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ISSN : 1226-9999(Print)
ISSN : 2287-7851(Online)
Korean J. Environ. Biol. Vol.42 No.3 pp.288-300
DOI : https://doi.org/10.11626/KJEB.2024.42.3.288

A report on 22 unrecorded Actinomycetota species isolated from freshwater environments in the Republic of Korea

Soo-Yeong Lee, Jaeduk Goh, Ahyoung Choi*
Biological Resources Research Department, Nakdonggang National Institute of Biological Resources (NNIBR), Sangju 37242, Republic of Korea
*Corresponding author Ahyoung Choi Tel. 054-530-0841 E-mail. aychoi@nnibr.re.kr

Contribution to Environmental Biology


▪ This study identifies 22 previously unrecorded Actinomycetota species in freshwater ecosystems of Korea, filling a crucial knowledge gap and aiding biodiversity preservation.


▪ These new Actinomycetota species offer the potential for developing antibiotics, bioactive compounds, and bioremediation agents, supporting sustainable practices and water quality improvement.


12/07/2024 24/08/2024 10/09/2024

Abstract


Freshwater environments are rich ecosystems that support diverse microbial communities, including members of the phylum Actinomycetota critical for nutrient cycling, organic matter decomposition, and water quality maintenance. Actinomycetota known to produce numerous bioactive secondary metabolites are valuable in biotechnology, medicine, and agriculture. Despite their significance, the diversity and distribution of Actinomycetota in freshwater habitats, especially in the Republic of Korea, are underexplored. This study aimed to report the isolation and characterization of 22 previously unrecorded bacterial species of Actinomycetota from various freshwater environments in Korea. Using standard dilution plating techniques on six different culture media, 22 bacterial strains were isolated, incubated, and characterized based on colony and cellular morphologies, Gram staining, and biochemical properties. Genomic DNA was extracted and the 16S rRNA gene was sequenced to determine species identity using the EzBioCloud service with a cutoff of 98.7% sequence similarity for classification as unreported species. These strains were phylogenetically diverse, belonging to two classes, ten orders, and eighteen genera. This study enhances our understanding of bacterial diversity in freshwater ecosystems and underscores the importance of exploring microbial diversity in underexplored habitats, potentially leading to discovery of novel bioactive compounds. Findings of this study contribute valuable insights into ecological roles and biotechnological potential of Actinomycetota in freshwater environments.


freshwater environments , unrecorded species , Actinomycetota , bacterial diversity

초록

    1. INTRODUCTION

    Freshwater environments are diverse ecosystems that harbor a wide range of microbial communities, including bacteria that play critical roles in nutrient cycling, organic matter decomposition, and maintaining water quality (Böckelmann et al. 2000;Ferreira et al. 2020). Among these bacterial communities, members of the phylum Actinomycetota are of particular interest due to their ecological significance and potential applications in biotechnology, medicine, and agriculture (Blin et al. 2021;Kim et al. 2021;Ngamcharungchit et al. 2023;Bruna et al. 2024). Actinomycetes are known for their ability to produce a vast array of secondary metabolites, including antibiotics, antifungals, and immunosuppressive agents, making them valuable resources for novel bioactive compounds (Goodfellow and Williams 1983;Comba et al. 2013;Donald et al. 2022). Furthermore, these species play crucial roles in biodegradation and pollutant removal, contributing to environmental remediation processes, that support sustainable environmental management (Bérdy 2005;Slemc et al. 2021;Behera and Das 2023).

    Despite their importance, the diversity and distribution of Actinomycetota in freshwater environments remain underexplored, particularly in regions such as Korea. Previous studies have primarily focused on marine environments and soil, leaving a significant knowledge gap regarding freshwater habitats (Bae et al. 2016;Lee et al. 2016;Heo et al. 2023). The unique physicochemical conditions of freshwater systems, such as rivers, lakes, and reservoirs, offer distinct niches that may support the growth of unrecorded or novel bacterial species.

    Actinomycetota is a bacterial phylum that underwent renaming from ‘Actinobacteria’ in 2021 (Oren and Garrity 2021). Representing a diverse phylum of Gram-positive bacteria, Actinomycetota are characterized by their high G+C content (Gao and Gupta 2012). They inhabit both terrestrial and aquatic environments and play a crucial role in various ecosystems (Servin et al. 2008). As of the time of this writing, taxonomic classification based on 16S rRNA gene sequences indicates that the phylum Actinomycetota comprises six classes with correct names: Acidimicrobiia, Actinomycetia, Coriobacteriia, Nitriliruptoria, Rubrobacteria, and Thermoleophilia. Actinomycetota stands out as one of the dominant bacterial phyla, boasting one of the largest bacterial genera, Streptomyces. Streptomyces and other Actinomycetota play crucial roles in soil health by contributing significantly to biological buffering (Donald et al. 2022).

    In this study, we report the isolation and characterization of 22 previously unrecorded bacterial species belonging to the phylum Actinomycetota from various freshwater environments in Korea. This research aims to contribute to the understanding of bacterial diversity in freshwater ecosystems and provide insights into the potential biotechnological applications of these newly identified species. The findings of this study highlight the importance of continued exploration of microbial diversity in underexplored habitats, which could lead to the discovery of novel bioactive compounds and enhance our understanding of microbial ecology.

    2. MATERIALS AND METHODS

    A total of 22 bacterial strains were obtained from various freshwater environments by employing the standard dilution plating technique on different culture media. The media used included marine agar 2216 (MA), Reasoner’s 2A (R2A), 1/10-diluted R2A (1/10 R2A), nutrient agar (NA), 1/10-diluted tryptic soy agar (1/10 TSA), and inorganic salts starch (ISP 4) agar. These media were selected based on their efficacy in supporting the growth of diverse bacterial communities, particularly Actinomycetota, which are known for their unique nutrient requirements. The isolates were incubated at 15-25°C for 14 days to accommodate a range of growth temperatures suitable for different bacterial species. After serial dilution spreading, each strain was isolated as a single colony, and the pure cultures were stored as 20% glycerol suspensions at -80°C. Table 1 provides details of the strain IDs, culture media, and incubation conditions.

    For 16S rRNA sequence analysis, genomic DNA was extracted from the isolates using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The 16S rRNA gene was amplified by PCR using universal bacterial primers 27F and 1492R and sequenced through Sanger sequencing (Weisburg et al. 1991). The nearly full-length 16S rRNA gene sequences obtained were identified using the “16S-based ID” service in EzBioCloud (Yoon et al. 2017) with a sequence similarity cutoff set at 98.7%. Strains showing ≥98.7% sequence similarity with known bacterial species not previously reported in Korea were classified as unreported species.

    Phylogenetic analyses involved aligning the 16S rRNA gene sequences of the isolates with those of reference-type strains using the Clustal_W program (Larkin et al. 2007), with manual verification via EzEditor (Jeon et al. 2014). Phylogenetic trees were constructed based on the aligned sequences using the neighbor-joining method (Saitou and Nei 1987) and the Kimura 2-parameter model (Kimura 1980) implemented in MEGA 7.0 software (Kumar et al. 2016). The robustness of the phylogenetic trees was tested through bootstrap analyses with 1,000 random re-samplings (Felsenstein 1985).

    The colony morphologies of the bacterial strains were examined on agar plates using a magnifying glass after cultivation to the stationary phase. Transmission electron microscopy further examined cellular morphology and size (Talos L120C; ThermoFisher Scientific, Waltham, MA, USA). Gram staining was performed using a Gram-staining kit (BioMérieux, Marcy-I’Étoile, France). Additionally, biochemical characteristics were assessed with API 20NE galleries (BioMérieux) following the provided protocols.

    3. RESULTS AND DISCUSSION

    The 16S rRNA gene sequence analyses conducted on approximately 1,000 bacterial strains obtained in this study revealed the presence of novel species or species previously unreported in Korea. Specifically, 22 strains exhibited ≥98.7% 16S rRNA gene sequence similarities with unrecorded Actinomycetota species in Korea. These strains belonged to one phylum, two classes, ten orders, and eighteen genera. The detailed taxonomic composition and identification results of these species are summarized in Table 1. The phylogenetic tree of the bacterial strains assigned to the phylum Actinomycetota is shown in Figure 1. This figure depicts the phylogenetic relationships between the isolates and closely related species. Transmission electron microscopic images of the isolates are provided in Figure 2.

    The identification of these 22 previously unrecorded Actinomycetota species enhances our understanding of microbial diversity in freshwater ecosystems. Actinomycetota, known for their role in nutrient cycling, organic matter decomposition, and maintenance of water quality, are critical components of these ecosystems. The discovery of these species suggests the presence of unique ecological niches within the freshwater environments of Korea, highlighting the importance of continued microbial exploration in underexplored habitats.

    Actinomycetota are renowned for their production of bioactive secondary metabolites, including antibiotics, antifungals, and immunosuppressive agents. The newly identified species hold considerable promise for biotechnological applications. For instance, the genus Streptomyces, well-known for its antibiotic production, includes newly discovered strains that could potentially yield novel antimicrobial compounds. Additionally, species from the genera Micromonospora and Nocardia have been previously noted for their roles in bioremediation, suggesting that the newly identified species may contribute to sustainable environmental practices by degrading pollutants and improving water quality. Future research should focus on investigating the functional properties and ecological roles of these bacteria to fully realize their potential benefits.

    In addition to the Actinomycetota species isolated from Korean freshwater environments, similar studies have been conducted in other parts of Asia, particularly in Japan and China. These studies provide valuable insights into the diversity and distribution of Actinomycetota across different freshwater ecosystems. For instance, a study conducted in Japan reported the isolation of novel Actinomycetota species from river sediments, highlighting the presence of unique microbial communities adapted to specific environmental conditions (Lipko and Belykh 2021). Similarly, research in China has revealed the presence of diverse Actinomycetota species in various freshwater habitats, including lakes and rivers, some of which have demonstrated significant potential for producing bioactive compounds (Chen et al. 2021;Wu et al. 2021). A comparison of the Actinomycetota species isolated from Korean freshwater with those from Japan and China reveals both similarities and distinct differences in the microbial communities. For example, while some genera such as Streptomyces and Micromonospora are commonly found across all three regions, species diversity may differ due to variations in environmental factors such as pH, temperature, and nutrient availability. Moreover, the unique geographic and climatic conditions of Korea may contribute to the presence of species not yet reported in Japan or China, underscoring the importance of exploring underexplored habitats to uncover novel microbial taxa. This comparison also emphasizes the importance of regional studies in understanding the global diversity of Actinomycetota and their potential applications. Continued exploration and characterization of Actinomycetota in different regions are essential for uncovering new species that may hold promise for biotechnological applications, such as the development of new antibiotics or bioremediation agents.

    3.1. Description of Catenulispora rubra LS9-103

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on 1/10 R2A are circular, translucent, substrate mycelium, and ivory after incubation for 3 days at 25°C. Positive for glucose fermentation, arginine dihydrolase, and urease, but nitrate reduction, indole production, esculin hydrolysis, gelatinase, and β-galactosidase. Phenylacetic acid is utilized but does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, and trisodium citrate. Strain LS9-103 displays the highest 16S rRNA gene sequence similarity with Catenulispora rubra Aac-30T (99.4%). Strain LS9-103 (=NNIBR2021641BA1680=FBCC-B8703) was isolated from riverside sediment collected in Gangneung-si, Gangwon-do, Korea. The GenBank accession number of the 16S rRNA gene sequence is OL773530.

    3.2. Description of Cellulomonas algicola 23ND14S-41

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on MA are circular, raised, entire, and yellow after incubation for 3 days at 25°C. Positive for glucose fermentation, esculin hydrolysis, gelatinase, and β-galactosidase, but nitrate reduction, indole production, arginine dihydrolase, and urease. D-Glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, and potassium gluconate are utilized. Does not utilize capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain 23ND14S-41 displays the highest 16S rRNA gene sequence similarity with Cellulomonas algicola TKZ-21T (99.9%). Strain 23ND14S-41 (=NNIBR 2023641BA2539=FBCC-B15847) was isolated from riverside sediment collected in Hadong-gun, Gyeongsangnam- do, Korea. The GenBank accession number of the 16S rRNA gene sequence is OR945205.

    3.3. Description of Populibacterium corticicola 23ND75W-61

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on MA are umbonate, and creamy-white after incubation for 3 days at 25°C. Positive for esculin hydrolysis, and β-galactosidas, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, and gelatinase. D-Glucose, L-arabinose, D-mannose, D-mannitol, and D-maltose are utilized. Does not utilize N-acetyl- glucosamine, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain 23ND75W-61 displays the highest 16S rRNA gene sequence similarity with Populibacterium corticicola 2D-4T (99.3%). Strain 23ND75W-61 (=NNIBR2023641BA2564=FBCC-B15808) was isolated from the surface water of a stream collected in Seosan-si, Chungcheongnam-do, Korea. The GenBank accession number of the 16S rRNA gene sequence is OR984989.

    3.4. Description of Intrasporangium flavum 21SJ05W-0M23

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on R2A are circular convex entire, and pale yellow after incubation for 3 days at 20°C. Positive for nitrate reduction, gelatinase, and β-galactosidas, but indole production, glucose fermentation, arginine dihydrolase, urease, and esculin hydrolysis. L-Arabinose, D-mannose, D-maltose, potassium gluconate, and adipic acid are utilized. Does not utilize D-glucose, D-mannitol, N-acetyl-glucosamine, capric acid, malic acid, trisodium citrate, and phenylacetic acid. Strain 21SJ05W-0M23 displays the highest 16S rRNA gene sequence similarity with Intrasporangium flavum MUSC 78T (98.7%). Strain 21SJ05W-0M23 (=NNIBR2021641BA1533=FBCC-B8168) was isolated from surface water collected in Imsil-gun, Jeollabuk- do, Korea. The GenBank accession number of the 16S rRNA gene sequence is MZ976827.

    3.5. Description of Ornithinimicrobium flavum 23ND49W-34

    Cells are Gram-stain-positive, non-flagellated, and oval-shaped. Colonies grown on MA are circular raised entire, and yellow after incubation for 3 days at 25°C. Positive for nitrate reduction, but negative for indole production, glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis, gelatinase, and β-galactosidas. D-Maltose, potassium gluconate, adipic acid, malic acid, trisodium citrate, and phenylacetic acid are utilized. Does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, and capric acid. Strain 23ND49W-34 displays the highest 16S rRNA gene sequence similarity with Ornithinimicrobium flavum CPCC 203535T (99.7%). Strain 23ND49W-34 (=NNIBR2023641BA2550= FBCC-B15836) was isolated from surface water collected in Namhae-gun, Gyeongsangnam-do, Korea. The GenBank accession number of the 16S rRNA gene sequence is OR945216.

    3.6. Description of Blastococcus aurantiacus HRW3-25

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, smooth, and orange after incubation for 3 days at 25°C. Positive for nitrate reduction, indole production, esculin hydrolysis, and β-galactosidase, but negative for glucose fermentation, arginine dihydrolase, urease, and gelatinas. Does not all utilize substrates: D-Glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain HRW3-25 displays the highest 16S rRNA gene sequence similarity with Blastococcus aurantiacus AT 7-1T (99.8%). Strain HRW3-25 (=NNIBR2017301BA1=FBCC-B3542) was isolated from surface water collected in Hanam-si, Gyeonggi-do, Korea. The GenBank accession number of the 16S rRNA gene sequence is MG818295.

    3.7. Description of Blastococcus endophyticus 6LW2-11

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, smooth, and cream after incubation for 3 days at 25°C. Positive for esculin hydrolysis, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, gelatinase, and β-galactosidas. D-Glucose and D-mannitol are utilized. Does not utilize L-arabinose, D-mannose, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain 6LW2-11 displays the highest 16S rRNA gene sequence similarity with Blastococcus endophyticus DSM 45413T (99.4%). Strain 6LW2-11 (=NNIBR2022641BA2177=FBCCB11910) was isolated from surface water collected in Yangyang-gun, Gangwon-do, Korea. The GenBank accession number of the 16S rRNA gene sequence is OP872590.

    3.8. Description of Kineosporia rhamnosa 22LW2-51

    Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on R2A are wrinkle, brittle, umbonate, substrate mycelium developed, and light orange after incubation for 3 days at 25°C. Positive for esculin hydrolysis, gelatinase, and β-galactosidase, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, and urease. D-Glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, adipic acid, and malic acid are utilized. Does not utilize capric acid, trisodium citrate, and phenylacetic acid. Strain 22LW2-51 displays the highest 16S rRNA gene sequence similarity with Kineosporia rhamnosa JCM 9954T (99.4%). Strain 22LW2-51 (=NNIBR 2022641BA2283=FBCC-B12124) was isolated from surface water collected in Yangyang-gun, Gangwondo, Korea. The GenBank accession number of the 16S rRNA gene sequence is OP999349.

    3.9. Description of Arenivirga flava 23ND25W-67

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on ISP4 are punctiform, flat, and white after incubation for 3 days at 20°C. Positive for esculin hydrolysis and β-galactosidase, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, and gelatinas. Does not all utilize substrates: D-Glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain 23ND25W-67 displays the highest 16S rRNA gene sequence similarity with Arenivirga flava HIs16-32T (99.5%). Strain 23ND25W-67 (=NNIBR 2023641BA615=FBCC-B13684) was isolated from surface water collected in Gumi-si, Gyeongsangbukdo, Korea. The GenBank accession number of the 16S rRNA gene sequence is OR984974.

    3.10. Description of Cryobacterium breve LW1-R25

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, smooth, and yellow after incubation for 3 days at 20°C. Positive for glucose fermentation, esculin hydrolysis, and β-galactosidase, but negative for nitrate reduction, indole production, arginine dihydrolase, urease, and gelatinase. D-Glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, malic acid, and phenylacetic acid are utilized. Does not utilize capric acid, adipic acid, trisodium citrate. Strain LW1-R25 displays the highest 16S rRNA gene sequence similarity with Cryobacterium breve TMT4-23T (99.3%). Strain LW1-R25 (=NNIBR 2022641BA232=FBCC-B9166) was isolated from surface water collected in Goseong-gun, Gangwondo, Korea. The GenBank accession number of the 16S rRNA gene sequence is OP872594.

    3.11. Description of Actinoplanes digitatis 22LW2-48

    Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on R2A are small, brittle, opaque, substrate mycelium developed, and light orange after incubation for 3 days at 25°C. Positive for esculin hydrolysis, gelatinase, and β-galactosidase, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, and urease. D-Glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, adipic acid, and malic acid are utilized. Does not utilize capric acid, trisodium citrate, and phenylacetic acid. Strain 22LW2-48 displays the highest 16S rRNA gene sequence similarity with Actinoplanes digitatis IFO 12512T (99.0%). Strain 22LW2-48 (=NNIBR 2022641BA2282=FBCC-B12123) was isolated from surface water collected in Yangyang-gun, Gangwondo, Korea. The GenBank accession number of the 16S rRNA gene sequence is OP999348.

    3.12. Description of Micromonospora globbae 23ND14S-8

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on 1/10 TSA are circular raised entire, and brown after incubation for 2 days at 25°C. Positive for esculin hydrolysis and β-galactosidase, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, and gelatinas. L-Arabinose, D-mannitol, D-maltose, potassium gluconate, adipic acid, malic acid, and trisodium citrate are utilized. Does not utilize D-glucose, D-mannose, N-acetyl-glucosamine, capric acid, and phenylacetic acid. Strain 23ND14S-8 displays the highest 16S rRNA gene sequence similarity with Micromonospora globbae WPS1-2T (99.8%). Strain 23ND14S-8 ( =NNIBR2023641BA2545 = FBCC-B15841) was isolated from riverside sediment collected in Hadong-gun, Gyeongsangnam-do, Korea. The GenBank accession number of the 16S rRNA gene sequence is OR945211.

    3.13. Description of Corynebacterium variabile SS-105

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on 1/10 TSA are circular raised entire, and white after incubation for 2 days at 25°C. Positive for glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis and β-galactosidase, but negative for nitrate reduction, indole production, and gelatinase. Utilize all substrates: D-Glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain SS-105 displays the highest 16S rRNA gene sequence similarity with Corynebacterium variabile DSM 20132T (99.5%). Strain SS-105 (=NNIBR2023641BA2542=FBCC-B15844) was isolated from insect intestine collected from fresh water in Jangseong-gun, Jeollanam-do, Korea. The GenBank accession number of the 16S rRNA gene sequence is OR945208.

    3.14. Description of Gordonia sputi SJ8

    Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, viscous, convex with entire edge, and mily orange after incubation for 3 days at 25°C. Positive for nitrate reduction, urease, and esculin hydrolysis but negative for indole production, glucose fermentation, arginine dihydrolase, gelatinase, and β-galactosidase. D-Glucose, D-mannose, malic acid, and phenylacetic acid are utilized. Does not utilize L-arabinose, D-mannitol, N-acetyl- glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, and trisodium citrate. Strain SJ8 displays the highest 16S rRNA gene sequence similarity with Gordonia sputi NBRC 100414T (99.9%). Strain SJ8 (=NNIBR2016301BA1=FBCC-B3259) was isolated from surface water collected in Gwangyang-si, Jeollanam-do, Korea. The GenBank accession number of the 16S rRNA gene sequence is KY319045.

    3.15. Description of Nocardia stercoris 21LS8-22

    Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, substrate mycelium, aerial mycelium, and yellowish white after incubation for 3 days at 25°C. Positive for nitrate reduction and urease, but negative for indole production, glucose fermentation, arginine dihydrolase, esculin hydrolysis, gelatinase, and β-galactosidas. Phenylacetic acid is utilized but does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, and trisodium citrate. Strain 21LS8-22 displays the highest 16S rRNA gene sequence similarity with Nocardia stercoris NEAU-LL90T (99.3%). Strain 21LS8-22 (=NNIBR 2021641BA1247=FBCC-B7675) was isolated from riverside sediment collected in Goseong-gun, Gangwon- do, Korea. The GenBank accession number of the 16S rRNA gene sequence is OL773524.

    3.16. Description of Nocardia rayongensis 23ND34S-94

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on 1/10 R2A are circular, raised, entire, and white after incubation for 2 days at 25°C. Positive for esculin hydrolysis and β-galactosidase, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, and gelatinase. L-Arabinose, malic acid, and trisodium citrate are utilized. Does not utilize D-glucose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, and phenylacetic acid. Strain 23ND34S-94 displays the highest 16S rRNA gene sequence similarity with Nocardia rayongensis RY45-3T (99.7%). Strain 23ND34S-94 (=NNIBR2023641BA2546= FBCC-B15840) was isolated from riverside sediment collected in Namhae-gun, Gyeongsangnam-do, Korea. The GenBank accession number of the 16S rRNA gene sequence is OR945212.

    3.17. Description of Mycobacterium gilvum SJW1-30

    Cells are Gram-stain-positive, non-flagellated, and short rod-shaped. Colonies grown on NA are circular, convex, and yellow after incubation for 3 days at 25°C. Positive for nitrate reduction and urease, but negative for indole production, glucose fermentation, arginine dihydrolase, esculin hydrolysis, gelatinase, and β-galactosidase. D-Mannitol and malic acid are utilized. Does not utilize D-glucose, L-arabinose, D-mannose, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, trisodium citrate, and phenylacetic acid. Strain SJW1-30 displays the highest 16S rRNA gene sequence similarity with Mycobacterium gilvum ATCC 43909T (99.3%). Strain SJW1-30 (=NNIBR2017301BA9=FBCC-B3425) was isolated from surface water collected in Gwangyang-si, Jeollanam- do, Korea. The GenBank accession number of the 16S rRNA gene sequence is MG818303.

    3.18. Description of Nocardioides allogilvus 21LS6-111

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on 1/10 R2A are circular, smooth, opaque, and pale yellow after incubation for 3 days at 25°C. Positive for urease, esculin hydrolysis, gelatinase, and β-galactosidase, but negative for nitrate reduction, indole production, glucose fermentation, and arginine dihydrolase. D-Glucose and phenylacetic acid are utilized. Does not utilize L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, and trisodium citrate. Strain 21LS6-111 displays the highest 16S rRNA gene sequence similarity with Nocardioides allogilvus CFH 30205T (99.4%). Strain 21LS6-111 (=NNIBR2021641BA147=FBCC-B6334) was isolated from riverside sediment collected in Goseong-gun, Gangwon-do, Korea. The GenBank accession number of the 16S rRNA gene sequence is OL773523.

    3.19. Description of Nocardioides lianchengensis 23ND01W-24

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on 1/10 TSA are circular, raised, entire, and white after incubation for 2 days at 25°C. Positive for glucose fermentation, esculin hydrolysis, and β-galactosidase, but negative for nitrate reduction, indole production, arginine dihydrolase, urease, and gelatinase. D-Glucose, L-arabinose, D-mannitol, potassium gluconate, and malic acid are utilized. Does not utilize D-mannose, N-acetyl-glucosamine, D-maltose, capric acid, adipic acid, trisodium citrate, and phenylacetic acid. Strain 23ND01W-24 displays the highest 16S rRNA gene sequence similarity with Nocardioides lianchengensis CGMCC 4.6858T (100.0%). Strain 23ND01W-24 (=NNIBR2023641BA2538= FBCC-B15848) was isolated from surface water collected in Hamyang-gun, Gyeongsangnam-do, Korea. The GenBank accession number of the 16S rRNA gene sequence is OR945204.

    3.20. Description of Nocardioides kandeliae 23ND54S-44

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on MA are circular, raised, entire, and white after incubation for 3 days at 25°C. Negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis, gelatinase, and β-galactosidase. D-Glucose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, and adipic acid are utilized. Does not utilize L-arabinose, D-mannose, capric acid, malic acid, trisodium citrate, and phenylacetic acid. Strain 23ND54S-44 displays the highest 16S rRNA gene sequence similarity with Nocardioides kandeliae BGMRC 2075T (99.3%). Strain 23ND54S-44 (=NNIBR2023641BA2552=FBCC-B15834) was isolated from riverside sediment collected in Samcheoksi, Gangwon-do, Korea. The GenBank accession number of the 16S rRNA gene sequence is OR945218.

    3.21. Description of Aestuariimicrobium soli 21SJ05S-R33

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on R2A are convex with entire edge, and ivory after incubation for 3 days at 20°C. Positive for nitrate reduction, esculin hydrolysis, and β-galactosidase, but negative for indole production, glucose fermentation, arginine dihydrolase, urease, and gelatinase. Does not utilize all substrates: D-Glucose, L-arabinose, D-mannose, D-mannitol, Nacetyl- glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain 21SJ05S-R33 displays the highest 16S rRNA gene sequence similarity with Aestuariimicrobium soli D6T (98.7%). Strain 21SJ05S-R33 (=NNIBR2021641BA1532=FBCC-B8167) was isolated from surface water collected in Imsil-gun, Jeollabuk- do, Korea. The GenBank accession number of the 16S rRNA gene sequence is MZ976826.

    3.22. Description of Solirubrobacter pauli 21SJ02S-54

    Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, smooth, and white after incubation for 3 days at 20°C. Positive for esculin hydrolysis, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, gelatinase, and β-galactosidase. Does not utilize all substrates: D-Glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain 21SJ02S-54 displays the highest 16S rRNA gene sequence similarity with Solirubrobacter pauli DSM 14954T (99.7%). Strain 21SJ02S-54 (=NNIBR2021641BA1655=FBCC-B8600) was isolated from riverside sediment collected in Imsil-gun, Jeollabuk-do, Korea. The GenBank accession number of the 16S rRNA gene sequence is OL677210.

    4. CONCLUSION

    This study successfully identified and characterized 22 previously unrecorded bacterial species from the phylum Actinomycetota in Korean freshwater environments. These findings significantly expand the known diversity of Actinomycetota and underscore the importance of exploring underexplored habitats for discovering novel microbial taxa. The newly identified species hold considerable promise for various biotechnological applications, such as the development of new antimicrobial agents and bioremediation strategies. Future research should focus on investigating the functional properties and ecological roles of these bacteria to fully realize their potential benefits.

    ACKNOWLEDGEMENTS

    This study was supported by a grant from the Nakdonggang National Institute of Biological Resources (NNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NNIBR20241103).

    CRediT authorship contribution statement

    SY Lee: Investigation, Data curation, Writing, Funding acquisition, Writing-Reviewing and editing. J Goh: Supervision. A Choi: Project administration, Conceptualization, Writing, Funding acquisition, Writing-Reviewing and editing.

    Declaration of Competing Interest

    The authors declare no conflict of interest.

    Figure

    KJEB-42-3-288_F1.gif

    Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing relationships of 22 strains isolated in this study and their relatives in the phylum Actinomycetota. Acidipila rosea AP8T (GenBank accession no. AB561884) was used as an outgroup. Bootstrap values (>70%) are shown at nodes. Bar, 0.02 substitutions per nucleotide position.

    KJEB-42-3-288_F2.gif

    Transmission electron micrographs of cells of strains isolated in this study. Scale bars are shown for each image. Strains: 1, LS9- 103; 2, 23ND14S-41; 3, 23ND75W-61; 4, 21SJ05W-0M23; 5, 23ND49W-34; 6, HRW3-25; 7, 6LW2-11; 8, 22LW2-51; 9, 23ND25W-67; 10, LW1-R25; 11, 22LW2-48; 12, 23ND14S-8; 13, SS-105; 14, SJ8; 15, 21LS8-22; 16, 23ND34S-94; 17, SJW1-30; 18, 21LS6-111; 19, 23ND01W-24; 20, 23ND54S-044; 21, 21SJ05S-R33; 22, 21SJ02S-54.

    Table

    Summary of 22 isolates belonging to the phylum Actinomycetota and their taxonomic affiliations, isolation sources, and culture conditions

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    Vol. 40 No. 4 (2022.12)

    Journal Abbreviation 'Korean J. Environ. Biol.'
    Frequency quarterly
    Doi Prefix 10.11626/KJEB.
    Year of Launching 1983
    Publisher Korean Society of Environmental Biology
    Indexed/Tracked/Covered By

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