Amycolatopsis mongoliensis sp. nov., a novel actinobacterium with antifungal activity isolated from a coal mining site in Mongolia.

A novel filamentous actinobacterium designated strain 4-36T showing broad-spectrum antifungal activity was isolated from a coal mining site in Mongolia, and its taxonomic position was determined using polyphasic approach. Optimum growth occurred at 30 °C, pH 7.5 and in the absence of NaCl. Aerial and substrate mycelia were abundantly formed on agar media. The colour of aerial mycelium was white and diffusible pigments were not formed. Phylogenetic analyses based on 16S rRNA gene sequence showed that strain 4-36T formed a distinct clade within the genus Amycolatopsis. The 16S rRNA gene sequence similarity showed that the strain was mostly related to Amycolatopsis lexingtonensis DSM 44544T and Amycolatopsis rifamycinica DSM 46095T with 99.3 % sequence similarity. However, the highest digital DNA-DNA hybridization value to closest species was 44.1 %, and the highest average nucleotide identity value was 90.2 %, both of which were well below the species delineation thresholds. Chemotaxonomic properties were typical of the genus Amycolatopsis, as the major fatty acids were C15 : 0, iso-C16 : 0 and C16 : 0, the cell-wall diamino acid was meso-diaminopimelic acid, the quinone was MK-9(H4), and the main polar lipids were diphosphatidylglycerol, phosphatidylmethanolamine and phosphatidylethanolamine. The in silico prediction of chemotaxonomic markers was also carried out by phylogenetic analysis. The genome mining for biosynthetic gene clusters of secondary metabolites in strain 4-36T revealed the presence of 34 gene clusters involved in the production of polyketide synthase, nonribosomal peptide synthetase, ribosomally synthesized and post-translationally modified peptide, lanthipeptide, terpenes, siderophore and many other unknown clusters. Strain 4-36T showed broad antifungal activity against several filamentous fungi. The phenotypic, biochemical and chemotaxonomic properties indicated that the strain could be clearly distinguished from other species of Amycolatopsis, and thus the name Amycolatopsis mongoliensis sp. nov. is proposed accordingly (type strain, 4-36T=KCTC 39526T=JCM 30565T).

Description of Chryseobacterium fluminis sp. nov., a keratinolytic bacterium isolated from a freshwater river.

A Gram-stain-negative, aerobic, rod-shaped bacterial strain, designated MMS21-Ot14T, was isolated from a freshwater river, and shown to represent a novel species of the genus Chryseobacterium on the basis of the results from a polyphasic approach. The 16S rRNA gene sequence analysis revealed that MMS21-Ot14T represented a member of the genus Chryseobacterium of the family Weeksellaceae and was closely related to Chryseobacterium hagamense RHA2-9T (97.52 % sequence similarity), Chryseobacterium gwangjuense THG A18T (97.46 %) and Chryseobacterium gregarium P 461/12T (97.27 %). The optimal growth of MMS21-Ot14T occurred at 25-30 °C, pH 6.0-7.0 and in the absence of NaCl. MMS21-Ot14T was capable of hydrolysing casein, starch, DNA, Tween 20 and tyrosine. The strain also showed keratinolytic activity with keratin azure and decolourising activity with remazol brilliant blue R (RBBR), which indicated potential ability to degrade keratin and lignin. The main polar lipids of MMS21-Ot14T were phosphatidylethanolamine, unidentified aminophospholipids, unidentified aminolipids, an unidentified phospholipid and several unidentified lipids. The predominant fatty acids of MMS21-Ot14T were iso-C15 : 0 and iso-C17 : 0 3-OH, and the major isoprenoid quinone was menaquinone 6 (MK-6). The whole genome of MMS21-Ot14T was 5 062 016 bp in length with a DNA G+C content of 37.7 %. The average nucleotide identity and digital DNA-DNA hybridisation values between MMS21-Ot14T and phylogenetically related members of the genus Chryseobacterium were well below the threshold values for species delineation. It is evident from the results of this study that MMS21-Ot14T should be classified as representing a novel species of the genus Chryseobacterium, for which the name Chryseobacterium fluminis sp. nov. (type strain, MMS21-Ot14T = KCTC 92255T = LMG 32529T) is proposed.

Description of Microcella humidisoli sp. nov. and Microcella daejeonensis sp. nov., isolated from riverside soil, reclassification of Marinisubtilis pacificus as Microcella pacifica comb. nov., and emended description of the genus Microcella.

Three Gram-positive, aerobic and rod shaped actinobacteria, designated strains MMS21-STM10T, MMS21-STM12T and MMS21-STM26, were isolated from riverside soil and subjected to polyphasic taxonomic analysis. The strains grew optimally at mesophilic temperatures (25-30 °C) and neutral to slightly alkaline pH (7-8), and NaCl was not required for growth. Best growth was observed on nutrient agar or marine agar media. The strains contained diphosphatidylglycerol, phosphatidylglycerol and a series of unidentified phospholipids, glycolipids and aminolipids, and anteiso-C15 : 0 and iso-C16 : 0 as the main fatty acids in common. The genome sizes ranged between 2.65 and 2.78 Mbp, and the DNA G+C contents between 70.4 and 72.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MMS21-STM10T showed highest sequence similarity of 98.3 % to Microcella putealis CV-2T, and MMS21-STM12T and MMS21-STM26 of 99.2-99.3 % to Microcella flavibacter WY83T, respectively. In the whole genome-based comparison using the orthologous average nucleotide identity and digital DNA-DNA hybridization, each of strains MMS21-STM10T and MMS21-STM12T could be separated from other species of Microcella. The genome analysis also indicated that both strains contained gene clusters involved in the biosynthesis of alkylresorcinol, microansamycin and carotenoids. The phenotypic characteristics again differentiated the strains from related species, and two new species of Microcella, Microcella humidisoli sp. nov. (type strain, MMS21-STM10T=KCTC 49773T=LMG 32522T) and Microcella daejeonensis sp. nov. (type strain, MMS21-STM12T=KCTC 49750T=LMG 32523T) are proposed accordingly. It was also evident that Marinisubtilis pacificus KN1116T should be reclassified as a new species of Microcella, and Microcella pacifica comb. nov. (type strain, KN1116T=CGMCC 1.17143T=KCTC 49299T) is proposed. In addition, an emended description of Microcella is proposed based on this study.

Targeted and Logical Discovery of Piperazic Acid-Bearing Natural Products Based on Genomic and Spectroscopic Signatures.

A targeted and logical discovery method was devised for natural products containing piperazic acid (Piz), which is biosynthesized from ornithine by l-ornithine N-hydroxylase (KtzI) and N-N bond formation enzyme (KtzT). Genomic signature-based screening of a bacterial DNA library (2020 strains) using polymerase chain reaction (PCR) primers targeting ktzT identified 62 strains (3.1%). The PCR amplicons of KtzT-encoding genes were phylogenetically analyzed to classify the 23 clades into two monophyletic groups, I and II. Cultivating hit strains in media supplemented with 15NH4Cl and applying 1H-15N heteronuclear multiple bond correlation (HMBC) along with 1H-15N heteronuclear single quantum coherence (HSQC) and 1H-15N HSQC-total correlation spectroscopy (HSQC-TOCSY) NMR experiments detected the spectroscopic signatures of Piz and modified Piz. Chemical investigation of the hit strains prioritized by genomic and spectroscopic signatures led to the identification of a new azinothricin congener, polyoxyperuin B seco acid (1), previously reported chloptosin (2) in group I, depsidomycin D (3) incorporating two dehydropiperazic acids (Dpz), and lenziamides A and B (4 and 5), structurally novel 31-membered cyclic decapeptides in group II. By consolidating the phylogenetic and chemical analyses, clade-structure relationships were elucidated for 19 of the 23 clades. Lenziamide A (4) inhibited STAT3 activation and induced G2/M cell cycle arrest, apoptotic cell death, and tumor growth suppression in human colorectal cancer cells. Moreover, lenziamide A (4) resensitized 5-fluorouracil (5-FU) activity in both in vitro cell cultures and the in vivo 5-FU-resistant tumor xenograft mouse model. This work demonstrates that the genomic and spectroscopic signature-based searches provide an efficient and general strategy for new bioactive natural products containing specific structural motifs.

Nocardioides okcheonensis sp. nov., isolated from riverside soil.

An actinobacterial strain designated MMS20-HV4-12T, displaying a high hydrolytic potential for various substrates, was isolated from a riverside soil sample and characterized by polyphasic taxonomic analysis. Growth occurred at 10-37 °C (optimum, 30°C), with NaCl concentrations of 0-4 % (optimum, 0 %) and at pH 7-9 (optimum, pH 8). MMS20-HV4-12T was catalase-positive, oxidase-negative, rod-shaped and formed creamy white-coloured colonies. Based on the results of 16S rRNA gene sequence analysis, MMS20-HV4-12T was found to be mostly related to the type strains of Nocardioides alpinus (98.3 % sequence similarity), Nocardioides furvisabuli (98.1 %) and Nocardioides zeicaulis (98.0 %). MMS20-HV4-12T showed optimal growth on Reaoner's 2A agar, forming white-coloured colonies. The diagnostic polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol, the major fatty acids were iso-C16 : 0, C17 : 1 ω8c and 10-methyl-C17 : 0, the major isoprenoid quinone was MK-8(H4), the diagnostic cell-wall sugar was galactose, and the cell-wall diamino acid was ll-diaminopimelic acid. The genome of MMS20-HV4-12T was 4.47 Mbp in size with a G+C content of 72.9 mol%. The genome based analysis indicated low relatedness between MMS20-HV4-12T and all compared species of Nocardioides, as the highest digital DNA-DNA hybridization and the orthologous average nucleotide identity values were 26.8 and 83.8% respectively. Based on genotypic, phenotypic and phylogenomic characterization, MMS20-HV4-12T evidently represents a novel species of genus Nocardioides, for which the name Nocardioides okcheonensis sp. nov. (type strain=MMS20-HV4-12T=KCTC 49651T=LMG 32360T) is proposed.

Brevibacillus humidisoli sp. nov., a moderately thermoalkaliphilic and halotolerant species isolated from riverside soil.

A Gram-stain-positive, aerobic, rod-shaped, motile, and endospore-forming bacterial strain designated MMS20-4M-10-YT was isolated from riverside soil and subjected to taxonomic characterization. Strain MMS20-4M-10-YT was moderately thermophilic, alkaliphilic and halotolerant, as the strain grew at 25-50 °C (optimum, 45 °C), at pH 7.0-10.0 (optimum, pH 8.0) and in the presence of 0-6 % NaCl (optimum, 0 %). Analysis of 16S rRNA gene sequences indicated that MMS20-4M-10-YT fell into a phylogenetic cluster belonging to the genus Brevibacillus. Strain MMS20-4M-10-YT showed the highest 16S rRNA gene sequence similarity to Brevibacillus marinus SCSIO 07484T (96.7 %). Based on the reults of orthologous average nucleotide identity analysis, MMS20-4M-10-YT was again mostly related to B. marinus SCSIO 07484T with 78.0 % identity, which also shared the highest average nucleotide identity of 68.0 %. In contrast, the digital DNA-DNA relatedness analysis indicated that Aneureibacillus migulanus DSM 2895T was the closest species with 29.5 % similarity. The genome-based analyses indicated that all compared species showed low genomic relatedness with MMS20-4M-10-YT. The major fatty acids of the strain were anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 0 and iso-C17 : 0, the major respiratory quinone was MK-7, the diagnostic polar lipids were phosphatidyl-N-methylethanolamine, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol, and diagnostic cell-wall diamino acid was meso-diaminopimelic acid, which was consistent with the general chemotaxonomic features of the genus. The total length of the genome was 4.91 Mbp and the DNA G+C content was 51.8 mol%. Based on both phenotypic and phylogenetic evidence, strain MMS20-4M-10-YT should be classified as representing a novel species of the genus Brevibacillus, for which a name Brevibacillus humidisoli sp. nov. (type strain=MMS20-4M-10-YT=KCTC 43333T=LMG 32359T) is proposed.

Flavobacterium humidisoli sp. nov., isolated from riverside soil.

A novel Gram-stain-negative, yellow-pigmented, non-motile and rod-shaped bacterial strain designated MMS21-Er5T was isolated and subjected to polyphasic taxonomic characterization. MMS21- Er5T could grow at 4-34 °C (optimum, 30 °C), at pH 6-8 (optimum, pH 7) and in the presence of 0-2% NaCl (optimum, 1 %). The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that MMS21- Er5T showed low levels of sequence similarities with other species, as the highest similarity of 97.83 % was observed with Flavobacterium tyrosinilyticum THG DN8.8T, then 97.68 % with 'Flavobacterium ginsengiterrae' DCY 55 and 97.63 % with Flavobacterium banpakuense 15F3T, which were well below the suggested cutoff for species distinction. The whole genome sequence of MMS21-Er5T consisted of a single contig of 5.63 Mbp, and the DNA G+C content was 34.06 mol%. The in-silico DNA-DNA hybridization and orthologous average nucleotide identity values were highest with Flavobacterium tyrosinilyticum KCTC 42726T (45.7 and 91.92% respectively). The predominant respiratory quinone for the strain was menaquinone-6 (MK-6), the major cellular fatty acid was iso-C15 : 0, and the diagnostic polar lipids were phosphatidylethanolamine and phosphatidyldiethanolamine. The combination of physiological and biochemical tests clearly distinguished the strain from related species of the genus Flavobacterium. On the basis of these results, strain MMS21-Er5T evidently represents a novel species of the genus Flavobacterium, for which the name Flavobacterium humidisoli sp. nov. is proposed (type strain=MMS21-Er5T=KCTC 92256T =LMG 32524T).

Agromyces seonyuensis sp. nov., isolated from island soil.

A Gram-positive, non-motile, pale yellow coloured actinobacterial strain designated MMS17-SY077T was isolated from island soil, and its taxonomic position was investigated using a polyphasic approach. Strain MMS17-SY077T grew optimally at 30 °C, at pH 7 and in the absence of NaCl on Reasoner's 2A agar. Based on the 16S rRNA gene sequence analysis, the strain was assigned to the genus Agromyces of the family Microbacteriaceae, and the most related species were Agromyces italicus DSM 16388T (98.8 % sequence similarity), Agromyces allii UMS-62T (98.1 %) and Agromyces terreus DS-10T (97.8 %). Strain MMS17-SY077T formed a distinct cluster within the Agromyces clade in the phylogenetic tree. Genome-based comparative analyses confirmed a clear distinction between the strain and neighbouring species, as the highest orthologous average nucleotide identity and digital DNA-DNA hybridization values with other related species were 77.2 and 21.4% respectively, which were far below the cutoffs for species distinction. The diagnostic polar lipids of MMS17-SY077T were diphosphatidylglycerol and phosphatidylglycerol, and unidentified glycolipids and an unidentified aminolipid were also present. The main isoprenoid quinones were menaquinones with 11 and 12 isoprene units (MK-11 and MK-12), and main fatty acids were anteiso-C15 : 0 (34.4 %) and iso-C16 : 0 (33.2 %). The whole-cell hydrolysates contained rhamnose, ribose and galactose as diagnostic sugars, and l-2,4-diaminobutyric acid as the major diamino acid. The DNA G+C content was 72.1 mol %. Based on phenotypic, chemotaxnomic and phylogenetic characterization, strain MMS17-SY077T should be classified as representing a new species of the genus Agromyces, for which the name Agromyces seonyunensis sp. nov. is proposed (type strain MMS17-SY077T=KCTC 49423T=LMG 31762T).

Streptomyces guryensis sp. nov. exhibiting antimicrobial activity, isolated from riverside soil.

A novel Gram-stain-positive, aerobic actinobacterial strain designated NR30T was isolated from riverside soil. A polyphasic approach was employed for the taxonomic characterization of NR30T. The strain developed extensively branched light brown to light pink substrate mycelia and light grey aerial mycelia, and produced spiny spores in loose spiral spore chains on ISP 3 and 4 agars. NR30T grew at 10-40°C (optimum, 30°C), at pH 6.0-9.0 (optimum, pH 8.0) and in the presence of 0-3 % NaCl (optimum, 0 %). Analysis of 16S rRNA gene sequences indicated that NR30T represents a member of the genus Streptomyces. NR30T shared the highest 16S rRNA gene sequence similarity with Streptomyces cyaneus NRRL B-2296T (98.6 %). On the basis of orthologous average nucleotide identity, NR30T was most closely related to Streptomyces panaciradicis NBRC 109811T with 86.3 % identity. The results of the digital DNA-DNA hybridization analysis also indicated low levels of relatedness with other species, as the highest value was observed with Streptomyces panaciradicis NBRC 109811T (31.1 %). The major fatty acids of the strain were anteiso-C15 : 0, C16 : 0, iso-C16 : 0 and anteiso-C17 : 0. The major respiratory quinones were MK-9(H8) and MK-9(H6). The diagnostic polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol mannoside. The major cell wall diamino acid was ll-diaminopimelic acid, and the characteristic whole-cell sugars were rhamnose, ribose and glucose. The DNA G+C content was 70.3 mol %. NR30T exhibited antimicrobial activity against several Gram-negative bacteria and yeasts. On the basis of the results of both phenotypic and phylogenetic analyses, strain NR30T evidently represents a novel species of the genus Streptomyces, and the name Streptomyces guryensis sp. nov. (type strain=NR30T =KCTC 49653T=LMG 32476T) is proposed accordingly.

Micromonospora antibiotica sp. nov. and Micromonospora humidisoli sp. nov., two new actinobacterial species exhibiting antimicrobial potential.

Two novel actinobacterial strains, designated MMS20-R2-23T and MMS20-R2-29T, were isolated from riverside soil and subjected to taxonomic characterization. Both strains were Gram-stain-positive, aerobic, non-motile and filamentous, and formed orange to strong orange-brown coloured colonies, which later turned black. Both strains grew optimally at mesophilic temperatures, neutral to slightly alkaline pH and in the absence of NaCl. Analysis of 16S rRNA gene sequences indicated that the two novel strains fell into phylogenetic clusters belonging to the genus Micromonospora. Strains MMS20-R2-23T and MMS20-R2-29T showed the highest 16S rRNA gene sequence similarity to Micromonospora phytophila SG15T (99.3 %) and Micromonospora humida MMS20-R1-14T (99.4 %), respectively. Based on the comparative genome analysis, strain MMS20-R2-23T had the highest orthologous average nucleotide identity (orthoANI) value of 92.70 % with Micromonospora matsumotoense DSM 44100T, and MMS20-R2-29T shared 94.99 % with Micromonospora wenchangensis CCTCC AA 2012002T. Besides, the digital DNA-DNA hybridization (dDDH) values of MMS20-R2-23T and MMS20-R2-29T with the same species were 47.6 and 59.2% respectively, which were also highest among the compared species, thus confirming the separation of each strain at species level from related species. The orthoANI and dDDH values between MMS20-R2-23T and MMS20-R2-29T were 92.18 and 44.9% respectively. The genomes of strains MMS20-R2-23T and MMS20-R2-29T were estimated as 7.56 Mbp and 7.13 Mbp in size, and the DNA G+C contents were 72.5 and 72.9 mol%, respectively. The chemotaxonomic properties of both strains were consistent with those of the genus. The novel strains showed antimicrobial activity against a broad range of microbes, in particular Gram-positive bacteria and yeasts. It is evident that each of the isolated strains merits recognition as representing novel species of Micromonospora, for which the names Micromonospora antibiotica sp. nov. (type strain=MMS20-R2-23T=KCTC 49542T=JCM 34495T) and Micromonospora humidisoli sp. nov. (type strain=MMS20-R2-29T=KCTC 49543T=JCM 34496T) are proposed.

Pseudomonas insulae sp. nov., isolated from island soil.

A novel Gram-stain-negative, rod-shaped, aerobic and motile bacterium designated strain UL073T was isolated from a forest soil of an island, and subjected to taxonomic characterization. Strain UL073T grew at 10-37 °C (optimum, 30 °C), at pH 5.0-10.0 (optimum, pH 7.0) and in the presence of 0-3 % NaCl (optimum, 0 %), respectively. Strain UL073T showed the highest sequence similarity to Pseudomonas lalkuanensis PE08T based on 16S rRNA gene analysis with a sequence similarity of 98.08 %, which was well below the suggested cutoff for species distinction. The 16S rRNA gene tree as well as the multilocus sequence analysis and genome-based trees indicated the independent taxonomic position of strain UL073T, and the orthologous average nucleotide identity and in silico DNA-DNA hybridization values between strain UL073T and related species were no higher than 84.7 and 28.3% respectively, thus confirming the distinctive taxonomic position of the strain. The chemotaxonomic properties were consistent with those of the genus, as the major fatty acids of the strain were a summed feature consisting of C18 : 1 ω7c/C18 : 1 ω6c (31.4 %), another summed feature consisting of C16 : 1 ω7c/C16 : 1 ω6c (23.1 %), and C16 : 0 (22.0 %), the major respiratory quinone was ubiquinone 9, and the major polar lipids were phosphatidylethanolamine and diphosphatidylglycerol. The genome size and DNA G+C content of strain UL073T were 4.87 Mbp and 65.9 mol%. On the basis of phenotypic and phylogenetic evidence, strain UL073T should be classified as representing a novel species of Pseudomonas, for which the name Pseudomonas insulae sp. nov. (type strain=UL073T=KCTC 82407T=JCM 34511T) is proposed.

Streptomyces montanisoli sp. nov., exhibiting antimicrobial activity, isolated from mountain soil around a decaying tree.

A Gram-stain-positive, aerobic actinobacterial strain designated MMS17-BM035T isolated from mountain soil around a decaying tree was subjected to taxonomic characterization. The isolate developed extensively branched substrate mycelia and white aerial hyphae on International Streptomyces Project 2 agar. Strain MMS17-BM035T grew at 15-34 °C (optimum, 30 °C), at pH 5.0-8.0 (optimum, pH 7.0) and in the presence of 0-6 % NaCl (optimum, 0 %). Analysis of 16S rRNA gene sequences indicated that MMS17-BM035T fell into a phylogenetic cluster belonging to the genus Streptomyces. MMS17-BM035T shared the highest sequence similarity of 99.45 % with Streptomyces fuscigenes JBL-20T, and no higher than 98.7 % with other species of Streptomyces. Based on the orthologous average nucleotide identity, MMS17-BM035T was again mostly related to S. fuscigenes JBL-20T with 84.14 % identity, and less than 80 % with other species. The digital DNA-DNA hybridization analysis also indicated low levels of relatedness with related species, as the highest value was observed with S. fuscigenes JBL-20T (28.8 %). The major fatty acids of the strain were anteiso-C15 : 0, a summed feature (consisting of C18 : 1 ω7c/C18 : 1 ω6c), iso-C15 : 0, C16 : 0 and C20 : 0. The major respiratory quinones were MK-9(H4) and MK-9(H6). The diagnostic polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositolmannoside. The major cell-wall diamino acid was ll-diaminopimelic acid, and the characteristic whole-cell sugars were glucose and ribose. The DNA G+C content was 72.1 mol%. Strain MMS17-BM035T exhibited antimicrobial activity against several Gram-positive bacteria and yeasts. Based on both phenotypic and phylogenetic evidences, strain MMS17-BM035T should be classified as representing a novel species, for which the name Streptomyces montanisoli sp. nov. (type strain=MMS17-BM035T=KCTC 49544T=JCM 34528T) is proposed.

Paenibacillus artemisiicola sp. nov. and Paenibacillus lignilyticus sp. nov., two new endophytic bacterial species isolated from plant roots.

Two Gram-positive, endospore-forming, rod-shaped bacterial strains designated MWE-103T and DLE-14T were isolated from plant roots. The 16S rRNA gene sequence analysis indicated that strain MWE-103T was closely related to Paenibaillus sacheonensis SY01T with a sequence similarity of 97.82 %, and strain DLE-14T to Paenibacillus rhizoryzae IZS3-5T with 99.09 % similarity. The orthologous average nucleotide identity and digital DNA-DNA hybridization values using whole genome data indicated that strains MWE-103T and DLE-14T could be readily distinguished from the mostly related species. Both strains grew at mesophilic temperature ranges, and grew best at pH 6 and in the absence of NaCl. The major fatty acid in both strains was anteiso-C15 : 0, but their relative proportions differed. The predominant quinone of both strains was menaquinone 7, the cell-wall diamino acid was meso-diaminopimelic acid, and the diagnostic polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol, which were consistent with those of related species. Amylase and cellulase activities were positive for both strains. Strain DLE-14T exhibited the potential for lignin degradation. The DNA G+C contents of strain MWE-103T and DLE-14T were 60.9 and 50.8 mol% respectively. The genomes of the two strains revealed potential plant-growth-promoting characteristics such as nitrogen fixation, siderophore production and phosphate solubilization. Based on phylogenetic and phenotypic evidence, strains MWE-103T and DLE-14T should each be recognized as a novel species of Paenibacillus, for which the names Paenibacillus artemisiicola sp. nov. (type strain: MWE-103T=KCTC 43287T=JCM 34503T) and Paenibacillus lignilyticus sp. nov. (type strain: DLE-14T=KCTC 43288T=JCM 34504T) are proposed.

Metabacillus bambusae sp. nov., isolated from bamboo grove soil.

A bacterial strain designated BG109T was isolated from bamboo grove soil, and subjected to polyphasic taxonomic characterization. BG109T is an aerobic, non-motile, Gram-stain-positive and endospore-forming bacterium. BG109T showed growth at 10-40 °C (optimum, 37 °C), at pH 4-10 (optimum, 8), and in the presence of 0-7 % NaCl concentration (optimum, 0-1 %). The predominant menaquinone of BG109T was MK-7, and the cell wall peptidoglycan contained major amounts of meso-diaminopimelic acid as the diagnostic diamino acid. The diagnostic polar lipids were diphosphatidylglycerol and phosphatidylglycerol, and unidentified phospholipids and glycolipids were also present. The major fatty acids were anteiso-C15 : 0, iso-C15 : 0, iso-C14 : 0, iso-C16 : 0 and C16 : 0. The chemotaxonomic properties of BG109T were generally consistent with those of members of the genus Metabacillus. BG109T shared highest 16S rRNA gene sequence similarities with 'Metabacillus elymi' KUDC1714 (99.26 %), Metabacillus sediminilitoris DSL-17T (98.17 %), Metabacillus litoralis SW-211T (98.16 %) and Metabacillus crassostreae JSM 100118T (97.13 %), all of which were well below the suggested cutoff level for species distinction. The genome level relatedness also confirmed the separation of BG109T from other species of the genus Metabacillus. Thus, it is evident that BG109T merits recognition as representing a novel species of the genus Metabacillus, for which the name Metabacillus bambusae sp. nov. is proposed. The type strain is BG109T (=KCTC 43190T=JCM 34515T).

Characterization of Klebsiella pneumoniae bacteriophages, KP1 and KP12, with deep learning-based structure prediction.

Concerns over Klebsiella pneumoniae resistance to the last-line antibiotic treatment have prompted a reconsideration of bacteriophage therapy in public health. Biotechnological application of phages and their gene products as an alternative to antibiotics necessitates the understanding of their genomic context. This study sequenced, annotated, characterized, and compared two Klebsiella phages, KP1 and KP12. Physiological validations identified KP1 and KP12 as members of Myoviridae family. Both phages showed that their activities were stable in a wide range of pH and temperature. They exhibit a host specificity toward K. pneumoniae with a broad intraspecies host range. General features of genome size, coding density, percentage GC content, and phylogenetic analyses revealed that these bacteriophages are distantly related. Phage lytic proteins (endolysin, anti-/holin, spanin) identified by the local alignment against different databases, were subjected to further bioinformatic analyses including three-dimensional (3D) structure prediction by AlphaFold. AlphaFold models of phage lysis proteins were consistent with the published X-ray crystal structures, suggesting the presence of T4-like and P1/P2-like bacteriophage lysis proteins in KP1 and KP12, respectively. By providing the primary sequence information, this study contributes novel bacteriophages for research and development pipelines of phage therapy that ultimately, cater to the unmet clinical and industrial needs against K. pneumoniae pathogens.

Micromonospora humida sp. nov., exhibiting antimicrobial potential, isolated from riverside soil.

An actinobacterial strain designated MMS20-R1-14T was isolated from a riverside soil sample. Colonies on agar plates were orange to strong orange brown in colour, which later became black. The cells grew at 10-40 °C (optimum, 37 °C), pH 5.0-11.0 (pH 8.0) and in the presence of 0-4 % NaCl (1 %). The 16S rRNA gene sequence of strain MMS20-R1-14T showed highest similarities to Micromonospora wenchangensis CCTCC AA 2012002T (99.51 %) and Micromonospora rifamycinica AM105T (99.37 %). The orthoANI values between strain MMS20-R1-14T and the two type strains were 95.72 and 90.99 %, and the digital DNA-DNA hybridization values were 63.6 and 40.8 %, respectively, thus confirming the distinction of strain MMS20-R1-14T from its mostly related species. The DNA G+C content of strain MMS20-R1-14T was 72.9 mol%. The strain contained meso-diaminopimelic acid as the major cell-wall amino acid, and the characteristic whole-cell sugars were arabinose, xylose, glucose, ribose and rhamnose. The main cellular fatty acids were C18 : 1 ω9c, iso-C15 : 0 and iso-C16 : 0, the diagnostic polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, and the predominant menaquinones were MK-10(H4) and MK-10(H6), all of which were consistent with those of Micromonospora. Strain MMS20-R1-14T showed antimicrobial activity against a range of bacterial and yeast species. The genome of the strain was found to contain 33 potential biosynthetic gene clusters for secondary metabolites, thus showing a high potential as a producer of bioactive compounds. On the basis of these phenotypic, genotypic and chemotaxonomic data, strain MMS20-R1-14T merits recognition as representing a novel species of the genus Micromonospora, for which the name Micromonospora humida sp. nov. (type strain=MMS20 R1-14T=KCTC 49541T=JCM 34494T) is proposed.

Pseudomonas guryensis sp. nov. and Pseudomonas ullengensis sp. nov., isolated from soil.

Two Gram-staining-negative, aerobic, rod-shaped bacteria designated strains SR9T and UL070T, were isolated from soil and subjected to taxonomic characterization. Strain SR9T grew at 10-37 °C (optimum 30 °C), at pH 4.0-10.0 (optimum pH 8.0) and in the presence of 0-1 % NaCl (optimum 0 %), and UL070T at 4-33 °C (optimum 30 °C), at pH 4.0-10.0 (optimum pH 7.0) and in the presence of 0-2 % NaCl (optimum 0 %), respectively. Strain UL070T was motile with flagella. Analysis of 16S rRNA gene sequences indicated that the two strains fell into phylogenetic clusters belonging to the genus Pseudomonas. Both strains SR9T and UL070T were mostly related to Pseudomonas campi S1-A32-2T with 99.70 and 99.01% sequence similarities, and the similarity between the two isolates was 98.90 %. The genome-based in silico analyses indicated that each of the strains SR9T and UL070T was clearly separated from other species of Pseudomonas, as the orthologous average nucleotide identity (OrthoANI) and the digital DNA-DNA hybridization (dDDH) values were no higher than 93.09 and 50.03% respectively with any related species, which were clearly below the cutoff for species distinction. The fatty acid profiles of the two strains mainly consisting of unsaturated components, the presence of ubiquinone 9 (Q-9) as the major respiratory quinone, and phosphatidylethanolamine (PE) and diphosphatidylglycerol (DPG) as the diagnostic polar lipids were consistent with their classification into Pseudomonas. The DNA G+C contents of strains SR9T and UL070T were 63.2 mol% and 63.6 mol% respectively. On the basis of both phenotypic and phylogenetic evidences, each of the isolated strains should be classified as a novel species, for which the names Pseudomonas guryensis sp. nov. (type strain=SR9T=KCTC 82228T=JCM 34509T) and Pseudomonas ullengensis sp. nov. (type strain=UL070T=KCTC 82229T=JCM 34510T) are proposed.

Comparison of 16S rRNA Gene Based Microbial Profiling Using Five Next-Generation Sequencers and Various Primers.

Microbial community analysis based on the 16S rRNA-gene is used to investigate both beneficial and harmful microorganisms in various fields and environments. Recently, the next-generation sequencing (NGS) technology has enabled rapid and accurate microbial community analysis. Despite these advantages of NGS based metagenomics study, sample transport, storage conditions, amplification, library preparation kits, sequencing, and bioinformatics procedures can bias microbial community analysis results. In this study, eight mock communities were pooled from genomic DNA of Lactobacillus acidophilus KCTC 3164T, Limosilactobacillus fermentum KCTC 3112T, Lactobacillus gasseri KCTC 3163T, Lacticaseibacillus paracasei subsp. paracasei KCTC 3510T, Limosilactobacillus reuteri KCTC 3594T, Lactococcus lactis subsp. lactis KCTC 3769T, Bifidobacterium animalis subsp. lactis KCTC 5854T, and Bifidobacterium breve KCTC 3220T. The genomic DNAs were quantified by droplet digital PCR (ddPCR) and were mixed as mock communities. The mock communities were amplified with various 16S rRNA gene universal primer pairs and sequenced by MiSeq, IonTorrent, MGIseq-2000, Sequel II, and MinION NGS platforms. In a comparison of primer-dependent bias, the microbial profiles of V1-V2 and V3 regions were similar to the original ratio of the mock communities, while the microbial profiles of the V1-V3 region were relatively biased. In a comparison of platform-dependent bias, the sequence read from short-read platforms (MiSeq, IonTorrent, and MGIseq-2000) showed lower bias than that of long-read platforms (Sequel II and MinION). Meanwhile, the sequences read from Sequel II and MinION platforms were relatively biased in some mock communities. In the data of all NGS platforms and regions, L. acidophilus was greatly underrepresented while Lactococcus lactis subsp. lactis was generally overrepresented. In all samples of this study, the bias index (BI) was calculated and PCA was performed for comparison. The samples with biased relative abundance showed high BI values and were separated in the PCA results. In particular, analysis of regions rich in AT and GC poses problems for genome assembly, which can lead to sequencing bias. According to this comparative analysis, the development of reference material (RM) material has been proposed to calibrate the bias in microbiome analysis.

Flavobacterium hydrocarbonoxydans sp. nov., isolated from polluted soil.

This paper presents a polyphasic taxonomic study of a Gram-stain-negative bacterium designated GA093T, a soil isolate capable of benzo(α)pyrene degradation. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain GA093T is a member of the genus Flavobacterium, and formed an independent phylogenetic line while clustering with the type strains of Flavobacterium hibernum, Flavobacterium branchiarum and Flavobacterium hydatis. Strain GA093T was facultatively anaerobic, and could grow at 4-33 °C (optimum, 30 °C), at pH 6-11 (optimum, pH 7) and in the presence of 0-2 % (w/v) NaCl (optimum, 0 %). Strain GA093T was capable of producing acid from various carbon sources, which was comparable to other related species of Flavobacterium. The strain contained MK-6 as the only isoprenoid quinone, iso-C15 : 0 as the major cellular fatty acid, phosphatidylethanolamine and phosphatidylinositol as diagnostic polar lipids, and sym-homospermidine as the major polyamine. The chemotaxonomic properties of strain GA093T were consistent with the general properties of Flavobacterium except the presence of phosphatidylinositol, which distinguished it from other related species. The total stretch of the obtained genome of GA093T was 5.05 Mbp, and the DNA G+C content was 34.79 mol%. The genome contained genes potentially related to the degradation of aromatic hydrocarbons. On the basis of the present polyphasic analysis, strain GA093T was found to have properties that distunguished it as representing a novel species of the genus Flavobacterium, for which the name Flavobacterium hydrocarbonoxydans sp. nov. is proposed. The type strain is GA093T (=KCTC 72594T=LMG 31760T).

Muriicola soli sp. nov., isolated from soil.

A Gram-stain-negative, oxidase-positive, catalase-positive, aerobic, orange-pigmented, rod-shaped and non-motile bacterium designated strain MMS17-SY002T was isolated from island soil. The isolate grew at 20-37 °C (optimum, 30 °C), at pH 6.0-9.5 (optimum, pH 7) and in the presence of 0.5-4.0 % (w/v) NaCl (optimum, 2.0 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MMS17-SY002T was mostly related to the genus Muriicola of the family Flavobacteriaceae and had highest sequence similarity of 96.82 % to Muriicola marianensis A6B8T and Muriicola jejuensis EM44T, but formed a distinct phylogenetic line within the genus. Chemotaxonomic analyses showed that menaquinone 6 was the predominant isoprenoid quinone, the major fatty acids were iso-C15 : 1 G and iso-C15 : 0, and the diagnostic polar lipid was phosphatidylethanolamine. The genomic DNA G+C content was 42.4 mol%. Strain MMS17-SY002T could be distinguished from related species by the combination of trypsin, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, ß-galactosidase and ß-glucosidase activities. The orthologous average nucleotide identity between the genomes of strain MMS17-SY002T and M. jejuensis and that between the strain and M. marianensis A6B8T were 73.26 and 73.33%, respectively, thus confirming the separation of the strain from related species at species level. Based on the phenotypic, phylogenetic, chemotaxonomic and genomic characterization, MMS17-SY002T should be recognized as a novel species of the genus Muriicola, for which the name Muriicola soli sp. nov. is proposed. The type strain is MMS17-SY002T (=KCTC 62790T=JCM 32370T).