Lee, Soon Dong

Two Gram-stain-positive, aerobic, non-spore-forming, non-motile, irregular rod-shaped actinobacteria, designated as D2-41T and D3-21, were isolated from soil samples collected in a natural cave in Jeju, Republic of Korea. Both of the isolates were shown to share 100 % 16S rRNA sequence identity. The cell wall contained meso-diaminopimelic acid, arabinose and galactose. The predominant menaquinone was MK-8(H2). The polar lipids contained phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, an unidentified aminolipid, an unidentified aminoglycolipid, an unidentified phospholipid and two unidentified lipids. The predominant fatty acids were C16 : 0 and summed feature 3 (C16 : 1  ω7c and/or iso-C15 : 0 2-OH). Mycolic acids of C30–C38 were present. The 16S rRNA gene trees showed that the organisms occupied a distinct position remotely located from recognized genera within the order Mycobacteriales, albeit with the 16S rRNA gene similarities of 97.0–97.1 % with Rhodococcus olei, Rhodococcus rhodnii and Rhodococcus triatomae. The genome sizes and DNA G+C contents of strains D2-41T and D3-21 were 4.77–4.88 Mbp and 69.8 mol%, respectively. Both of the isolates shared an average nucleotide identity of 99.4 % and digital DNA–DNA hybridization of 95.2 % to each other, revealing that strains D2-41T and D3-21 belonged to the same species. In the core genome-based phylogenomic tree, both of the isolates were found to be closely associated with members of the genus Tomitella. However, strains D2-41T and D3-21 revealed the highest amino acid identity values (mean 66.5 %, range 66.2–67.0 % with the genus Prescottella of the family Nocardiaceae, followed by the genus Tomitella (mean 64.1 %, range 63.6–64.7 %) of the family Tomitellaceae. Based on the combined data obtained here, the novel isolates belong to a new genus of the new family for which the name Speluncibacter jeojiensis gen. nov. sp. nov. is proposed, with Speluncibacteraceae fam. nov. The type strain is strain D2-41T (=KACC 17930T=DSM 101875T).

The taxonomic relationships of 10 strains isolated from seaweeds collected from two beaches in Republic of Korea were studied by sequencing and analyses of 16S rRNA genes and whole genomes. For the construction of a more reliable and robust 16S rRNA gene phylogeny, the authentic and nearly complete 16S rRNA gene sequences of all the Microbacterium type strains were selected through pairwise comparison of the sequences contained in several public databases including the List of Prokaryotic names with Standing in Nomenclature (LPSN). The clustering of the ten study strains into five distinct groups was apparent in this single gene-based phylogenetic tree. In addition, the 16S rRNA gene sequences of a few type strains were shown to be incorrectly listed in LPSN. An overall phylogenomic clustering of the genus Microbacterium was performed with a total of 113 genomes by core genome analysis. As a result, nine major (≥ three type strains) and eight minor (two type strains) clusters were defined mostly at gene support index of 92 and mean intra-cluster OrthoANIu of >80.00%. All of the study strains were assigned to a Microbacterium liquefaciens clade and distributed further into four subclusters in the core genome-based phylogenetic tree. In vitro phenotypic assays for physiological, biochemical, and chemotaxonomic characteristics were also carried out with the ten study strains and seven closely related type strains. Comparison of the overall genomic relatedness indices (OGRI) including OrthoANIu and digital DNA–DNA hybridization supported that the study strains constituted four new species of the genus Microbacterium. In addition, some Microbacterium type strains were reclassified as members of preexisting species. Moreover, some of them were embedded in a new genus of the family Microbacteriaceae based on their distinct separation in the core genome-based phylogenetic tree and amino acid identity matrices. Based on the results here, four new species, namely, Microbacterium aurugineum sp. nov., Microbacterium croceum sp. nov., Microbacterium galbinum sp. nov., and Microbacterium sufflavum sp. nov., are described, along with the proposal of Paramicrobacterium gen. nov. containing five reclassified Microbacterium species from the “Microbacterium agarici clade”, with Paramicrobacterium agarici gen. nov., comb. nov. as the type species.