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

Abstract

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.

Keywords: lanthanide, methylotroph, XoxF, methanol dehydrogenase, Methylobacterium species.

Figure 1

A schematic of the methylotrophy pathway. Dashed lines indicate possible direct oxidations.

Figure 2

Molecular phylogenetic analysis of MDH-like proteins found in strain 22A (in boldface) and other related sequences. The evolutionary history was inferred by using the maximum likelihood method based on the Jones-Taylor-Thornton (JTT) matrix-based model [48]. The tree is drawn to scale. Evolutionary analyses were conducted in MEGA7 [49]. Bar, average number of amino acid substitutions per site.

Figure 3

Left panel, growth of single-gene-remaining mutants of MDH-like genes and a mutant without any MDH-like gene (re-0). Right panel, growth of the suppression mutants. Cultivation was done in 96-well plates under shaking at 300 rpm. The results are presented as the average ± SD (technical triplicates).

Figure 4

(A) Growth of strain 22A wild-type and ΔmxbD on (left panel) succinate and (right panel) methanol in the presence/absence of La³⁺. Circles, wild-type (WT); squares, ΔmxbD; filled symbols, in the presence of La³⁺; open symbols, in the absence of La³⁺. The results are presented as the average ± SD (technical triplicates). (B) Quantification of mxaF and xoxF1 expression in strain 22A wild-type and ΔmxbD, grown on methanol in the presence/absence of La³⁺. ΔmxbD in +La condition was grown on methanol plus succinate. The results are presented as the average ± SD (biological triplicates). The compact letter display indicates statistically significant differences (p < 0.05, ANOVA and Tukey’s multiple comparison test) among the means for each gene. (C) Growth of ΔxoxF1ΔmxbD complemented by wild-type and mutated mxbD on methanol in the presence/absence of La³⁺. Open circle, vector control; closed circle, ΔxoxF1ΔmxbD (mxbD-wt); squares, ΔxoxF1ΔmxbD (mxbD-re-mxaFSup); triangles, ΔxoxF1ΔmxbD (mxbD-ΔxoxF1Sup). The results are presented as the average ± SD (technical triplicates).

Figure 5

(A) Purification of XoxF1-His expressed in strain 22A. SDS-PAGE of crude cell extract, flow-through fraction through Ni-NTA column, and purified MDH in eluate fractions. An arrow indicates the purified XoxF1-His. (B) The activity of XoxF1-His against methanol and formaldehyde of varying concentrations. The data were used to calculate kinetic parameters. The results are presented as the average ± SD (technical triplicates).

Figure 6

Hgd activity in the cell-free extracts of the wild-type, Δfae1Δfae2, ΔxoxF1SupΔfae1Δfae2, ΔmxaFΔfae1Δfae2, and Δhgd, grown on methanol plus succinate in the (A) presence and (B) absence of La³⁺. Filled bar, +La³⁺; open bar, -La³⁺; n.d., not detected. The results are presented as the average ± SD (biological triplicates). Statistical tests were done with ANOVA and Tukey’s multiple comparison test independently for each dataset (presence/absence of La³⁺), and p values for the comparisons with the wild-type data are shown.

Figure 7

Growth of various formaldehyde oxidation-pathway mutants constructed in the wild-type, ΔxoxF1Sup, and ΔmxaF backgrounds, in methanol and methanol plus succinate liquid medium in the presence and absence of La³⁺. FD-3 (Δfae1Δfae2Δhgd), FD-4 (Δfae1Δfae2ΔmchΔhgd), FD-5 (ΔxoxF1Δfae1Δfae2ΔmchΔhgd), FD-6 (ΔexaFΔfae1Δfae2ΔmchΔhgd), and FD-7 (ΔxoxF1ΔexaFΔfae1Δfae2ΔmchΔhgd) correspond to all three genetic backgrounds, except that FD-5 to FD-7 were constructed only for the mxaF background. The results are presented as the average ± SD (technical triplicates).