Kim, Song-Gun

The order Sphingomonadales strains are globally distributed in various biomes and are renowned for their biodegradable and biosynthesis capabilities. At present, it consists of 4 families and 49 genera making it the third largest order within the class Alphaproteobacteria. However, their taxonomy remains complex, especially due to polyphyly in the family Sphingomonadaceae. In this study, we collected 429 Sphingomonadales type strain genomes, reconstructed robust phylogenomic relationships, and proposed delineation thresholds at the genus and family levels based on average amino acid identities (AAI) and evolutionary distances (ED). Based on the maximum-likelihood and Bayesian phylogenomic trees reconstructed by two molecular sets determined by orthologous sequence identity and the Genome Taxonomy Database, the consensus degree values were all higher than 90%, revealing that those phylogenomic trees had similar topological structures. By confirming monophyletic taxa and determining stable nodes, we reclassified the order Sphingomonadales into thirteen families including nine novel ones. AAI calculations indicated that the average intra-family AAI values ranged from 0.62 to 0.84, while inter-family ones were 0.51 to 0.60. ED summaries demonstrated that the average and median intra-family ED values were 0.16 to 0.57, and inter-family ones ranged from 0.50 to 1.22. Comparisons of AAI and ED values calculated by using genomic and phylogenetic analyses supported that those 13 families were significantly separated with p values < 2.2×10−16. Thus, it was speculated that the AAI and ED thresholds for distinguishing different families were <0.6 and >0.5, respectively. Additionally, we reclassified 163 species into new genera with their phylogenetic topologies, according to the previous genus AAI and ED boundaries of 0.7 and 0.4. Our study is the first genomic-based study of the order Sphingomonadales and will promote further insights into the evolution of this order.

Strain IT6T, a thermoacidophilic and facultative methane-oxidizing bacterium, was isolated from a mud–water mixture collected from Pisciarelli hot spring in Pozzuoli, Italy. The novel strain is white when grown in liquid or solid media and forms Gram-negative rod-shaped, non-flagellated, non-motile cells. It conserves energy by aerobically oxidizing methane and hydrogen while deriving carbon from carbon dioxide fixation. Strain IT6T had three complete pmoCAB operons encoding particulate methane monooxygenase and genes encoding group 1d and 3b [NiFe] hydrogenases. Simple carbon–carbon substrates such as ethanol, 2-propanol, acetone, acetol and propane-1,2-diol were used as alternative electron donors and carbon sources. Optimal growth occurred at 50–55°C and between pH 2.0–3.0. The major fatty acids were C18 : 0, C15 : 0 anteiso, C14 : 0 iso, C16 : 0 and C14 : 0, and the main polar lipids were phosphatidylethanolamine, aminophospholipid, phosphatidylglycerol, diphosphatidylglycerol, some unidentified phospholipids and glycolipids, and other unknown polar lipids. Strain IT6T has a genome size of 2.19 Mbp and a G+C content of 40.70 mol%. Relative evolutionary divergence using 120 conserved single-copy marker genes (bac120) and phylogenetic analyses based on bac120 and 16S rRNA gene sequences showed that strain IT6T is affiliated with members of the proposed order ‘Methylacidiphilales’ of the class Verrucomicrobiia in the phylum Verrucomicrobiota . It shared a 16S rRNA gene sequence identity of >96 % with cultivated isolates in the genus ' Methylacidiphilum ' of the family 'Methylacidiphilaceae’, which are thermoacidophilic methane-oxidizing bacteria. ‘ Methylacidiphilum sp.’ Phi (100 %), ‘Methylacidiphilum infernorum’ V4 (99.02 %) and ‘ Methylacidiphilum sp.’ RTK17.1 (99.02 %) were its closest relatives. Its physiological and genomic properties were consistent with those of other isolated ‘ Methylacidiphilum ’ species. Based on these results, we propose the name Methylacidiphilum caldifontis gen. nov., sp. nov. to accommodate strain IT6T (=KCTC 92103T=JCM 39288T). We also formally propose that the names Methylacidiphilaceae fam. nov. and Methylacidiphilales ord. nov. to accommodate the genus Methylacidiphilum gen. nov.