Rhodoferax potami sp. nov. and Rhodoferax mekongensis sp. nov., isolated from the Mekong River in Thailand.

Four newly discovered Gram-stain-negative bacteria, designated as BL00010T, BL00058, D8-11T and BL00200, were isolated from water samples collected at three hydrological monitoring stations (namely Chiang Saen, Chiang Khan and Nong Khai) located along the Mekong River in Thailand. An investigation encompassing phenotypic, chemotaxonomic and genomic traits was conducted. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that all four isolates represented members of the genus Rhodoferax. These isolates were closely related to Rhodoferax bucti KCTC 62564T with a similarity of 99.59%. The major fatty acids of the four novel isolates included C16:0 and C16:1ω7c and/or C16 : 1ω6c, whereas the major respiratory quinone was identified as ubiquinone Q-8. In addition, phosphatidylethanolamine was identified as a major polar lipid in these bacteria. The genomes of BL00010T, BL00058, D8-11T and BL00200 were similar in size (3.88-4.01 Mbp) and DNA G+C contents (59.5, 59.3, 59.5 and 59.3 mol%, respectively). In contrast to R. bucti KCTC 62564T and Rhodoferax aquaticus KCTC 32394T, the newly discovered species possessed several genes involved in nitrite and nitrile metabolism, which may be related to their unique adaptation to nitrile-rich environments. From the results of the pairwise analysis of average nucleotide identity of the whole genome and digital DNA-DNA hybridisation, it was evident that BL00010T and D8-11T represented two novel species, for which we propose the nomenclature Rhodoferax potami sp. nov., with the type strain BL00010T (TBRC 17198T = NBRC 116413T), and Rhodoferax mekongensis sp. nov., with the type strain D8-11T (TBRC 17307T = NBRC 116415T).

Commensalibacter nepenthis sp. nov. and Commensalibacter oyaizuii sp. nov., from fluid in a Nepenthes pitcher cup and a butterfly (Junonia lemonias) in Thailand.

Two novel Gram-negative, aerobic, rod-shaped, non-motile bacteria, strains TBRC 10068T and TBRC 16381T, were isolated from a fluid sample from a close-pitcher cup (Nepenthes gracilis) and an insect sample (Junonia lemonias), respectively. Comparing the 16S rRNA gene sequences with those found in EzBioCloud's publicly available databases revealed that the two strains exhibited a close genetic relationship with Commensalibacter intestini A911T; the calculated sequence similarities were 98.56 and 97.70  %, respectively. The average nucleotide identity and digital DNA-DNA hybridization values of the two Commensalibacter strains, as well as those of their closely related type strains, were found to be lower than the species demarcation threshold of 95 and 70 %, respectively. The phylogenomic analysis of strains TBRC 10068T and TBRC 16381T showed that they belong to the genus Commensalibacter. However, they formed distinct lineages separate from all other strains of Commensalibacter by use of 81 bacterial core genes. In addition, the comparative genomic analysis revealed that the core orthologues of strains TBRC 10068T and TBRC 16381T, compared to the closely related type strains of Commensalibacter species, had distinct genetic profiles. Strain TBRC 10068T contained 163 unique genes, whereas strain TBRC 16381T contained 83. The three Commensalibacter species possessed Q-9 as the primary isoprenoid quinone homologue. The results of a polyphasic taxonomic investigation indicated that strains TBRC 10068T and TBRC 16381T represent two separate new species within the genus Commensalibacter. The species were designated as Commensalibacter nepenthis sp. nov. with the type strain TBRC 10068T (=KCTC 92798T) and Commensalibacter oyaizuii sp. nov. with the type strain TBRC 16381T (=KCTC 92799T).

Leeia speluncae sp. nov., isolated from Stegodon Sea Cave in Thailand.

A novel Gram-stain-negative, obligately aerobic, short rod-shaped and motile bacterium, designated strain BC00092T, was isolated from brackish ground water collected in Stegodon Sea Cave located at Satun UNESCO Global Geopark, Satun Province, Thailand. The phylogenetic analysis of BC00092T based on 16S rRNA gene sequences revealed that the strain represented a member of the genus Leeia and was closely related to Leeia oryzae DSM 17879T (96.68 %) and Leeia aquatica IMCC25680T (94.89 %). The average nucleotide identity and digital DNA-DNA hybridization values calculated from the whole-genome sequences between BC00092T and closely related type strains of species within the family Leeiaceae were lower than the species demarcation threshold values of 95 and 70 %, respectively. Moreover, five conserved signature indels of members of the family Leeiaceae were found in the protein sequences from the annotated assembled genome of BC00092T. According to the results of the polyphasic taxonomic study, strain BC00092T represents a novel species within the genus Leeia, for which the name Leeia speluncae sp. nov. is proposed. The type strain is BC00092T (TBRC 13508T = KCTC 92111T).

Novosphingobium kaempferiae sp. nov., a phosphate-solubilizing bacterium isolated from stem of Kaempferia marginata Carey.

A Gram-stain-negative, aerobic, rod-shaped, non-spore-forming and yellow-pigment-producing bacterium, designated as Sx8-5T, was isolated from stem tissue of Kaempferia marginata Carey in Kanchanaburi Province, Thailand. The strain exhibited tricalcium phosphate solubilizing activity. Its taxonomic position was investigated using a polyphasic approach. Sx8-5T grew at 25-37 °C (optimum 30 °C), pH 6-9 (optimum 7) and with 0 and 1% NaCl (optimum 0 %). According to the 16S rRNA gene phylogeny, Sx8-5T represents a member of genus Novosphingobium and shared the highest sequence similarities to Novosphingobium barchaimii LL02T of 99.4 % and shared sequence similarities with other species of the genus Novosphingobium of less than 99.4 %. The whole-genome size was 5.7 Mb, comprised of one contig, with a DNA G+C content of 66 %. The average nucleotide identity using BLASTn (ANIb) or MUMMER (ANIm) values for whole genome comparisons between Sx8-5T and Novosphingobium barchaimii LL02T and six closely related type strains were 72.33-82.14 % and 83.82-87.38 %, respectively, and the digital DNA-DNA hybridization (dDDH) values ranged from 21.0 to 28.6% when compared with the type strains of the members of the genus Novosphingobium. Major fatty acids were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C16 : 0 and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), respectively. Polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, unidentified phospholipids and unidentified polar lipids. The major isoprenoid quinone was Q-10. According to results obtained using a polyphasic approach, Sx8-5T represents a novel species of the genus Novosphingobium, the name Novosphingobium kaempferiae sp. nov. is proposed. The type strain is Sx8-5T (=JCM 35076T =TBRC 15600T).

Neokomagataea anthophila sp. nov., an osmotolerant acetic acid bacterium isolated in Thailand and emended description of the genus Neokomagataea.

A novel Gram-stain-negative, rod-shaped, non-motile, aerobic bacterium isolated from a sea bean flower [Canavalia rosea (Sw.) DC.] collected in Surat Thani Province, Thailand, and designated as AH18T was characterized on the basis of polyphasic taxonomy. The phylogenetic analysis of 16S rRNA gene revealed that strain AH18T represented a member of the genus Neokomagataea. In the 16S rRNA gene sequence analysis, the strain's closest phylogenetic neighbour was Neokomagataea thailandica TBRC 376T. The draft genome size of strain AH18T was 2613495 bp, and its DNA G+C content was 52.0 mol%. The strain showed 90.3 and 76.3% pairwise-determined whole-genome average nucleotide identity and 39.8 and 19.6% digital DNA-DNA hybridization values with N. thailandica TBRC 376T and N. tanensis TBRC 7768T, respectively. The 16S rRNA gene sequences and phylogenomic analysis revealed that the strain clustered with the members of the genus Neokomagataea but was located in a distinct branch closely related to N. thailandica TBRC 376T. The predominant cellular fatty acids of the strain were summed feature 8 (C18:1 ω6c and/or C18:1 ω7c), C16:0 and C18:1 2OH (>5%). The major respiratory ubiquinone was Q-10. In addition, strain AH18T was substantiated by differences in several physiological characteristics and by MALDI-TOF profiling. On the basis of the results obtained from phenotypic, chemotaxonomic, phylogenetic and genomic analyses, the strain clearly represented a novel species within the genus Neokomagataea, for which the name Neokomagataea anthophila sp. nov. (AH18T=TBRC 2177T=NBRC 115156T) is proposed. An emended description of the genus Neokomagataea is also given.

Acetobacter garciniae sp. nov., an acetic acid bacterium isolated from fermented mangosteen peel in Thailand.

Two isolates, MS16-SU-2T and MS18-SU-3, obtained from fermented mangosteen peel in vinegar were suggested to constitute a new species assignable to the genus Acetobacter based on the results of 16S rRNA gene sequencing. The two isolates showed the highest sequence similarity (98.58%) to Acetobacter tropicalis NBRC 16470T and Acetobacter senegalensis LMG 23690T. However, the calculated similarity values were lower than the threshold for species demarcation. The phylogenetic analysis showed that the branches of the two isolates were separated from other Acetobacter species, and the two isolates constituted a new species in the genus Acetobacter. The genomic DNA of isolate MS16-SU-2T was sequenced. The assembled genome of the isolate was analysed, and the results showed that the highest average nucleotide identity value of 75.9 % was with Acetobacter papayae JCM 25143T and the highest digital DNA-DNA hybridization value of 25.1 % was with Acetobacter fallax LMG 1636T, which were lower than the cutoff values for species delineation. The phylogenetic tree based on the genome sequences showed that the lineage of isolate MS16-SU-2T was most closely related to A. papayae JCM 25143T and Acetobacter suratthaniensis TBRC 1719T, but separated from the branches of these two species. In addition, the two isolates could be distinguished from the type strains of closely related species by their phenotypic characteristics and MALDI-TOF profiles. Therefore, the two isolates, MS16-SU-2T (=TBRC 12339T=LMG 32243T) and MS18-SU-3 (=TBRC 12305), can be assigned to an independent species within the genus Acetobacter, and the name of Acetobacter garciniae sp. nov. is proposed for the two isolates.

Donghicola mangrovi sp. nov., a member of the family Rhodobacteraceae isolated from mangrove forest in Thailand.

Two novel Gram-stain-negative, rod-shaped and non-motile bacterial strains, designated B5-SW-15T and C2-DW-16, were isolated from water collected in mangrove forests in Ranong Province, Thailand. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains B5-SW-15T and C2-DW-16 belonged to the genus Donghicola and were most closely related to Donghicola tyrosinivorans DSM 100212T (98.2 and 98.1 %, respectively) and Donghicola eburneus DSM 29127T (97.7 and 97.6 %, respectively). The average nucleotide identity and digital DNA-DNA hybridization values between strain B5-SW-15T, strain C2-DW-16 and related species were 95.8 and 71.6 % (to strain C2-DW-16), 76.8 and 21.3 % (to D. tyrosinivorans DSM 100212T) and 80.3 and 24.2 % (to D. eburneus DSM 29127T), respectively. The predominant cellular fatty acids (>5 %) were summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c), C16 : 0 and C12 : 1 3-OH. Ubiquinone Q-10 was the sole respiratory quinone. DNA G+C contents of the isolates were 61.0 and 61.2 mol% based on whole genome sequences. Strains B5-SW-15T and C2-DW-16 contained aminolipid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol as the major polar lipids. On the basis of the results from phenotypic, chemotaxonomic and phylogenetic analyses, strains B5-SW-15T and C2-DW-16 constitute a novel species of the genus Donghicola in the family Rhodobacteraceae for which the name Donghicola mangrovi sp. nov. is proposed. The type strain is B5-SW-15T (=BCC 56522T=TBRC 9562T=KCTC 72743T).

Gluconobacter aidae sp. nov., an acetic acid bacterium isolated from tropical fruits in Thailand.

Two bacterial strains, isolates AC10T and AC20, which were reported in a previous study on the diversity of acetic acid bacteria in Thailand, were subjected to a taxonomic study. The phylogenetic analysis based on the 16S rRNA gene sequences showed that the two isolates were located closely to the type strains of Gluconobacter oxydans and Gluconobacter roseus. However, the two isolates formed a separate cluster from the type strains of the two species. The genomic DNA of isolate AC10T was sequenced. The assembled genomes of the isolate were analysed for average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH). The results showed that the highest ANI and dDDH values between isolate AC10T and G. oxydans DSM 3503T were 91.15 and 68.2 %, which are lower than the suggested values for species delineation. The genome-based tree was reconstructed and the phylogenetic lineage based on genome sequences showed that the lineage of isolate AC10T was distinct from G. oxydans DSM 3503T and its related species. The two isolates were distinguished from G. oxydans and their relatives by their phenotypic characteristics and MALDI-TOF profiles. Therefore, the two isolates, AC10T (=BCC 15749T=TBRC 11329T=NBRC 103576T) and AC20 (=BCC 15759=TBRC 11330=NBRC 103579), can be assigned to an independent species within the genus Gluconobacter, and the name Gluconobacter aidae sp. nov. is proposed for the two isolates.

A functionally critical single nucleotide polymorphism in the gene encoding the membrane-bound alcohol dehydrogenase found in ethanol oxidation-deficient Gluconobacter thailandicus.

The Gluconobacter thailandicus strains NBRC3254, NBRC3255, NBRC3256, NBRC3257, and NBRC3258 are naturally deficient in the ethanol-oxidizing respiratory chain because they do not produce the cytochrome subunit of the membrane-bound alcohol dehydrogenase (ADH). Draft genomes of G. thailandicus strains NBRC3255 and NBRC3257 indicated that the adhB gene encoding the cytochrome subunit contains four base differences when compared to a closely related gene in the public database One of the nucleotide differences results in an Opal codon at the -19th tryptophan (Trp) in the signal sequence for translocation to the periplasmic space (here, the position of +1st residue is assigned to the N-terminal amino acid residue after signal peptide cleavage), while the other differences result in one missense and two silent amino acid alterations. All five of the G. thailandicus strains were shown to have the Trp(-19)Opal alteration. Ethanol oxidation and ADH activities in NBRC3255 were restored by transformation with a derivative of the endogenous adhB gene, of which the -19th Opal codon was altered to encode Trp. These results indicate that this sequence is a functionally critical single nucleotide polymorphism in the cytochrome subunit. Comparative genomic analyses between the draft genomes of NBRC3255 and NBRC3257 revealed that although the two genomes are closely related, they both have a significant number of unique open reading frames. We suggest that the closely related NBRC3255 and NBRC3257 diverged from a common ancestor having the mutation in the adhB gene, whereas no additional functionally critical mutation occurred in the adhB pseudogene over the course of evolution.