Lanthanide-Dependent Methylotrophs of the Family Beijerinckiaceae: Physiological and Genomic Insights

Abstract

Methylotrophic bacteria use methanol and related C₁ compounds as carbon and energy sources. Methanol dehydrogenases are essential for methanol oxidation, while lanthanides are important cofactors of many pyrroloquinoline quinone-dependent methanol dehydrogenases and related alcohol dehydrogenases. We describe here the physiological and genomic characterization of newly isolated Beijerinckiaceae bacteria that rely on lanthanides for methanol oxidation. A broad physiological diversity was indicated by the ability to metabolize a wide range of multicarbon substrates, including various sugars, and organic acids, as well as diverse C₁ substrates such as methylated amines and methylated sulfur compounds. Methanol oxidation was possible only in the presence of low-mass lanthanides (La, Ce, and Nd) at submicromolar concentrations (>100 nM). In a comparison with other Beijerinckiaceae, genomic and transcriptomic analyses revealed the usage of a glutathione- and tetrahydrofolate-dependent pathway for formaldehyde oxidation and channeling methyl groups into the serine cycle for carbon assimilation. Besides a single xoxF gene, we identified two additional genes for lanthanide-dependent alcohol dehydrogenases, including one coding for an ExaF-type alcohol dehydrogenase, which was so far not known in Beijerinckiaceae Homologs for most of the gene products of the recently postulated gene cluster linked to lanthanide utilization and transport could be detected, but for now it remains unanswered how lanthanides are sensed and taken up by our strains. Studying physiological responses to lanthanides under nonmethylotrophic conditions in these isolates as well as other organisms is necessary to gain a more complete understanding of lanthanide-dependent metabolism as a whole.IMPORTANCE We supplemented knowledge of the broad metabolic diversity of the Beijerinckiaceae by characterizing new members of this family that rely on lanthanides for methanol oxidation and that possess additional lanthanide-dependent enzymes. Considering that lanthanides are critical resources for many modern applications and that recovering them is expensive and puts a heavy burden on the environment, lanthanide-dependent metabolism in microorganisms is an exploding field of research. Further research into how isolated Beijerinckiaceae and other microbes utilize lanthanides is needed to increase our understanding of lanthanide-dependent metabolism. The diversity and widespread occurrence of lanthanide-dependent enzymes make it likely that lanthanide utilization varies in different taxonomic groups and is dependent on the habitat of the microbes.

Keywords: Beijerinckiaceae; lanthanides; methanol dehydrogenases; methylotrophy.

FIG 1

Phylogenetic relationship, average nucleotide identities (ANI), and phylogenomic analysis of soft coal slag methylotrophs and related Beijerinckiaceae. (A) An unrooted maximum-likelihood tree of 16S rRNA gene sequences of related Beijerinckiaceae was calculated using the Tamura-Nei model (114) and bootstrapping (n = 100) (105). Bootstrap support is indicated by circles placed on the respective nodes. Colored circles show the metabolic grouping of the corresponding organisms. The grouping is based on available literature information for M. palustris (55), M. silvestris (49), M. tundrae (50), M. acidiphila (115), M. aurea (11), M. palsarum (12), M. gorgona (13), M. stellata (116), M. polaris (59), M. ligni (14), and Beijerinckia spp. (117, 118). The scale bar refers to nucleotide substitutions. (B) A triangular matrix of pairwise ANI between soft coal slag methylotrophs and genome-sequenced relatives (assembly levels: complete, chromosome, scaffold). NCBI assembly accession numbers are given in parentheses. The color code refers to ANI in percentages. (C) A phylogenomic tree was calculated using a set of 270 shared protein sequences encoded by single-copy core genes. Alignments for individual gene products were concatenated, and the concatenated alignment was subjected to treeing using fasttree (version 2.1.8) (74) as described in Materials and Methods. Local support values are indicated by circles placed on nodes. The scale bar indicates amino acid substitutions.

FIG 2

Screening of C₁ metabolism modules in Beijerinckiaceae and lanthanide availability in solidifying agents and sampled slag material. (A) The presence of selected key genes of C₁ metabolism modules (methane oxidation, methanol oxidation, and methylamine utilization) in isolated soft coal slag methylotrophs was assessed by PCR and screening of sequenced genomes by using profile hidden Markov models (pHMMs) (pmoA [particulate methane monooxygenase], mmoX [soluble methane monooxygenase], mxaF [MxaF methanol dehydrogenase], xoxF [XoxF methanol dehydrogenase], mauA [methylamine dehydrogenase], and gmaS [gamma-glutamylmethylamide synthetase]). Information for related Beijerinckiaceae was deduced from available literature for M. palustris (55), M. silvestris (49), M. tundrae (50), M. acidiphila (115), M. aurea (11), M. palsarum (12), M. gorgona (13), M. stellata (116), M. polaris (59), M. ligni (14), and Beijerinckia spp. (117, 118). Whenever possible, available genome sequences of Beijerinckiaceae (assembly levels: chromosome, complete, scaffold) were used to complement literature information by screening genomes using the aforementioned set of pHMMs. Filled and empty symbols indicate the presence of the respective gene based on PCR genotyping as described in the respective references and genome screening (this study). N.A., not available (the presence of genes was not tested by PCR previously, nor is a representative genome for a pHMM-based screening yet available. (B) Lanthanide availability in solidifying agents was determined by microwave digestion followed by ICP-MS, b.d., below detection limit. (C) The bioavailable fraction of lanthanides present in the sampled slag material was assessed by total digestion plus ICP-MS to determine the absolute quantities of lanthanides present (see Table S4 in the supplemental material) and by sequential digestion plus ICP-MS. We define bioavailable here as fractions I (mobile) and II (exchangeable) according to the sequential digestion method of Zeien and Brümmer (53, 89). The nomenclature for the sampling sites is according to Wegner and Liesack (42). RH1 and RH2 refer to slag deposition sites from which the slag material that was used for isolation was collected. RHRef refers to undisturbed forest soil collected from the surroundings of sites RH1 and RH2.

FIG 3

Morphology of methylotrophic isolates from soft coal slag. (A) Light microscopy of Beijerinckiaceae bacterium RH CH11 was done using differential interference contrast (DIC). Arrows point to refractile cytoplasmic inclusions. (B) Transmission electron microscopy (TEM) of Beijerinckiaceae bacterium RH AL1 grown on solid medium with methanol as a carbon source and without lanthanide supplementation for 2 weeks revealed contrast-rich polar bodies (P) partially surrounded by vesicle-like structures (V). (C) TEM analysis of Beijerinckiaceae bacterium RH AL1 during exponential growth in liquid culture with methanol as a carbon source and with additional lanthanide supplementation (1 μM lanthanum) revealed bright polar bodies, which are characteristic of PHB-storing vacuoles (PHB).

FIG 4

Carbon utilization by soft coal slag methylotrophs. The carbon utilization range was assessed by growing isolates in triplicates in MM2 medium with different carbon sources (see the text in the supplemental material). Growth was assessed against negative controls not supplemented with any carbon source. Information about other Beijerinckiaceae was taken from the literature for M. palustris (55), M. silvestris (49), M. tundrae (50), M. acidiphila (115), M. aurea (11), M. palsarum (12), M. gorgona (13), M. stellata (116), M. polaris (59), M. ligni (14), and Beijerinckia spp. (117, 118). N.A., data not available/not tested.

FIG 5

Methylotrophic growth dependent on lanthanide concentration (0 nM to 10 μM) (A) and species (La, Ce, Nd, Dy, Ho, Er, and Yb) (B). Soft coal slag methylotrophs were grown in MM2 medium (pH 5 to 5.5) supplemented with methanol (1% [vol/vol]) as a carbon source. Cultures were grown in triplicates (n = 3); error bars represent standard deviation. Ln³⁺, lanthanides; La, lanthanum; Ce, cerium; Nd, neodymium; Dy, dysprosium; Ho, holmium; Er, erbium; Yb, ytterbium.

FIG 6

Graphical representation of the genomic potential of Beijerinckiaceae bacterium RHAL1 (A) and gene expression of genes encoding products that might be involved in lanthanide-dependent metabolism (B). Methanol oxidation-related gene products are highlighted in orange, nitrogen metabolism-related gene products in blue, and sulfur metabolism-related gene products in yellow. Abbreviations: XoxF, XoxF-MDH; LanM, lanmodulin; ExaF, ExaF PQQ ADH; Gfa, glutathione-dependent formaldehyde-activating enzyme; FrmAB, glutathione-dependent formaldehyde dehydrogenase, S-formylglutathione hydrolase; Fhs, formate-tetrahydrofolate ligase; FolD, bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase and 5,10-methylene-tetrahydrofolate cyclohydrolase; Fdh, formate dehydrogenase; NMG, N-methylglutamate pathway; SsuD, alkanesulfonate monooxygenase; SsuA, alkanesulfonate transporter; H4F, tetrahydrofolate; ED pathway, Entner-Doudoroff pathway; PHB, polyhydroxybutyrate; PolyP, polyphosphate; FMD, formamidase; NTL, nitrilase; CynS, cyanase; CA, carbonic anhydrase; NIR, nitrite reductase; SSP, simple sugar permease; Chv, multiple sugar-binding periplasmic receptor Chv; Ggu, multiple sugar transport system; Rbs, ribose transporter; Kps, capsular polysaccharide transporter. A list of genes, gene products, and their gene expression is given in Table S7 in the supplemental material. Gene expression data for all genes in the genome can be found in Table S8 in the supplemental material. Homologs of gene products linked to lanthanide-dependent metabolism in Beijerinckiaceae were identified by blastp (79) queries of the amino acid sequences of the corresponding gene products in M. extorquens AM1 against the amino acid sequences of all gene products of the genome of RH AL1. Detailed blast results are given in Table S9 in the supplemental material. RPKM, reads per kilobase million; LutAEF, ABC transporter linked to lanthanide uptake into the cytoplasm (LutA, ABC transporter cytoplasmic binding component; LutE, ABC transporter ATP binding component; LutF, ABC transporter membrane component); LutH, TonB-dependent receptor involved in lanthanide uptake into the periplasm; LanM, lanthanide-binding protein lanmodulin; XoxJ, periplasmic binding protein potentially involved in XoxF activation; XoxG, c_L-type cytochrome accepting electrons from XoxF; XoxD, MxaD homolog interacting with XoxF and XoxG. The color code indicates the expression of genes encoding gene products linked to lanthanide-dependent metabolism. Dotted lines indicate a low degree of homology (≤40%) based on amino acid sequence identity. The presented model for lanthanide uptake and utilization was adapted based on work from the laboratories of Cotruvo (37, 39, 71), Martinez-Gomez and Skovran (57), and Vorholt (40).