Speluncibacter jeojiensis gen. nov. sp. nov., a novel bacterium of the order Mycobacteriales isolated from a cave and a proposal of Speluncibacteraceae fam. nov.

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).

Microbacterium aquilitoris sp. nov. and Microbacterium gwkjiense sp. nov., isolated from beach.

Three Gram-reaction-positive bacterial strains, designated KSW-18T, KSW2-22, and KSW4-11T, were isolated from seawater, and two dried seaweed samples collected at Gwakji Beach in Jeju, Republic of Korea, respectively, and their taxonomic positions were examined by a polyphasic approach. The 16S rRNA gene phylogeny showed that strain KSW4-11T was tightly associated with Microbacterium oleivorans NBRC 103075T, while strains KSW-18T and KSW2-22 formed a distinctive subline at the base of a clade including the above two strains. The three isolates showed high sequence similarity with one another (99.7-99.9%; 1-4 nt differences) and Microbacterium oleivorans (99.8-99.9%; 1-3 nt differences). The chemotaxonomic features were typical for the genus Microbacterium; Lysine as the diagnostic diamino acid and N-glycolylated muramic acid of the peptidoglycans, the predominant menaquinones of MK-11, MK-10 and MK-12, the major fatty acids of anteiso-C15:0 and anteiso-C17:0, and the major polar lipids including diphosphatidylglycerol, phosphatidylglycerol, and two or three unidentified glycolipids. In core genome-based phylogenetic tree, strains KSW-18T and KSW2-22 were closely associated with Microbacterium oleivorans NBRC 103075T, while strain KSW4-11T formed a distinctive subline at the base of a clade including the above three strains, in contrast to the 16S rRNA gene tree. Strains KSW-18T and KSW2-22 shared an OrthoANIu of 98.6% and a digital DNA-DNA hybridization of 87.6% with each other, representing that they were strains of a species, while the OrthoANIu and digital DNA-DNA hybridization values between strains KSW-18T and KSW4-11T, and between both of these isolates and all members of the genus Microbacterium were ≤86.5% and ≤30.7%, respectively. The analyses of overall genomic relatedness indices and phenotypic distinctness support that the three isolates represent two new species of the genus Microbacterium. Based on the results obtained here, Microbacterium aquilitoris sp. nov. (type strain KSW-18T = KCTC 49623T = NBRC 115222T) and Microbacterium gwkjiense sp. nov. (type strain KSW4-11T = KACC 23321T = DSM 116380T) are proposed.

Actinomadura terrae sp. nov. and Actinomadura litoris OS3-89, isolated from rhizosphere soil of cactus.

Two mycelium-forming actinobacterial strains, designated OS3-83 T and OS3-89, were isolated from rhizosphere soil of a cactus (Opuntia ficus-indica) sampled on Mara Island, Jeju, Republic of Korea. Both of the isolates were found to grow at 20-37 °C, pH 6.0-10.0 and with 0-2 % (w/v) NaCl. Their taxonomic positions were investigated by a polyphasic approach. Strains OS3-83T and OS3-89 were most closely related to the type strain of Actinomadura litoris (99.5 % and 98.9 % 16S rRNA gene sequence similarity, respectively). Both of the isolates shared 99.2 % sequence similarity to each other. Morphological and chemotaxonomic characteristics supported the affiliation of the two isolates to the genus Actinomadura. 16S rRNA gene phylogeny exhibited that strain OS3-83T formed a tight cluster with A. litoris, while strain OS3-89 occupied a position located remotely from A. litoris. Nevertheless, phylogenomic analysis based on 92 core gene sequences showed that both of the isolates formed a tight clade with A. litoris. The values of average nucleotide identity and digital DNA-DNA hybridization between strain OS3-83T and the closest relative, A. litoris, were 92.2 and 46.2 %, respectively, whereas strain OS3-89 shared an average nucleotide identity value of 97.5 % and a digital DNA-DNA hybridization value of 76.9 % with A. litoris. These results strongly suggested that strain OS3-83T (=KACC 19752T=NBRC 114688T) represents a novel species and strain OS3-89 (=KACC 19753=NBRC 114400) is a strain of A. litoris. On the basis of the data obtained here, strain OS3-83T is considered to represent a new species of the genus Actinomadura, for which the name Actinomadura terrae sp. nov. is proposed.

Four new Microbacterium species isolated from seaweeds and reclassification of five Microbacterium species with a proposal of Paramicrobacterium gen. nov. under a genome-based framework of the genus Microbacterium.

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.

Aeromicrobium stalagmiti sp. nov., isolated from a lava cave.

A Gram-reaction-positive, strictly aerobic, non-sporulating, non-motile, rod-shaped bacterium, designated YC3-14T, was isolated from pieces of stalagmite collected in a lava cave in Jeju, Republic of Korea. Cells showed growth at 15-35 °C, pH 6.0-9.0 and with 0-3 % (w/v) NaCl. Colonies of the cells were circular, smooth, convex and cream in colour. A 16S rRNA gene-based neighbour-joining tree indicated that the organism belonged to the genus Aeromicrobium and formed a sublineage between an Aeromicrobium endophyticum-Aeromicrobium fastidiosum cluster and an Aeromicrobium yanjiei-Aeromicrobium chenweiae cluster. The highest 16S rRNA gene similarity values of strain YC3-14T were with the type strains of A. yanjiei (99.2 %), A. endophyticum (99.1 %), A. fastidiosum (98.8 %), A. ginsengisoli (98.8 %) and A. chenweiae (98.7 %). The cell-wall peptidoglycan contained ll-diaminopimelic acid as the diagnostic diamino acid. The major menaquinone was MK-9(H4). The predominant fatty acids were C18 : 0.10-methyl, C18 : 1 ω9c and C16 : 0. The polar lipids comprised diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, an unidentified phospholipid and two unidentified lipids. The G+C content of the genome DNA was 69.9 mol%. These chemotaxonomic features of the isolate were typical for the genus Aeromicrobium. The genome-based phylogeny showed the same tree topology as the 16S rRNA gene phylogeny. The average nucleotide identity (≤84.5 %) and digital DNA-DNA hybridization (≤27.5 %) values supported that the isolate belongs to a novel species of the genus Aeromicrobium. On the basis of data obtained by a polyphasic approach, strain YC3-14T (=KCTC 49469T=NBRC 114653T) represents a novel species of the genus Aeromicrobium, for which the name Aeromicrobium stalagmiti sp. nov. is proposed.

Vagococcus allomyrinae sp. nov. and Enterococcus larvae sp. nov., isolated from larvae of Allomyrina dichotoma.

The taxonomic positions of two novel strains isolated from larvae of an insect (Allomyrina dichotoma) collected in Jeju, Republic of Korea, were determined by a polyphasic approach. Strain BWB3-3T was closely related to the type strain of Vagococcus salmoninarum, having 97.2 % 16S rRNA gene sequence similarity, whereas strain BWM-S5T formed an independent cluster within the genus Enterococcus in the 16S rRNA gene phylogeny and the closest relative was the type strain of Enterococcus canis (98.1 % sequence similarity). The core gene analysis supported the phylogenetic positions of the isolates revealed by 16S rRNA gene phylogeny. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain BWB3-3T and the type strain of V. salmoninarum were 73.2 and 20.0 %, respectively, whereas strain BWM-S5 T showed an ANI value of 70.9 % with the type strain of Enterococcus canis. The dDDH values between strain BWM-S5T and all the type strains of Enterococcus species were ≤25.1 %. On the basis of the results obtained here, the two isolates are considered to constitute two novel species of the family Enterococcaceae, for which the names Vagococcus allomyrineae sp. nov. and Enterococcus larvae sp. nov. are proposed, with the type strains BWB3-3T (=KCTC 43277T=CCM 9080T) and BWM-S5T (=KACC 22156T=CCM 9075T), respectively.

Tenebrionicola larvae gen. nov., sp. nov., isolated from larvae of mealworm Tenebrio molitor L., and a proposal to transfer Erwinia teleogrylli Liu et al. 2016 to a new genus Entomohabitans as Entomohabitans teleogrylli comb. nov.

Two enterobacterial strains, designated YMB-R21T and YMB-R22, were isolated from larvae of mealworm Tenebrio molitor L. and examined for their taxonomic characteristics. A 16S rRNA gene-based neighbour-joining tree showed that the two isolates formed two distinct sublineages within the family Enterobacteriaceae and were separated from other genera of the family. The isolates showed 16S rRNA gene sequence similarity of 98.9 % to each other and ≤96.5 % to members of the order Enterobacteriales. The phylogenomic analysis based on 92 singly-copy core genes showed that the two isolates belonged to the family Enterobacteriaceae and formed a distinct sublineage at a position located remotely from the genera of the family. The loosely associated members were the type strain of Erwinia teleogrylli and members of the genus Shimwellia. Average nucleotide identity and digital DNA-DNA hybridization values showed that the isolates represented members of a novel species in the family Enterobacteriaceae. The values of amino acid identity between the two isolates and the closest relatives were 74.5-75.0 % with the type strain of E. teleogrylli and 74.5-74.8 % with the type strains of two Shimwellia species, while E. teleogrylli showed the amino acid identity values of 76.3-76.5 % with two Shimwellia species. Based on the results obtained here, we propose a new genus Tenebrionicola with the description of Tenebrionicola larvae sp. nov. (type strain YMB-R21T=KCTC 82597T=CCM 9152T and strain YMB-R22=KCTC 82598=CCM 9153), with the transfer of Erwinia teleogrylli Liu et al. 2016 to a new genus Entomohabitans as Entomohabitans teleogrylli comb. nov. (type strain SCU-B244T=CGMCC 1.12772T=DSM 28222T=KCTC 42022T).

Jinshanibacter allomyrinae sp. nov., isolated from larvae of Allomyrina dichotoma, proposal of Insectihabitans xujianqingii gen. nov., comb. nov. and emended descriptions of the genera Jinshanibacter, Limnobaculum and Pragia.

Taxonomic positions of four Gram-negative bacterial strains, which were isolated from larvae of two insects in Jeju, Republic of Korea, were determined by a polyphasic approach. Strains CWB-B4, CWB-B41 and CWB-B43 were recovered from larvae of Protaetia brevitarsis seulensis, whereas strain BWR-B9T was from larvae of Allomyrina dichotoma. All the isolates grew at 10-37 °C, at pH 5.0-9.0 and in the presence of 4 % (w/v) NaCl. The 16S rRNA gene phylogeny showed that the four isolates formed two distinct sublines within the order Enterobacteriales and closely associated with members of the genus Jinshanibacter. The first group represented by strain CWB-B4 formed a tight cluster with Jinshanibacter xujianqingii CF-1111T (99.3 % sequence similarity), whereas strain BWR-B9T was most closely related to Jinshanibacter zhutongyuii CF-458T (99.5 % sequence similarity). The 92 core gene analysis showed that the isolates belonged to the family Budviciaceae and supported the clustering shown in 16S rRNA gene phylogeny. The genomic DNA G+C content of the isolates was 45.2 mol%. A combination of overall genomic relatedness and phenotypic distinctness supported that three isolates from Protaetia brevitarsis seulensis are different strains of Jinshanibacter xujianqingii, whereas one isolate from Allomyrina dichotoma represents a new species of the genus Jinshanibacter. On the basis of results obtained here, Jinshanibacter allomyrinae sp. nov. (type strain BWR-B9T=KACC 22153T=NBRC 114879T) and Insectihabitans xujianqingii gen. nov., comb. nov. are proposed, with the emended descriptions of the genera Jinshanibacter, Limnobaculum and Pragia.

Rouxiella aceris sp. nov., isolated from tree sap and the emended description of the genus Rouxiella.

Polyphasic taxonomic studies were performed for the seven strains, which were isolated from sap extracted from Acer pictum in Mt. Halla in Jeju, Republic of Korea. Cells of all the isolates were Gram-reaction-negative, facultatively anaerobic, short rods and contained the major isoprenoid quinone of Q-8, the predominant fatty acids of C16:0 and C17:0 cyclo and the major polar lipids including phosphatidylethanolamine, phosphatidylglycerol and an unidentified aminophospholipid. The G + C contents of the genomic DNAs were 50.6-51.3%.The 16S rRNA gene-based phylogeny exhibited that the seven isolates formed two distinct sublines within the family Yersiniaceae. In the 92 core gene analysis, strain SAP-1T formed a subline at the base of radiation of the genus Rouxiella and its assignment to the genus Rouxiella was supported by high amino acid identity values (82.0-83.4%), albeit with sharing low 16S rRNA gene identities (96.0-96.9%). The average nucleotide identity and digital DNA-DNA hybridisation values together with phenotypic differences showed that strains SAP-1T, SAP-7, SAP-8 and SAP-13 belonged to a new species of the genus Rouxiella, while strains SAP-2, SAP-3 and SAP-27 were strains of Rouxiella silvae. On the basis of data obtained here, Rouxiella aceris sp. nov. (type strain, SAP-1T = KCTC 72599T = CCM 9078T) is proposed, with the emended description of the genus Rouxiella.

Hongsoonwoonella zoysiae gen. nov., sp. nov., a new member of the family Stappiaceae isolated from a tidal mudflat.

A Gram stain-negative bacterial strain, designated SY4-7T, was isolated from rhizosphere mudflat of a halophyte (Zoysia sinica) collected around Seonyu Island, Republic of Korea. Cells of the organism were strictly aerobic, non-sporulating, non-motile rods and grew at 20-42 °C, pH 6-8 and 1-6% (w/v) NaCl. The 16S rRNA gene-based phylogenetic analyses revealed that strain SY4-7T formed an independent cluster separated from the recognized genera of the family Stappiaceae, which was also supported by phylogenomic analysis-based 92-core gene sequences. The type stains of the phylogenetically closest relatives were Stappia indica (95.6% sequence similarity), Stappia stellulata (95.1%) and Roseibium hamelinense (95.1%). The isoprenoid quinone was Q-10. The polar lipids consisted of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, three unidentified aminophospholipids, an unidentified phosphoglycolipid, an unidentified aminolipid, two unidentified phospholipids and an unidentified lipid. The major cellular fatty acids are C18:1ω7c and C19:1 cyclo ω8c. The G + C content of the genomic DNA is 60.7%. Discrimination of the organism from all the recognized genera of the family Stappiaceae was apparent by the chemotaxonomic and phylogenetic features. Based on the results presented here, strain SY4-7T (= KCTC 72226T = NBRC 113902T) represents a novel species of a new genus in the family Stappiaceae, for which the name Hongsoonwoonella zoysiae sp. nov. is proposed.

Nitratireductor kimnyeongensis sp. nov., isolated from seaweed.

An aerobic, Gram-negative, catalase-positive, oxidase-positive bacterium was isolated from a dried seaweed sample collected from Kimnyeong Beach in Jeju, Republic of Korea. The cells of the organism, designated strain KY 101(T), were rods (0.4-0.5x1.2-2.7 mum) and motile by means of flagella. The colonies of the cells were 0.5-1 mm in diameter, smooth, circular, convex and light yellow in colour. The isolate showed growth at 10-40 degrees C, pH 6.1-12.1 and in the presence of 7 % NaCl. The major fatty acid was summed feature C(18 : 1)omega7c/C(18 : 1)omega9c/C(18 : 1)omega12t and the DNA G+C content was 60.4 mol%. 16S rRNA gene sequence studies showed that the organism was phylogenetically related to the family Phyllobacteriaceae, with Nitratireductor aquibiodomus (99.1 % sequence similarity) as the closest neighbour. The level of DNA-DNA relatedness between strain KY 101(T) and N. aquibiodomus DSM 15645(T) was 18.4-23.2 % (duplicate measurements). The combination of phenotypic differences and DNA-DNA hybridization data supports the suggestion that the isolate represents a novel species of the genus Nitratireductor, for which the name Nitratireductor kimnyeongensis sp. nov. is proposed. The type strain is strain KY 101(T) (=KACC 11904(T)=JCM 14851(T)).