support specific metabolisms, and so far, no other redox proteins involved in lanthanide-dependent methanotrophy have been demonstrated. The research presented here provides enzyme activity-level data on lanthanide-dependent methanotrophy in a model methanotroph. Additionally, we identify a second protein important for lanthanide-dependent metabolism in this organism, XoxG(4), a novel cytochrome. XoxG(4) appears to have multiple functions in methanotrophy, one function as an electron acceptor from XoxF and another function remaining unknown. On the basis of the dramatic phenotype of the XoxG(4) mutant, this function must be crucial for methanotrophy.

Current Trends in Methylotrophy.

Methylotrophy is a field of study dealing with microorganisms capable of utilization of compounds devoid of carbon-carbon bonds (C1 compounds). In this review, we highlight several emerging trends in methylotrophy. First, we discuss the significance of the recent discovery of lanthanide-dependent alcohol dehydrogenases for understanding both the occurrence and the distribution of methylotrophy functions among bacteria, and then we discuss the newly appreciated role of lanthanides in biology. Next, we describe the detection of other methylotrophy pathways across novel bacterial taxa and insights into the evolution of methylotrophy. Further, data are presented on the occurrence and activity of aerobic methylotrophs in hypoxic and anoxic environments, questioning the prior assumptions on niche separation of aerobic and anaerobic methylotrophy. The concept of communal function in aerobic methane oxidation is also briefly discussed. Finally, we review recent research in engineering methylotrophs for biotechnological applications as well as recent progress in engineering synthetic methylotrophy.

Genomic characterization of methylotrophy of Oharaeibacter diazotrophicus strain SM30T.

Oharaeibacter diazotrophicus strain SM30T, isolated from rice rhizosphere, is an aerobic, facultative lanthanide (Ln3+)-utilizing methylotroph and diazotroph that belongs to the Methylocystaceae family. In this research, the complete genome sequence of strain SM30T was determined, and its methylotrophy modules were characterized. The genome consists of one chromosome and two plasmids, comprising a total of 5,004,097 bp, and the GC content was 71.6 mol%. A total of 4497 CDSs, 67 tRNA, and 9 rRNA were encoded. Typical alpha-proteobacterial methylotrophy genes were found: pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) (mxaF and xoxF1-4), methylotrophy regulatory proteins (mxbDM and mxcQE), PQQ synthesis, H4F pathway, H4MPT pathway, formate oxidation, serine cycle, and ethylmalonyl-CoA pathway. SDS-PAGE and subsequent LC-MS analysis, and qPCR analysis revealed that MxaF and XoxF1 were the dominant MDH in the absence or presence of lanthanum (La3+), respectively. The growth of MDH gene-deletion mutants on alcohols and qPCR results indicated that mxaF and xoxF1 are also involved in ethanol and propanol oxidation, xoxF2 participates in methanol oxidation in the presence of La3+, while xoxF3 was associated with methanol and ethanol oxidation in the absence of La3+, implying that XoxF3 is a calcium (Ca2+)-binding XoxF. Four Ln3+ such as La3+, cerium (Ce3+), praseodymium (Pr3+), and neodymium (Nd3+) served as cofactors for XoxF1 by supporting ΔmxaF growth on methanol. Some heavier lanthanides inhibited growth of SM30 on methanol. This study contributes to the understanding of the function of various XoxF-type MDHs and their roles in methylotrophs.

Comparative Genomics and Mutational Analysis Reveals a Novel XoxF-Utilizing Methylotroph in the Roseobacter Group Isolated From the Marine Environment.

The Roseobacter group comprises a significant group of marine bacteria which are involved in global carbon and sulfur cycles. Some members are methylotrophs, using one-carbon compounds as a carbon and energy source. It has recently been shown that methylotrophs generally require a rare earth element when using the methanol dehydrogenase enzyme XoxF for growth on methanol. Addition of lanthanum to methanol enrichments of coastal seawater facilitated the isolation of a novel methylotroph in the Roseobacter group: Marinibacterium anthonyi strain La 6. Mutation of xoxF5 revealed the essential nature of this gene during growth on methanol and ethanol. Physiological characterization demonstrated the metabolic versatility of this strain. Genome sequencing revealed that strain La 6 has the largest genome of all Roseobacter group members sequenced to date, at 7.18 Mbp. Multilocus sequence analysis (MLSA) showed that whilst it displays the highest core gene sequence similarity with subgroup 1 of the Roseobacter group, it shares very little of its pangenome, suggesting unique genetic adaptations. This research revealed that the addition of lanthanides to isolation procedures was key to cultivating novel XoxF-utilizing methylotrophs from the marine environment, whilst genome sequencing and MLSA provided insights into their potential genetic adaptations and relationship to the wider community.