Aerobic Methoxydotrophy: Growth on Methoxylated Aromatic Compounds by Methylobacteriaceae

Abstract

Pink-pigmented facultative methylotrophs have long been studied for their ability to grow on reduced single-carbon (C1) compounds. The C1 groups that support methylotrophic growth may come from a variety of sources. Here, we describe a group of Methylobacterium strains that can engage in methoxydotrophy: they can metabolize the methoxy groups from several aromatic compounds that are commonly the product of lignin depolymerization. Furthermore, these organisms can utilize the full aromatic ring as a growth substrate, a phenotype that has rarely been described in Methylobacterium. We demonstrated growth on p-hydroxybenzoate, protocatechuate, vanillate, and ferulate in laboratory culture conditions. We also used comparative genomics to explore the evolutionary history of this trait, finding that the capacity for aromatic catabolism is likely ancestral to two clades of Methylobacterium, but has also been acquired horizontally by closely related organisms. In addition, we surveyed the published metagenome data to find that the most abundant group of aromatic-degrading Methylobacterium in the environment is likely the group related to Methylobacterium nodulans, and they are especially common in soil and root environments. The demethoxylation of lignin-derived aromatic monomers in aerobic environments releases formaldehyde, a metabolite that is a potent cellular toxin but that is also a growth substrate for methylotrophs. We found that, whereas some known lignin-degrading organisms excrete formaldehyde as a byproduct during growth on vanillate, Methylobacterium do not. This observation is especially relevant to our understanding of the ecology and the bioengineering of lignin degradation.

Comprehensive Phylogenomics of Methylobacterium Reveals Four Evolutionary Distinct Groups and Underappreciated Phyllosphere Diversity

“…For example, the evolutionary distinction between groups A and D, and their importance in Methylobacterium diversity, could not have been revealed without a thorough investigation of diversity in the phyllosphere, from which the majority of candidate species from groups A, B, and D were isolated. A recent survey of Methylobacterium in metagenomes from various biomes ( Lee et al 2022 ) also suggested the association of groups A (represented by M. pseudosasicola and M. radiotolerans in Lee et al 2022 study), B and especially D (represented by M. gossipiicola and Methylobacterium sp. Leaf 88) with the aerial part of plants.…”

“…On the contrary, groups B and C included most Methylobacterium model species frequently used in the lab and isolated from anthropogenic environments. While group B is occasionally identified on and isolated from the surface of leaves ( Leducq et al 2022 ; Lee et al 2022 ), group C is rarely, if ever, found in the phyllosphere, and seems to be more widespread in soil and in aquatic environments, often in association with plant roots ( Lee et al 2022 ). Interestingly, authors from a recent study estimated that Rhizobiales common ancestor likely had a free-living lifestyle, while Methylobacterium groups A, B and D’s common ancestor likely had a plant-associated lifestyle (node 1 in fig.…”

“…The ancestral lifestyle of Methylobacterium , and more widely, of Methylobacteriaceae , is more unclear. The isolation source of group C genomes, as well as the two sister genera of Methylobacterium , Enterovirga and Microvirga , and their survey in metagenomes ( Lee et al 2022 ) indicate that these three groups are mostly found with soils, sometimes in association with the rhizosphere. These observations suggest that Methylobacteriaceae and Methylobacterium ’s ancestors inhabited soils, and were occasionally associated with plants, for instance in the rhizosphere, and that Methylobacterium groups A/B/D’s association with the phyllosphere occurred after divergence from group C. The exact origin and nature of this association is an open question, but the smaller genome size, gene copy number, GC content and to a lower extent, mobile element number, we observed in group A/B/D in comparison with group C could be the genomic signatures of a progressive specialization to life on plants ( Nishida 2012 ; Levy et al 2018 ), among other things through the evolution of metabolic pathways in response to contrasted nutrient availability between the soil and the phyllosphere ( Lee et al 2022 ; Alessa et al 2021 ).…”

“…The isolation source of group C genomes, as well as the two sister genera of Methylobacterium , Enterovirga and Microvirga , and their survey in metagenomes ( Lee et al 2022 ) indicate that these three groups are mostly found with soils, sometimes in association with the rhizosphere. These observations suggest that Methylobacteriaceae and Methylobacterium ’s ancestors inhabited soils, and were occasionally associated with plants, for instance in the rhizosphere, and that Methylobacterium groups A/B/D’s association with the phyllosphere occurred after divergence from group C. The exact origin and nature of this association is an open question, but the smaller genome size, gene copy number, GC content and to a lower extent, mobile element number, we observed in group A/B/D in comparison with group C could be the genomic signatures of a progressive specialization to life on plants ( Nishida 2012 ; Levy et al 2018 ), among other things through the evolution of metabolic pathways in response to contrasted nutrient availability between the soil and the phyllosphere ( Lee et al 2022 ; Alessa et al 2021 ). For instance, some genes involved in the metabolism of aromatic compounds resulting from lignin degradation are present in Microvirga and Methylobacterium group C, but absent from other Methylobacterium groups ( Lee et al 2022 ), suggesting that these functions essential for ground lifestyle were lost in A/B/D group after they divergence with group C. Inversely, Methylobacterium from group A/B/D arbor a larger panoply of genes allowing the use of methanol, available in the phyllosphere, than group C, while most of these pathways are absent from Microvirga ( Alessa et al 2021 ), suggesting that the transition from soil to phyllosphere lifestyle in Methylobacterium also coincided with the acquisition and diversification of methylotrophic pathways.…”

Lanthanide-dependent isolation of phyllosphere methylotrophs selects for a phylogenetically conserved but metabolically diverse community

“…Recently, facultative methylotrophy has expanded to include species capable of growth on sugars within the extorquens clade (21) and species capable of growth on methoxylated aromatic acids within the nodulans and aquaticums clades of Methylobacterium (22). Methylotrophic growth on the latter is predicted to occur via an initial demethoxylation of the methoxy group that is released as formaldehyde to generate protocatechuic acid; formaldehyde can be assimilated or dissimilated via methylotrophic pathways (22). The capability to utilize different carbon sources, such as methanol, ethanol, and multicarbon substrates , may explain why methylotrophs are so well-suited to life in the phyllosphere (2,5,23) where metabolite availability changes dynamically (2).…”