Comprehensive Comparative Genomics and Phenotyping of Methylobacterium Species

Abstract

The pink-pigmented facultative methylotrophs (PPFMs), a major bacterial group found in the plant phyllosphere, comprise two genera: Methylobacterium and Methylorubrum. They have been separated into three major clades: A, B (Methylorubrum), and C. Within these genera, however, some species lack either pigmentation or methylotrophy, which raises the question of what actually defines the PPFMs. The present study employed a comprehensive comparative genomics approach to reveal the phylogenetic relationship among the PPFMs and to explain the genotypic differences that confer their different phenotypes. We newly sequenced the genomes of 29 relevant-type strains to complete a dataset for almost all validly published species in the genera. Through comparative analysis, we revealed that methylotrophy, nitrate utilization, and anoxygenic photosynthesis are hallmarks differentiating the PPFMs from the other Methylobacteriaceae. The Methylobacterium species in clade A, including the type species Methylobacterium organophilum, were phylogenetically classified into six subclades, each possessing relatively high genomic homology and shared phenotypic characteristics. One of these subclades is phylogenetically close to Methylorubrum species; this finding led us to reunite the two genera into a single genus Methylobacterium. Clade C, meanwhile, is composed of phylogenetically distinct species that share relatively higher percent G+C content and larger genome sizes, including larger numbers of secondary metabolite clusters. Most species of clade C and some of clade A have the glutathione-dependent pathway for formaldehyde oxidation in addition to the H4MPT pathway. Some species cannot utilize methanol due to their lack of MxaF-type methanol dehydrogenase (MDH), but most harbor an XoxF-type MDH that enables growth on methanol in the presence of lanthanum. The genomes of PPFMs encode between two and seven (average 3.7) genes for pyrroloquinoline quinone-dependent alcohol dehydrogenases, and their phylogeny is distinctly correlated with their genomic phylogeny. All PPFMs were capable of synthesizing auxin and did not induce any immune response in rice cells. Other phenotypes including sugar utilization, antibiotic resistance, and antifungal activity correlated with their phylogenetic relationship. This study provides the first inclusive genotypic insight into the phylogeny and phenotypes of PPFMs.

Comprehensive phylogenomics of Methylobacterium reveals four evolutionary distinct groups and underappreciated phyllosphere diversity

“…Group C always formed the most basal group of Methylobacterium , confirming previous observations (Green and Ardley, 2018; Alessa et al ., 2021; Leducq et al ., 2022). Group B regrouped clades B, formerly Methylorubrum (Green and Ardley, 2018) and B2 (Alessa et al . 2021; corresponding to clade A4 in Leducq et al .…”

“…The 24 newly assembled genomes had 41 to 405 scaffolds (depth: 188-304x) for a total size (5-7Mb) and average GC content (67-70%) in the expected range for Methylobacterium genomes (Dataset S2). We annotated 184 genomes de novo , excluding 29 genomes that were not published at the time of the analysis (Alessa et al ., 2021; Bijlani et al ., 2021) through the same pipeline (RAST) and after excluding hypothetical proteins, repeat and mobile elements, we identified 9,970 unique gene annotations (i.e., regardless of copy number: Dataset S3), with on average 2637 (SD: 210) unique gene annotations per genome. We identified 893 candidate core genes, i.e., genes that were present in a single copy in at least 90% of Methylobacteriaceae genomes.…”

“…We first defined the Methylobacteriaceae core genome from 184 genomes, excluding 29 genomes that were not yet published nor annotated at the time of the analysis (Alessa et al ., 2021; Bijlani et al ., 2021). In these 184 genomes, we identified 9,970 unique gene annotations (i.e., regardless copy number: Dataset S3), with on average 2637 ± 210 unique gene annotations per genome.…”

“…The Methylobacterium reclassification has been pointed out as problematic, because of the low phylogenetic resolution of the 16S rRNA gene, and because no genus name was proposed for strains that were not retained in Methylorubrum or Methylobacterium , which could potentially render either new genus as paraphyletic (Hördt et al ., 2020; Leducq et al ., 2022). Accordingly, the taxonomy of Methylobacterium was reexamined by coupling genome-wide DNA-DNA hybridization and phenotypic information for 63 strains, each representative of a described species (Alessa et al ., 2021). Alessa et al .…”

“…Although more than 60 Methylobacterium species have been described so far (Green and Ardley, 2018; Chen et al ., 2019; Feng et al ., 2020; Jia et al ., 2020; Kim, Chhetri, Kim, Lee, et al ., 2020; Kim, Chhetri, Kim, Kim, et al ., 2020; Ten et al ., 2020; Jiang et al ., 2020; Pascual et al ., 2020; Alessa et al ., 2021), available genomic and phenotypic information was until recently limited to a few model species, mostly from groups B and C, and mostly isolated from anthropogenically impacted environments, and in rare cases from plants (Marx et al ., 2012; Tani et al ., 2015; Minami et al ., 2016; Morohoshi and Ikeda, 2016; Belkhelfa et al ., 2018). Surveys of Methylobacterium diversity associated with plants have mainly focused on the rhizosphere, especially in crop species (Sy et al ., 2001; Jourand et al ., 2004; Grossi et al ., 2020).…”