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.

Comprehensive Comparative Genomics and Phenotyping of Methylobacterium Species

“…The second group (G2), represented by the XoxF2 from strain AM1, is found mostly in clades A and C. The third group (G3) contains XoxF1 from Mb. aquaticum strain 22A, an Ln 3+ -dependent MDH that also participates in the regulation of mxaF in this strain ( Masuda et al, 2018 ; Yanpirat et al, 2020 ). This group is found distinctively in clade C members.…”

“…Group 6 is found mostly in clade C1 members and contains a homolog of ExaF found in Mb. aquaticum strain 22A ( Yanpirat et al, 2020 ). Group 7 includes PQQ-ADH type 6a and is found mostly in members of clade C1.…”

Unravelling Formaldehyde Metabolism in Bacteria: Road towards Synthetic Methylotrophy

“…Methylene-H 4 MPT is further converted to methenyl-H 4 MPT in the reaction catalyzed by methylene-H 4 MPT dehydrogenase and followed by several reactions leading to the formation of CO 2 [41]. A comprehensive analysis was performed to elucidate the distribution of the H 4 MPTdependent pathway among methylotrophic bacteria, where it was shown that it is present not only in α-proteobacteria that possess the serine cycle for formaldehyde assimilation but also methylotrophic γand β-proteobacteria, which assimilate formaldehyde through the RuMP cycle (Table 1, Section 3.1) [39,43,44,51,52]. The H 4 MPT-dependent formaldehyde dissimilation pathway was detected neither in Amycolatopsis methanolica nor in B. methanolicus MGA3 [39].…”

“…S-Formyl-GSH is then hydrolyzed by S-formyl-GSH hydrolase (Fgh) to GSH and formate, with the latter being subsequently oxidized to CO 2 (Figure 2) [54]. This pathway is present and functional in different α-proteobacteria, for example, Methylobacterium aquaticum 22A, P. denitrificans, Rhodobacter sphaeroides, and Rhodopseudomonas acidophila (Table 1) [40,51,54,[57][58][59][60].…”

“…The PQQ-containing formaldehyde dehydrogenase from M. capsulatus Bath is a membrane-bound enzyme coupled to the electron transport chain via a b-type cytochrome or a quinone. Furthermore, the PQQ-lanthanide (Ln)-dependent methanol dehydrogenases (Mdh) XoxF1 from M. extorquens AM1, M. aquaticum 22A, or Methylacidiphilum fumariolicum SolV catalyze the oxidation of formaldehyde in vitro [51,[66][67][68]. However, while in M. aquaticum 22A XoxF1 was shown to be functional as a formaldehyde detoxification strategy in vivo, the M. extorquens AM1-derived XoxF1 does not exhibit that function, and therefore M. extorquens AM1 relies on the activity of the H 4 MPT-dependent pathway to maintain formaldehyde levels below the toxicity threshold [51,68].…”