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Comparative Genome analysis of the Genus Curvibacter and the Description of Curvibacter microcysteis sp. nov. and Curvibacter cyanobacteriorum sp. nov., Isolated from Fresh Water during the Cyanobacterial Bloom Period
1Cell Factory Research Centre, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea
2Department of Environmental Biotechnology, KRIBB School of Biotechnology, University of Science and Technology, Daejeon 34113, Republic of Korea
J. Microbiol. Biotechnol. 2023; 33(11): 1428-1436
Published November 28, 2023 https://doi.org/10.4014/jmb.2306.06017
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
The genus
Cyanobacteria play an essential role in the carbon and nutrient cycles [5]. However, they cause harm to ecosystems and humans through the production of cyanotoxins [6]. The excessive proliferation of cyanobacteria, also known as cyanobacterial bloom, destroys biodiversity and degrades drinking water quality [6]. During the study of the role of bacterial community in the formation of cyanobacterial blooms, three new strains, RS43T, HBC28, and HBC61T, were isolated from a freshwater reservoir during the cyanobacterial bloom period. The present study aimed to determine their taxonomic status using polyphasic approach. In addition, the first comparative genomic study for the genus
Materials and Methods
Isolation and Cultivation
Strains RS43T, HBC28, and HBC61T were isolated from Daechung Reservoir (36° 22' 33.7'' N, 127° 38' 20.6'' E) according to the procedures previously described [7]. Briefly, the sample was diluted by the standard ten-fold serial dilution method and was spread on agar plates of Reasoner’s 2A (R2A). After incubation at 25°C for 1 week under aerobic conditions, colonies were picked and purified two times by repeated subculture. The strains were then preserved in R2A medium containing 20% glycerol (v/v) at –80°C. Considering the close phylogenetic relationships,
Molecular Identification, Genome Sequencing, and Pangenome Analysis
The whole genomic DNA was extracted using FastDNA Spin Kit for Soil (MB Biomedicals, USA). For strain identification, the 16S rRNA gene was amplified and sequenced with the universal bacterial primers of 27F, 518F, 785F, 805R, 907R, and 1492R [8-10]. Pairwise comparisons of the almost-complete 16S rRNA gene sequences of three strains and those of related taxa were performed on the latest updated EzBioCloud (www.ezbiocloud.net)[11] and NCBI blast (www.ncbi.nlm.nih.gov). The draft genomes of three strains were sequenced using the Illumina MiSeq (Macrogen Inc., South Korea) platform and annotated by Prokka v.1.12 and Rapid Annotation using Subsystems Technology (RAST). The metabolic pathways of strains RS43T, HBC28, and HBC61T were reconstructed using BlastKOALA [12]. The G+C content was calculated from the whole genome sequence. For comparative genome analysis, the genomes of
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Table 1 . General genome characteristics of RS43T, HBC28, and HBC61T and related taxa.
Species Strain Accession No. Size (Mb) G+C (%) Gene Protein Curvibacter microcysteis RS43T JAQSIN000000000 4.89 65.22 4,382 4,297 Curvibacter microcysteis HBC28 JAQSIO000000000 4.82 65.29 4,399 4,256 Curvibacter cyanobacteriorum HBC61T JAQSIP000000000 4.85 67.15 4,266 4,185 Curvibacter gracilis ATCC BAA-807T JADZ00000000 6.75 66.01 6,214 6,036 Curvibacter lanceolatus ATCC 14669T ARLO00000000 6.83 65.55 6,367 6,157 Curvibacter delicatus NBRC 14919T BCWP00000000 3.78 63.51 3,678 3,543
Phylogeny
The 16S rRNA gene sequences of three strains and other closely related strains obtained from the GenBank/EMBL/DDBJ databases were aligned using the MUSCLE algorithm. To identify the phylogenetic location of three strains, phylogenetic trees were constructed by using maximum-likelihood [15], neighbor-joining [16], and minimum-evolution [17] methods with the software MEGA version 11 [18]. The confidence levels and distance matrices of phylogenetic trees were determined with 1000 bootstrap replicates and Kimura’s two-parameter model [19], respectively. To provide cogent evidence for the assignment of strains RS43T, HBC28, and HBC61T as novel species, the ANI and dDDH values were calculated using the OrthoANI algorithm and the Genome-to-Genome Distance Calculator (GGDC 2.0) [20], respectively. The two-way average amino acid identity (AAI) was calculated using AAI calculator (http://enve-omics.ce.gatech.edu/aai/) and percentage of conserved proteins (POCP) values were by using EDGAR 3.0 platform [13]. A phylogenomic tree was built utilizing the Type (Strain) Genome Server (TYGS) [21].
Phenotypic and Biochemical Properties
Morphological characteristics of strains RS43T, HBC28, and HBC61T were visualized under a CM-20 transmission electron microscope (Philips) after 3-day incubation at 25°C on R2A medium. Cell motility was identified using the hanging-drop method [22]. A Gram stain kit (Becton Dickinson, USA) was used to determine the Gram reaction. Catalase and oxidase tests were done using 3% (v/v) H2O2 and 1% (w/v) N, N, N’, N’-tetramethyl-1,4-phenylenediamine, respectively. The growth of three strains on R2A agar, Luria–Bertani agar (LBA, Difco, USA), tryptone soy agar (TSA, Difco, USA), and nutrient agar (NA, Difco, USA) was investigated at 25°C for five days. Growth tolerance ranges for temperature, pH, and NaCl were assessed by observing colony growth on R2A agar plates after 5-day incubation. The growth temperature was evaluated at 4, 10, 17, 20, 25, 30, 37, 40, and 60°C. Growth at different pH (4.0–12.0 with an interval of 1.0) was assessed by adjusting the pH using the buffer system [7]. The tolerance range for NaCl was determined by culturing strains on R2A agar plates supplemented with varying amounts of NaCl [0.0, 0.1, 0.3, 0.5, 1.0, 1.5, and 2.0% (w/v)]. Biochemical characteristics and enzyme activities were inspected using API ZYM and API 20NE kits (bioMérieux, France). Hydrolysis of Tween 20 (1.0%, w/v), Tween 80 (1.0%, w/v), and skim milk (0.5%, w/v) was tested after 3-day incubation [23].
Chemotaxonomic Characterization
For chemotaxonomic analysis, all strains were grown on R2A for 3 days at 25°C. After harvesting the cells, the fatty acids were saponified, methylated, extracted, and analyzed according to the protocol recommended by MIDI Microbial Identification System (version 6.1, MIDI) [24]. Polar lipids were extracted from lyophilized cells, separated and identified as described previously [25–28]. Quinones were extracted and purified with a mixture of chloroform and methanol (2:1) for 3–4 h and analyzed by HPLC [29].
Results and Discussion
Phylogenetic Characteristics
The 16S rRNA gene sequences of strains RS43T, HBC28, and HBC61T determined by the Sanger sequencing were 1474, 1484, and 1471 bp in length, respectively. Strains RS43T, HBC28, and HBC61T were most closely related to
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Fig. 1. Neighbor-joining phylogenetic tree based on the 16S rRNA gene sequences depicting the phylogenetic placements of three strains among the related species. Bootstrap values above 50% are shown at branch points. Closed circles indicate that the corresponding nodes were also recovered in the maximum-likelihood and minimum-evolution methods, whereas the open circles indicate nodes recovered with neighbor-joining and maximum-likelihood algorithms.
Genomic Characteristics
The draft genome sequences of strains RS43T, HBC28, and HBC61T had a total nucleotide length of 4,895,745 bp (24 contigs), 4,822,995 bp (21 contigs), and 4,848,818 bp (19 contigs), respectively, with the sequencing depth of 148×, 147×, and 147×, respectively. Overall, the draft genome met the criteria required for the taxonomic purposes proposed [30]. The G+C contents of strains RS43T and HBC28 were estimated to be 65.22 % and 65.29 %, which correspond with the G+C content range (62.2–66.0 mol%) of members of the genus
The genomic features of
Whole-Genome-Based Phylogeny
The AAI and POCP values for the three strains with the type species of the genus,
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Fig. 2. Heat map of AAI (A), POCP (B), ANI (C), and dDDH (D) from pairwise genome comparisons. AAI, average amino acid identity; POCP, percentage of conserved proteins; ANI, average nucleotide identity; dDDH, digital DNA– DNA hybridization.
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Fig. 3. Phylogenomic tree inferred with FastME 2.1.6.1 from Genome BLAST Distance Phylogeny (GBDP) distances calculated from genome sequences. The branch lengths are adjusted with the GBDP distance formula d5. The GBDP pseudo-bootstrap support values from 100 replications exceeding 60% are displayed above the branches. The average support across branches is 94.6%.
Comparative Genomic Analysis
The distribution of specific genomic regions of
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Fig. 4. Comparative genomic analysis. Circular view of the genome of
Curvibacter species (A). The number (B) and percentage (C) of common core genes across allCurvibacter species. Core genome (D) and pangenome (E) profiles ofCurvibacter species. The fitted exponential Heaps’ law function is represented by the red curve while the upper and lower boundaries of the 95% confidence internal are indicated by the green and blue curves, respectively.
The core gene proportion of all strains of
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Fig. 5. Functional genome analysis of
Curvibacter species. The distribution of core, dispensable, and singletons (A). Gene clusters predicted in the genomes ofCurvibacter species involved in secondary metabolite synthesis (B), Metabolism of cofactors and vitamins and xenobiotics biodegradation in the genomes ofCurvibacter species (C).
Aromatic compounds, which constitute approximately 25% of the world's biomass, are the second most prevalent group of organic compounds in nature after carbohydrates [44]. They are considered one of the most persistent pollutants in the environment [45]. The metabolic capacities of
Phenotypic Characteristics
The cells of strains RS43T, HBC28, and HBC61T were observed to be Gram-negative, rod-shaped with flagella (Fig. S3), and catalase- and oxidase-positive. The colonies appeared circular, smooth, convex, and colorless with a diameter of 1–2 mm after 3 days of growth on R2A agar medium at 25°C. These strains grew well on R2A and NA media but not on LBA and TSA media. They were positive for hydrolysis of Tween 80, but negative for hydrolysis of Tween 20 and skim milk. Growth occurred within the pH range of 5.5 to 10. Several phenotypic features distinguish strain RS43T and strain HBC61T, confirming they belong to two different species. For instance, strain HBC61T assimilated D-mannitol and gluconate, exhibited growth at 40°C, and was able to tolerate NaCl concentration up to 1% (w/v), whereas strain RS43T did not. Table 2 provides further details on the additional phenotypic features distinguishing strains RS43T, HBC28, and HBC61T from their closely related strains.
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Table 2 . Differential phenotypic characteristics of strains RS43T, HBC28, and HBC61T and their phylogenetic neighbors.
Characteristic 1 2 3 4 5 6 Hydrolysis of Tween 20 - - - + - - Optimal growth range pH 7.5-8.5 7.5-8.5 6.0-8.0 5.0-8.0a neutrophilicb 5.5-8.5c Temperature (°C) 20-30 20-30 20-37 25-30a 20-30b 30-32c Biochemical characteristics (API ZYM, API 20NE) Esterase (C4) + + + + + - Esterase lipase (C8) + + + + + - Cystine arylamidase - - - + + - Reduction of nitrates to nitrites - - - + + + Urea hydrolysis - + + + + + Assimilation of D-Glucose - - - + - - D-Mannitol - - + - - - Gluconate - - + + - - Taxa: 1, strain RS43T; 2, HBC28; 3, HBC61T; 4,
Curvibacter gracilis KCTC42831T; 5,Curvibacter lanceolatus KCTC42829T; 6,Curvibacter delicatus KCTC 42828T. +, positive; −, negative. aData were obtained from [3]; bData were obtained from [4]; cData were obtained from [46].
Chemotaxonomic Characteristics
The fatty acid profile of strains RS43T, HBC28, and HBC61T showed a similar pattern to those of the reference strains (Table 3). All strains had major fatty acids (> 10% of total fatty acids) that included summed feature 3 (C16:1
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Table 3 . Fatty acid compositions (%) of strains RS43T, HBC28, and HBC61T and their phylogenetic neighbors.
Fatty Acids 1 2 3 4 5 6 C8:0 3-OH 4.4 4.2 3.7 3.7 ND 4.2 C12:0 4.4 3.7 3.6 3.3 3.7 ND C14:0 ND ND ND ND TR ND C16:0 14.3 15.9 16.2 20.8 20.1 33.2 C17:1 ω6c ND ND ND 1.44 TR ND C17:0 cyclo ND ND ND ND ND 5.8 C18:1 ω9c ND ND TR TR ND 1.1 C18:1 ω7c 11-methyl 1.03 TR TR ND TR TR Summed Feature 3* 42.8 43.3 43.1 42.6 43.1 39.0 Summed Feature 8* 32.6 31.6 30.4 26.3 29.5 15.7 Taxa: 1, strain RS43T; 2, HBC28; 3, HBC61T; 4,
Curvibacter gracilis KCTC42831T; 5,Curvibacter lanceolatus KCTC42829T; 6,Curvibacter delicatus KCTC 42828T. Major fatty acids (> 10.0%) are highlighted as bold type. TR, trace amount (< 1%); ND, not detected. *Summed features refer to fatty acids that cannot be distinctly differentiated from another fatty acid and these fatty acids are grouped as one feature with a single percentage of the total. Summed feature 3 contained C16:1ω 6c and/or C16:1ω 7c . Summed feature 8 contained C18:1ω 7c and/or C18:1ω 6c .
Description of Curvibacter microcysteis sp. nov.
The type strain RS43T (=KCTC 92793T = LMG 32714T) was isolated from fresh water. The 16S rRNA gene sequence and the genomic sequence of strain RS43T have been deposited under the GenBank/EMBL/DDBJ accession numbers OQ642155 and JAQSIN000000000, respectively.
Description of Curvibacter cyanobacteriorum sp. nov.
Cells are rod-shaped, 1.2–2.7 μm in length and 0.7–0.9 μm in width. Colonies grown on R2A agar are smooth, convex, and colorless with a 1–2 mm diameter. Motile with monotrichous flagellum. Growth was observed at the temperature range of 10–40°C, up to 1% NaCl, and pH 5.5–10. Catalase and oxidase are positive. Able to hydrolyze Tween 80. Positive for esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase, hydrolysis of urea, and assimilation of D-mannitol and potassium gluconate. The major fatty acids were summed feature 3 (C16:1
The type strain HBC61T (=KCTC 92794T = LMG 32713T) was isolated from fresh water. The 16S rRNA gene sequence and the genomic sequence of strain HBC61T have been deposited under the GenBank/EMBL/DDBJ accession numbers OQ642154 and JAQSIP000000000, respectively.
Conclusions
Considering chemotaxonomic, phylogenetic, and phenotypic characteristics, strains RS43T, HBC28, and HBC61T belonged to the genus
Supplemental Materials
Acknowledgments
This research was supported by Korea Environment Industry & Technology Institute (KEITI) through Aquatic Ecosystem Conservation Research Program (2022003050004) and the National Research Foundation of Korea (2023R1A2C1003308) and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5252322).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2023; 33(11): 1428-1436
Published online November 28, 2023 https://doi.org/10.4014/jmb.2306.06017
Copyright © The Korean Society for Microbiology and Biotechnology.
Comparative Genome analysis of the Genus Curvibacter and the Description of Curvibacter microcysteis sp. nov. and Curvibacter cyanobacteriorum sp. nov., Isolated from Fresh Water during the Cyanobacterial Bloom Period
Ve Van Le1, So-Ra Ko1, Mingyeong Kang1,2, Seonah Jeong1, Hee-Mock Oh1,2, and Chi-Yong Ahn1,2*
1Cell Factory Research Centre, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea
2Department of Environmental Biotechnology, KRIBB School of Biotechnology, University of Science and Technology, Daejeon 34113, Republic of Korea
Correspondence to:Chi-Yong Ahn, cyahn@kribb.re.kr
Abstract
The three Gram-negative, catalase- and oxidase-positive bacterial strains RS43T, HBC28, and HBC61T, were isolated from fresh water and subjected to a polyphasic study. Comparison of 16S rRNA gene sequence initially indicated that strains RS43T, HBC28, and HBC61T were closely related to species of genus Curvibacter and shared the highest sequence similarity of 98.14%, 98.21%, and 98.76%, respectively, with Curvibacter gracilis 7-1T. Phylogenetic analysis based on genome sequences placed all strains within the genus Curvibacter. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between the three strains and related type strains supported their recognition as two novel genospecies in the genus Curvibacter. Comparative genomic analysis revealed that the genus possessed an open pangenome. Based on KEGG BlastKOALA analyses, Curvibacter species have the potential to metabolize benzoate, phenylacetate, catechol, and salicylate, indicating their potential use in the elimination of these compounds from the water systems. The results of polyphasic characterization indicated that strain RS43T and HBC61T represent two novel species, for which the name Curvibacter microcysteis sp. nov. (type strain RS43T =KCTC 92793T=LMG 32714T) and Curvibacter cyanobacteriorum sp. nov. (type strain HBC61T =KCTC 92794T =LMG 32713T) are proposed.
Keywords: Curvibacter, cyanobacterial bloom, Microcystis
Introduction
The genus
Cyanobacteria play an essential role in the carbon and nutrient cycles [5]. However, they cause harm to ecosystems and humans through the production of cyanotoxins [6]. The excessive proliferation of cyanobacteria, also known as cyanobacterial bloom, destroys biodiversity and degrades drinking water quality [6]. During the study of the role of bacterial community in the formation of cyanobacterial blooms, three new strains, RS43T, HBC28, and HBC61T, were isolated from a freshwater reservoir during the cyanobacterial bloom period. The present study aimed to determine their taxonomic status using polyphasic approach. In addition, the first comparative genomic study for the genus
Materials and Methods
Isolation and Cultivation
Strains RS43T, HBC28, and HBC61T were isolated from Daechung Reservoir (36° 22' 33.7'' N, 127° 38' 20.6'' E) according to the procedures previously described [7]. Briefly, the sample was diluted by the standard ten-fold serial dilution method and was spread on agar plates of Reasoner’s 2A (R2A). After incubation at 25°C for 1 week under aerobic conditions, colonies were picked and purified two times by repeated subculture. The strains were then preserved in R2A medium containing 20% glycerol (v/v) at –80°C. Considering the close phylogenetic relationships,
Molecular Identification, Genome Sequencing, and Pangenome Analysis
The whole genomic DNA was extracted using FastDNA Spin Kit for Soil (MB Biomedicals, USA). For strain identification, the 16S rRNA gene was amplified and sequenced with the universal bacterial primers of 27F, 518F, 785F, 805R, 907R, and 1492R [8-10]. Pairwise comparisons of the almost-complete 16S rRNA gene sequences of three strains and those of related taxa were performed on the latest updated EzBioCloud (www.ezbiocloud.net)[11] and NCBI blast (www.ncbi.nlm.nih.gov). The draft genomes of three strains were sequenced using the Illumina MiSeq (Macrogen Inc., South Korea) platform and annotated by Prokka v.1.12 and Rapid Annotation using Subsystems Technology (RAST). The metabolic pathways of strains RS43T, HBC28, and HBC61T were reconstructed using BlastKOALA [12]. The G+C content was calculated from the whole genome sequence. For comparative genome analysis, the genomes of
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Table 1 . General genome characteristics of RS43T, HBC28, and HBC61T and related taxa..
Species Strain Accession No. Size (Mb) G+C (%) Gene Protein Curvibacter microcysteis RS43T JAQSIN000000000 4.89 65.22 4,382 4,297 Curvibacter microcysteis HBC28 JAQSIO000000000 4.82 65.29 4,399 4,256 Curvibacter cyanobacteriorum HBC61T JAQSIP000000000 4.85 67.15 4,266 4,185 Curvibacter gracilis ATCC BAA-807T JADZ00000000 6.75 66.01 6,214 6,036 Curvibacter lanceolatus ATCC 14669T ARLO00000000 6.83 65.55 6,367 6,157 Curvibacter delicatus NBRC 14919T BCWP00000000 3.78 63.51 3,678 3,543
Phylogeny
The 16S rRNA gene sequences of three strains and other closely related strains obtained from the GenBank/EMBL/DDBJ databases were aligned using the MUSCLE algorithm. To identify the phylogenetic location of three strains, phylogenetic trees were constructed by using maximum-likelihood [15], neighbor-joining [16], and minimum-evolution [17] methods with the software MEGA version 11 [18]. The confidence levels and distance matrices of phylogenetic trees were determined with 1000 bootstrap replicates and Kimura’s two-parameter model [19], respectively. To provide cogent evidence for the assignment of strains RS43T, HBC28, and HBC61T as novel species, the ANI and dDDH values were calculated using the OrthoANI algorithm and the Genome-to-Genome Distance Calculator (GGDC 2.0) [20], respectively. The two-way average amino acid identity (AAI) was calculated using AAI calculator (http://enve-omics.ce.gatech.edu/aai/) and percentage of conserved proteins (POCP) values were by using EDGAR 3.0 platform [13]. A phylogenomic tree was built utilizing the Type (Strain) Genome Server (TYGS) [21].
Phenotypic and Biochemical Properties
Morphological characteristics of strains RS43T, HBC28, and HBC61T were visualized under a CM-20 transmission electron microscope (Philips) after 3-day incubation at 25°C on R2A medium. Cell motility was identified using the hanging-drop method [22]. A Gram stain kit (Becton Dickinson, USA) was used to determine the Gram reaction. Catalase and oxidase tests were done using 3% (v/v) H2O2 and 1% (w/v) N, N, N’, N’-tetramethyl-1,4-phenylenediamine, respectively. The growth of three strains on R2A agar, Luria–Bertani agar (LBA, Difco, USA), tryptone soy agar (TSA, Difco, USA), and nutrient agar (NA, Difco, USA) was investigated at 25°C for five days. Growth tolerance ranges for temperature, pH, and NaCl were assessed by observing colony growth on R2A agar plates after 5-day incubation. The growth temperature was evaluated at 4, 10, 17, 20, 25, 30, 37, 40, and 60°C. Growth at different pH (4.0–12.0 with an interval of 1.0) was assessed by adjusting the pH using the buffer system [7]. The tolerance range for NaCl was determined by culturing strains on R2A agar plates supplemented with varying amounts of NaCl [0.0, 0.1, 0.3, 0.5, 1.0, 1.5, and 2.0% (w/v)]. Biochemical characteristics and enzyme activities were inspected using API ZYM and API 20NE kits (bioMérieux, France). Hydrolysis of Tween 20 (1.0%, w/v), Tween 80 (1.0%, w/v), and skim milk (0.5%, w/v) was tested after 3-day incubation [23].
Chemotaxonomic Characterization
For chemotaxonomic analysis, all strains were grown on R2A for 3 days at 25°C. After harvesting the cells, the fatty acids were saponified, methylated, extracted, and analyzed according to the protocol recommended by MIDI Microbial Identification System (version 6.1, MIDI) [24]. Polar lipids were extracted from lyophilized cells, separated and identified as described previously [25–28]. Quinones were extracted and purified with a mixture of chloroform and methanol (2:1) for 3–4 h and analyzed by HPLC [29].
Results and Discussion
Phylogenetic Characteristics
The 16S rRNA gene sequences of strains RS43T, HBC28, and HBC61T determined by the Sanger sequencing were 1474, 1484, and 1471 bp in length, respectively. Strains RS43T, HBC28, and HBC61T were most closely related to
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Figure 1. Neighbor-joining phylogenetic tree based on the 16S rRNA gene sequences depicting the phylogenetic placements of three strains among the related species. Bootstrap values above 50% are shown at branch points. Closed circles indicate that the corresponding nodes were also recovered in the maximum-likelihood and minimum-evolution methods, whereas the open circles indicate nodes recovered with neighbor-joining and maximum-likelihood algorithms.
Genomic Characteristics
The draft genome sequences of strains RS43T, HBC28, and HBC61T had a total nucleotide length of 4,895,745 bp (24 contigs), 4,822,995 bp (21 contigs), and 4,848,818 bp (19 contigs), respectively, with the sequencing depth of 148×, 147×, and 147×, respectively. Overall, the draft genome met the criteria required for the taxonomic purposes proposed [30]. The G+C contents of strains RS43T and HBC28 were estimated to be 65.22 % and 65.29 %, which correspond with the G+C content range (62.2–66.0 mol%) of members of the genus
The genomic features of
Whole-Genome-Based Phylogeny
The AAI and POCP values for the three strains with the type species of the genus,
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Figure 2. Heat map of AAI (A), POCP (B), ANI (C), and dDDH (D) from pairwise genome comparisons. AAI, average amino acid identity; POCP, percentage of conserved proteins; ANI, average nucleotide identity; dDDH, digital DNA– DNA hybridization.
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Figure 3. Phylogenomic tree inferred with FastME 2.1.6.1 from Genome BLAST Distance Phylogeny (GBDP) distances calculated from genome sequences. The branch lengths are adjusted with the GBDP distance formula d5. The GBDP pseudo-bootstrap support values from 100 replications exceeding 60% are displayed above the branches. The average support across branches is 94.6%.
Comparative Genomic Analysis
The distribution of specific genomic regions of
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Figure 4. Comparative genomic analysis. Circular view of the genome of
Curvibacter species (A). The number (B) and percentage (C) of common core genes across allCurvibacter species. Core genome (D) and pangenome (E) profiles ofCurvibacter species. The fitted exponential Heaps’ law function is represented by the red curve while the upper and lower boundaries of the 95% confidence internal are indicated by the green and blue curves, respectively.
The core gene proportion of all strains of
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Figure 5. Functional genome analysis of
Curvibacter species. The distribution of core, dispensable, and singletons (A). Gene clusters predicted in the genomes ofCurvibacter species involved in secondary metabolite synthesis (B), Metabolism of cofactors and vitamins and xenobiotics biodegradation in the genomes ofCurvibacter species (C).
Aromatic compounds, which constitute approximately 25% of the world's biomass, are the second most prevalent group of organic compounds in nature after carbohydrates [44]. They are considered one of the most persistent pollutants in the environment [45]. The metabolic capacities of
Phenotypic Characteristics
The cells of strains RS43T, HBC28, and HBC61T were observed to be Gram-negative, rod-shaped with flagella (Fig. S3), and catalase- and oxidase-positive. The colonies appeared circular, smooth, convex, and colorless with a diameter of 1–2 mm after 3 days of growth on R2A agar medium at 25°C. These strains grew well on R2A and NA media but not on LBA and TSA media. They were positive for hydrolysis of Tween 80, but negative for hydrolysis of Tween 20 and skim milk. Growth occurred within the pH range of 5.5 to 10. Several phenotypic features distinguish strain RS43T and strain HBC61T, confirming they belong to two different species. For instance, strain HBC61T assimilated D-mannitol and gluconate, exhibited growth at 40°C, and was able to tolerate NaCl concentration up to 1% (w/v), whereas strain RS43T did not. Table 2 provides further details on the additional phenotypic features distinguishing strains RS43T, HBC28, and HBC61T from their closely related strains.
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Table 2 . Differential phenotypic characteristics of strains RS43T, HBC28, and HBC61T and their phylogenetic neighbors..
Characteristic 1 2 3 4 5 6 Hydrolysis of Tween 20 - - - + - - Optimal growth range pH 7.5-8.5 7.5-8.5 6.0-8.0 5.0-8.0a neutrophilicb 5.5-8.5c Temperature (°C) 20-30 20-30 20-37 25-30a 20-30b 30-32c Biochemical characteristics (API ZYM, API 20NE) Esterase (C4) + + + + + - Esterase lipase (C8) + + + + + - Cystine arylamidase - - - + + - Reduction of nitrates to nitrites - - - + + + Urea hydrolysis - + + + + + Assimilation of D-Glucose - - - + - - D-Mannitol - - + - - - Gluconate - - + + - - Taxa: 1, strain RS43T; 2, HBC28; 3, HBC61T; 4,
Curvibacter gracilis KCTC42831T; 5,Curvibacter lanceolatus KCTC42829T; 6,Curvibacter delicatus KCTC 42828T. +, positive; −, negative. aData were obtained from [3]; bData were obtained from [4]; cData were obtained from [46]..
Chemotaxonomic Characteristics
The fatty acid profile of strains RS43T, HBC28, and HBC61T showed a similar pattern to those of the reference strains (Table 3). All strains had major fatty acids (> 10% of total fatty acids) that included summed feature 3 (C16:1
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Table 3 . Fatty acid compositions (%) of strains RS43T, HBC28, and HBC61T and their phylogenetic neighbors..
Fatty Acids 1 2 3 4 5 6 C8:0 3-OH 4.4 4.2 3.7 3.7 ND 4.2 C12:0 4.4 3.7 3.6 3.3 3.7 ND C14:0 ND ND ND ND TR ND C16:0 14.3 15.9 16.2 20.8 20.1 33.2 C17:1 ω6c ND ND ND 1.44 TR ND C17:0 cyclo ND ND ND ND ND 5.8 C18:1 ω9c ND ND TR TR ND 1.1 C18:1 ω7c 11-methyl 1.03 TR TR ND TR TR Summed Feature 3* 42.8 43.3 43.1 42.6 43.1 39.0 Summed Feature 8* 32.6 31.6 30.4 26.3 29.5 15.7 Taxa: 1, strain RS43T; 2, HBC28; 3, HBC61T; 4,
Curvibacter gracilis KCTC42831T; 5,Curvibacter lanceolatus KCTC42829T; 6,Curvibacter delicatus KCTC 42828T. Major fatty acids (> 10.0%) are highlighted as bold type. TR, trace amount (< 1%); ND, not detected. *Summed features refer to fatty acids that cannot be distinctly differentiated from another fatty acid and these fatty acids are grouped as one feature with a single percentage of the total. Summed feature 3 contained C16:1ω 6c and/or C16:1ω 7c . Summed feature 8 contained C18:1ω 7c and/or C18:1ω 6c ..
Description of Curvibacter microcysteis sp. nov.
The type strain RS43T (=KCTC 92793T = LMG 32714T) was isolated from fresh water. The 16S rRNA gene sequence and the genomic sequence of strain RS43T have been deposited under the GenBank/EMBL/DDBJ accession numbers OQ642155 and JAQSIN000000000, respectively.
Description of Curvibacter cyanobacteriorum sp. nov.
Cells are rod-shaped, 1.2–2.7 μm in length and 0.7–0.9 μm in width. Colonies grown on R2A agar are smooth, convex, and colorless with a 1–2 mm diameter. Motile with monotrichous flagellum. Growth was observed at the temperature range of 10–40°C, up to 1% NaCl, and pH 5.5–10. Catalase and oxidase are positive. Able to hydrolyze Tween 80. Positive for esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase, hydrolysis of urea, and assimilation of D-mannitol and potassium gluconate. The major fatty acids were summed feature 3 (C16:1
The type strain HBC61T (=KCTC 92794T = LMG 32713T) was isolated from fresh water. The 16S rRNA gene sequence and the genomic sequence of strain HBC61T have been deposited under the GenBank/EMBL/DDBJ accession numbers OQ642154 and JAQSIP000000000, respectively.
Conclusions
Considering chemotaxonomic, phylogenetic, and phenotypic characteristics, strains RS43T, HBC28, and HBC61T belonged to the genus
Supplemental Materials
Acknowledgments
This research was supported by Korea Environment Industry & Technology Institute (KEITI) through Aquatic Ecosystem Conservation Research Program (2022003050004) and the National Research Foundation of Korea (2023R1A2C1003308) and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5252322).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Table 1 . General genome characteristics of RS43T, HBC28, and HBC61T and related taxa..
Species Strain Accession No. Size (Mb) G+C (%) Gene Protein Curvibacter microcysteis RS43T JAQSIN000000000 4.89 65.22 4,382 4,297 Curvibacter microcysteis HBC28 JAQSIO000000000 4.82 65.29 4,399 4,256 Curvibacter cyanobacteriorum HBC61T JAQSIP000000000 4.85 67.15 4,266 4,185 Curvibacter gracilis ATCC BAA-807T JADZ00000000 6.75 66.01 6,214 6,036 Curvibacter lanceolatus ATCC 14669T ARLO00000000 6.83 65.55 6,367 6,157 Curvibacter delicatus NBRC 14919T BCWP00000000 3.78 63.51 3,678 3,543
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Table 2 . Differential phenotypic characteristics of strains RS43T, HBC28, and HBC61T and their phylogenetic neighbors..
Characteristic 1 2 3 4 5 6 Hydrolysis of Tween 20 - - - + - - Optimal growth range pH 7.5-8.5 7.5-8.5 6.0-8.0 5.0-8.0a neutrophilicb 5.5-8.5c Temperature (°C) 20-30 20-30 20-37 25-30a 20-30b 30-32c Biochemical characteristics (API ZYM, API 20NE) Esterase (C4) + + + + + - Esterase lipase (C8) + + + + + - Cystine arylamidase - - - + + - Reduction of nitrates to nitrites - - - + + + Urea hydrolysis - + + + + + Assimilation of D-Glucose - - - + - - D-Mannitol - - + - - - Gluconate - - + + - - Taxa: 1, strain RS43T; 2, HBC28; 3, HBC61T; 4,
Curvibacter gracilis KCTC42831T; 5,Curvibacter lanceolatus KCTC42829T; 6,Curvibacter delicatus KCTC 42828T. +, positive; −, negative. aData were obtained from [3]; bData were obtained from [4]; cData were obtained from [46]..
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Table 3 . Fatty acid compositions (%) of strains RS43T, HBC28, and HBC61T and their phylogenetic neighbors..
Fatty Acids 1 2 3 4 5 6 C8:0 3-OH 4.4 4.2 3.7 3.7 ND 4.2 C12:0 4.4 3.7 3.6 3.3 3.7 ND C14:0 ND ND ND ND TR ND C16:0 14.3 15.9 16.2 20.8 20.1 33.2 C17:1 ω6c ND ND ND 1.44 TR ND C17:0 cyclo ND ND ND ND ND 5.8 C18:1 ω9c ND ND TR TR ND 1.1 C18:1 ω7c 11-methyl 1.03 TR TR ND TR TR Summed Feature 3* 42.8 43.3 43.1 42.6 43.1 39.0 Summed Feature 8* 32.6 31.6 30.4 26.3 29.5 15.7 Taxa: 1, strain RS43T; 2, HBC28; 3, HBC61T; 4,
Curvibacter gracilis KCTC42831T; 5,Curvibacter lanceolatus KCTC42829T; 6,Curvibacter delicatus KCTC 42828T. Major fatty acids (> 10.0%) are highlighted as bold type. TR, trace amount (< 1%); ND, not detected. *Summed features refer to fatty acids that cannot be distinctly differentiated from another fatty acid and these fatty acids are grouped as one feature with a single percentage of the total. Summed feature 3 contained C16:1ω 6c and/or C16:1ω 7c . Summed feature 8 contained C18:1ω 7c and/or C18:1ω 6c ..
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