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References

  1. Ryu SH, Lee DS, Park M, Wang Q, Jang HH, Park W, et al. 2008. Caenimonas koreensis gen. nov., sp. nov., isolated from activated sludge. Int. J. Syst. Evol. Microbiol. 58: 1064-1068.
    Pubmed CrossRef
  2. Kim SJ, Weon HY, Kim YS, Moon JY, Seok SJ, Hong SB, et al. 2012. Caenimonas terrae sp. nov., isolated from a soil sample in Korea, and emended description of the genus Caenimonas Ryu et al. 2008. J. Microbiol. 50: 864-868.
    Pubmed CrossRef
  3. Parte AC. 2018. LPSN - List of prokaryotic names with standing in nomenclature (Bacterio. net), 20 years on. Int. J. Syst. Evol. Microbiol. 68: 1825-1829.
    Pubmed CrossRef
  4. Dahal RH, Lee H, Chaudhary DK, Kim DY, Son J, Kim J, et al. 2021. Caenimonas soli sp. nov., isolated from soil. Arch. Microbiol. 203: 1123-1129.
    Pubmed CrossRef
  5. Xu J, Sheng M, Yang Z, Qiu J, Zhang J, Zhang L, He J. 2020. Caenimonas sedimenti sp. nov., isolated from sediment of the wastewater outlet of an agricultural chemical plant. Curr. Microbiol. 77: 3767-3772.
    Pubmed CrossRef
  6. Xu L, Han Y, Yi M, Yi H, Guo E, Zhang A. 2019. Shift of millet rhizosphere bacterial community during the maturation of parent soil revealed by 16S rDNA high-throughput sequencing. Appl. Soil Ecol. 135: 157-165.
    CrossRef
  7. Tindall BJ, Rosselló-Móra R, Busse HJ, Ludwig W, Kämpfer P. 2010. Notes on the characterization of prokaryote strains for taxonomic purposes. Int. J. Syst. Evol. Microbiol. 60: 249-266.
    Pubmed CrossRef
  8. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173: 697-703.
    Pubmed PMC CrossRef
  9. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J. 2017. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int. J. Syst. Evol. Microbiol. 67: 1613-1617.
    Pubmed PMC CrossRef
  10. Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425.
    Pubmed CrossRef
  11. Felsenstein J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol. 17: 368-376.
    Pubmed CrossRef
  12. Nei M, Kumar S, Takahashi K. 1998. The optimization principle in phylogenetic analysis tends to give incorrect topologies when the number of nucleotides or amino acids used is small. Proc. Natl. Acad. Sci. USA 95: 12390-12397.
    Pubmed PMC CrossRef
  13. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35: 1547-1549.
    Pubmed PMC CrossRef
  14. Kimura M. 1983. The neutral theory of molecular evolution. Cambridge University Press, Cambridge.
    CrossRef
  15. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30: 2114-2120.
    Pubmed PMC CrossRef
  16. Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. 2015. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31: 3210-3212.
    Pubmed CrossRef
  17. Aziz RK, Bartels D, Best A, DeJongh M, Disz T, Edwards RA, et al. 2008. The RAST server: rapid annotations using subsystems technology. BMC Genomics 9: 75.
    Pubmed PMC CrossRef
  18. Blin K, Shaw S, Steinke K, Villebro R, Ziemert N, Lee SY, et al. 2019. AntiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res. 47: W81-W87.
    Pubmed PMC CrossRef
  19. Davis JJ, Wattam AR, Aziz RK, Brettin T, Butler R, Butler RM, et al. 2020. The PATRIC bioinformatics resource center: expanding data and analysis capabilities. Nucleic Acids Res. 48: D606-D612.
    Pubmed PMC CrossRef
  20. Meier-Kolthoff JP, Göker M. 2019. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat. Commun. 10: 2182.
    Pubmed PMC CrossRef
  21. Yoon SH, Ha SM, Lim J, Kwon S, Chun J. 2017. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek 110: 1281-1286.
    Pubmed CrossRef
  22. Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. 2013. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14: 60.
    Pubmed PMC CrossRef
  23. Smibert R, Krieg NR. 1994. Phenotypic characterization, pp. 607-654. In: Gerhardt P, Murray R, Wood W, Krieg N (eds), Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington DC, USA.
    CrossRef
  24. Bauer AW, Kirby WM, Sherris JC, Turck M. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45: 493-496.
    Pubmed CrossRef
  25. Sasser M. Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101.
  26. Minnikin DE, O'Donnell AG, Goodfellow M, Alderson G, Athalye M, Schaal A, et al. 1984. An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J. Microbiol. Methods 2: 233-241.
    CrossRef
  27. Tindall BJ, Sikorski J, Smibert RA, Krieg NR. 2007. Phenotypic characterization and the principles of comparative systematics, pp. 330-393. In: Reddy CA, Beveridge TJ, Breznak JA, Marzluf G, Schmidt TM, Snyder LR (eds), Methods for General and Molecular Microbiology, 3rd Ed. American Society for Microbiology, Washington DC, USA.
    CrossRef
  28. Kates M. 1972. Techniques of lipidology. Elsevier, New York, USA.
    CrossRef
  29. Oren A, Duker S, Ritter S. 1996. The polar lipid composition of Walsby's square bacterium. FEMS Microbiol. Lett. 138: 135-140.
    CrossRef
  30. Tamaoka J. 1986. Analysis of bacterial menaquinone mixtures by reverse-phase high-performance liquid chromatography. Methods Enzymol. 123: 251-256.
    Pubmed CrossRef
  31. Busse J, Auling G. 1988. Polyamine pattern as a chemotaxonomic marker within the Proteobacteria. Syst. Appl. Microbiol. 11: 1-8.
    CrossRef
  32. Sharrar AM, Crits-Christoph A, Méheust R, Diamond S, Starr EP, Banfield JF. 2020. Bacterial secondary metabolite biosynthetic potential in soil varies with phylum, depth, and vegetation type. mBio 11: e00416-20.
    Pubmed PMC CrossRef
  33. Hibbing ME, Fuqua C, Parsek MR, Peterson SB. 2010. Bacterial competition: surviving and thriving in the microbial jungle. Nat. Rev. Microbiol. 8: 15-25.
    Pubmed PMC CrossRef
  34. Helfrich EJN, Lin GM, Voigt CA, Clardy J. 2019. Bacterial terpene biosynthesis: challenges and opportunities for pathway engineering. Beilstein J. Org. Chem. 15: 2889-2906.
    Pubmed PMC CrossRef
  35. Agrawal S, Acharya D, Adholeya A, Barrow CJ, Deshmukh SK. 2017. Nonribosomal peptides from marine microbes and their antimicrobial and anticancer potential. Front. Pharmacol. 8: 828.
    Pubmed PMC CrossRef
  36. Zhang J, Du L, Liu F, Xu F, Hu B, Venturi V, et al. 2014. Involvement of both PKS and NRPS in antibacterial activity in Lysobacter enzymogenes OH11. FEMS Microbiol. Lett. 355: 170-176.
    Pubmed PMC CrossRef
  37. Schöner TA, Gassel S, Osawa A, Tobias NJ, Okuno Y, Sakakibara Y, et al. 2016. Aryl polyenes, a highly abundant class of bacterial natural products, are functionally related to antioxidative carotenoids. Chembiochem 17: 247-253.
    Pubmed CrossRef
  38. Hegemann JD, Zimmermann M, Xie X, Marahiel MA. 2015. Lasso peptides: an intriguing class of bacterial natural products. Acc. Chem. Res. 48: 1909-1919.
    Pubmed CrossRef
  39. Ding YP, Khan IU, Li MM, Xian WD, Liu L, Zhou EM, et al. 2019. Calidifontimicrobium sediminis gen. nov., sp. nov., a new member of the family Comamonadaceae. Int. J. Syst. Evol. Microbiol. 69: 434-440.
    Pubmed CrossRef
  40. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR, da Costa MS, et al. 2018. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int. J. Syst. Evol. Microbiol. 68: 461-466.
    Pubmed CrossRef
  41. Kim M, Oh HS, Park SC, Chun J. 2014. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int. J. Syst. Evol. Microbiol. 64: 346-351.
    Pubmed CrossRef
  42. Auch AF, von Jan M, Klenk HP, Göker M. 2010. Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand. Genomic Sci. 2: 117-134.
    Pubmed PMC CrossRef
  43. Richter M, Rosselló-Móra R. 2009. Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl. Acad. Sci. USA 106: 19126-19131.
    Pubmed PMC CrossRef
  44. Busse HJ. 2011, pp. 239-259. Polyamines. Rainey F, Oren A (eds). Academic Press, Methods in Microbiology.
    CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2022; 32(5): 575-581

Published online May 28, 2022 https://doi.org/10.4014/jmb.2201.01023

Copyright © The Korean Society for Microbiology and Biotechnology.

Caenimonas aquaedulcis sp. nov., Isolated from Freshwater of Daechung Reservoir during Microcystis Bloom

Ve Van Le1,2, So-Ra Ko1, Sang-Ah Lee3, Mingyeong Kang1,2, 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
3Environmental Safety Groups, Korea Institute of Science and Technology (KIST) Europe, Saarbrücken 66123, Germany

Correspondence to:Chi-Yong Ahn,      cyahn@kribb.re.kr

Received: January 20, 2022; Revised: March 23, 2022; Accepted: March 23, 2022

Abstract

A Gram-stain-negative, white-coloured, and rod-shaped bacterium, strain DR4-4T, was isolated from Daechung Reservoir, Republic of Korea, during Microcystis bloom. Strain DR4-4T was most closely related to Caenimonas terrae SGM1-15T and Caenimonas koreensis EMB320T with 98.1% 16S rRNA gene sequence similarities. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain DR4-4T and closely related type strains were below 79.46% and 22.30%, respectively. The genomic DNA G+C content was 67.5%. The major cellular fatty acids (≥10% of the total) were identified as C16:0, cyclo C17:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c), and summed feature 8 (C18:1ω7c and/or C18:1ω6c). Strain DR4-4T possessed phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylglycerol as the main polar lipids and Q-8 as the respiratory quinone. The polyamine profile was composed of putrescine, cadaverine, and spermidine. The results of polyphasic characterization indicated that the isolated strain DR4-4T represents a novel species within the genus Caenimonas, for which the name Caenimonas aquaedulcis sp. nov. is proposed. The type strain is DR4-4T (=KCTC 82470T =JCM 34453T).

Keywords: Caenimonas aquaedulcis, Microcystis aeruginosa, freshwater, polyphasic characterization

Introduction

The genus Caenimonas, a member of the family Comamonadaceae, was first proposed by Ryu et al. [1] and amended by Kim et al. [2]. Since that formal description, the genus has encompassed three species with validly published names, including Caenimonas koreensis, Caenimonas terrae, and Caenimonas soli, and one non-validly published name species ‘Caenimonas sedimenti’ (https://lpsn.dsmz.de) [3]. Chemotaxonomically, members of this genus have ubiquinone-8 as major isoprenoid quinone and C18:1 ω7c, C16:1 ω7c and/or iso-C15:0 2-OH, and C16:0 as main fatty acids [1]. The ecology of the genus Caenimonas has been reported from activated sludge [1], soil [2, 4], and sediment [5]. Additionally, they are highly distributed in mellow soil, suggesting their potential application for fertilizer production to enhance soil quality [6]. In this study, we used a polyphasic approach to ascertain the taxonomic position of strain DR4-4T and proposed it as a novel species within the genus Caenimonas.

Materials and Methods

Strains and Culture Conditions

Strain DR4-4T was isolated from a freshwater sample collected from the Janggye site, Daechung Reservoir, Korea (GPS location: 36° 22′ 33.7′′ N, 127° 38′ 20.6′′ E) in September 2019. The strain was successfully purified by re-streaking a single colony onto R2A plates. The stock cultures were preserved in R2A supplemented with 20%glycerol (v/v) at -80°C. For taxonomic analysis, C. koreensis EMB320T (=KCTC 12616T), C. terrae SGM1-15T (=KACC 13365T) C. soli S4T (=KCTC 72742T), and ‘Caenimonas sedimenti’ HX9-20T (=KCTC 72473T) were selected as reference strains. All reference strains were procured from Korean Collection for Type Cultures (KCTC), except C. terrae KACC 13365T obtained from Korean Agricultural Culture Collection (KACC). Since all strains could grow optimally on R2A, their chemotaxonomic and phenotypic features were characterized on R2A at 30°C after 3 days of incubation [7].

Phylogenetic Analysis Based on 16S rRNA Gene Sequences

Genomic DNA was extracted from the pure cultures with FastDNA Spin DNA-extraction kit (MP Biomedicals). The 16S rRNA gene was amplified and sequenced using the universal primer set 27F (5’-AGAGTTTGATCATGGCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’) [8]. The nearly full-length 16S rRNA gene sequence of strain DR4-4T (1,402 bp) was compared to those of valid species using the EzBioCloud server [9]. Phylogenetic trees derived from 16S rRNA gene sequence of strain DR4-4T and related type strains were reconstructed by neighbor-joining (NJ) [10], maximum–likelihood (ML) [11] and minimum evolution (ME) [12] algorithms in MEGA X software [13]. The best substitution model for the ML tree was determined by the model test in MEGA X software with the lowest Bayesian information criterion scores. Accordingly, the ML tree was reconstructed using Kimura's two-parameter model [14] with a gamma distribution and invariant sites (K2+G+I). For the NJ and ME trees, the evolutionary distances were computed using the Kimura 2-parameter method [14]. The reliability of phylogenetic trees was estimated using a bootstrap procedure with 1000 replications.

Genome Sequencing and Phylogenomic Analysis

Genome sequencing was performed on an Illumina MiSeq (Macrogen Inc.) and assembled de novo using SPAdes v3.12.0. Low-quality and adapter sequences were eliminated using Trimmomatic (v0.36) [15] to avoid biases in data analysis. The Benchmarking Universal Single-Copy Orthologous (BUSCO, v3.0) was used to measure assembly completeness [16]. The assembled genome was annotated by Prokka v.1.12 and RAST 2.0 (Rapid Annotation using Subsystem Technology) [17]. The putative secondary metabolite biosynthetic gene clusters in the genome of DR4-4T were identified by the antiSMASH server [18]. The circular genomic map was visualized using PATRIC web service [19]. The phylogenomic tree of strain DR4-4T and closely related taxa collected from GenBank database was constructed by the Type (Strain) Genome Server (TYGS) [20]. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were computed from whole-genome sequences using the ANI [21] and Genome-to-Genome Distance Calculator (GGDC 2.1) formula 2 [22], respectively. The two-way average amino acid identity (AAI) scores were obtained using the AAI calculator which was developed by Kostas lab (http://enve-omics.ce.gatech.edu/aai/).

Phenotypic and Biochemical Analyses

Morphological characteristics of strain DR4-4T were observed after growth on R2A for 3 days under a phase-contrast light microscope (Nikon Eclipse 80i) and a transmission electron microscope (CM20, Philips). Motility was tested using the hanging drop method. The Gram staining reaction was done with a Gram-stain kit (Becton Dickinson). Growth on different media was assessed using TSA, NA, and LBA (Difco) after 5 days of incubation at 25°C. The optimal pH for cellular growth was tested from pH 4.0 to pH 12.0 in intervals of 0.5 using the following buffer systems: phosphate-citrate (pH 4.0–6.5), Tris-HCl (pH 7.0–9.0), NaHCO3–NaOH (pH 9.5–11), and Na2HPO4–NaOH (pH 11.5–12). The effect of temperature on growth was assessed at 4, 10, 17, 20, 25, 30, 37, 40, and 60°C, respectively. The sensitivities of strain DR4-4T to salt were tested in R2A supplemented with 0.5-11.0%sodium chloride in intervals of 0.5%. Growth was measured at an optical density of 600 nm. The presence of activity of oxidase and catalase in bacterial cells were detected using 1% (w/v) N, N, N’, N’-tetramethyl-1,4-phenylenediamine and 3% (v/v) H2O2, respectively. Starch, lipids, carboxymethyl cellulose, and skim milk hydrolysis were performed according to Smibert and Krieg [23]. Enzyme activities and production of acid from carbon source were determined using API ZYM kit and API 50 CH kit (bioMérieux), respectively, following the manufacturer’s instructions. Other biochemical tests were performed with API 20NE (bioMérieux). The sensitivity of strain DR4-4T to antibiotics was performed using the Kirby-Bauer disc diffusion method [24] with antibiotic discs containing the following amounts (μg/disc): amikacin (30), ampicillin (10), amoxicillin (10), cefadroxil (30), cefoperazone (75), ceftazidime (30), ceftriaxone (30), chloramphenicol (30), ciprofloxacin (5), cloxacillin (1), erythromycin (15), gentamicin (10), nalidixic acid (10), netillin (30), nitrofurantoin (300), norfloxacin (10), penicillin(10), tobramycin (10), and vancomycin (30).

Chemotaxonomic Analyses

For chemotaxonomic analysis, strain DR4-4T and reference strains were grown at 30°C for 3 days. Cellular fatty acids were extracted according to Sasser [25], analyzed by gas chromatography, and identified using the TSBA 6 database of the Microbial Identification System. Polar lipid extraction from freeze-dried biomass was done according to Minnikin et al. [26]. The extracted polar lipids were separated on the thin layer chromatography (TLC) plates with chloroform/methanol/water (65:25:4, v/v/v) and chloroform/methanol/acetic acid/water (80:12:15:4, v/v/v/v) as mobile phases for the first and second chromatography dimension, respectively [27]. Total polar lipids were identified by staining with 5% molybdophosphoric acid in ethanol [28, 29]. Molybdenum blue, α-naphthol, and ninhydrin reagents were used to visualize phospholipids, glycolipids, and amino lipids, respectively. Respiratory quinones were extracted from agitating wet culture pellets using chloroform/methanol (2:1, v/v) for 3–4 h and analyzed by HPLC [30]. Polyamines were extracted and analyzed according to Busse and Auling [31].

Results and Discussion

16S rRNA Phylogeny

Based on 16S rRNA gene sequence similarity, strain DR4-4 T was most closely related to C. terrae SGM1-15T (98.1%) and C. koreensis EMB320T (98.1%). In phylogenetic trees, DR4-4T formed a distinct branch within the genus Caenimonas, supporting the assignment of this strain to the genus Caenimonas (Figs. 1, S1 and S2).

Figure 1. Neighbor-joining phylogenetic tree based on the 16S rRNA gene sequences showing the relationship of strain DR4-4T to other members of the family Comamonadaceae. Burkholderia cepacia ATCC 25416T (GenBank accession No. NR114491) was used as an outgroup. Bootstrap values (≥50%) based on 1000 replications were indicated at branch nodes. Filled circles at nodes indicate that the corresponding nodes were recovered in the trees reconstructed with three algorithms (NJ, ME, and ML methods) whereas the nodes with empty circles were recovered by two algorithms. Bar, 0.01 nucleotide substitutions per nucleotide position.

Genomic and Phylogenomic Analyses

The genome of strain DR4-4T contained 3 contigs with a total length of 4,521,559 bp, an N50 length of 3,928,830 bp, and an L50 value of 1 (Fig. S3A). The completeness of the genome was 96.62%. The similarity level between 16S rRNA gene sequences retrieved from whole-genome data (1,523 bp) and that from Sanger sequencing (1,402 bp) was 100%, suggesting the authenticity of the genome assembly. The assembled genome sequence comprised 4,331 coding sequences, 43 tRNA genes, 1 tmRNA gene, and 3 rRNA genes. Most of the identified genes were involved in fundamental cellular processes such as metabolism, protein processing, energy, stress response, defense, and virulence (Fig. S3B). Microbes can produce secondary metabolites that play vital roles in interactions with their neighbors, such as competition, cooperation, and co-evolution [32, 33]. Notably, strain DR4-4T possessed five putative secondary metabolite biosynthetic gene clusters responsible for the synthesis of terpene, arylpolyene, lassopeptide, nonribosomal peptide synthetase (NRPS), and NRPS-like, type I polyketide synthase (T1PKS) (Fig. S4). These compounds have been reported to be related to defense mechanisms [34-38]. Among the five predicted secondary metabolite regions, terpene and lassopeptide exhibited no similarity with any known gene clusters. This finding highlighted that strain DR4-4T could produce such novel valuable natural compounds, which may be beneficial to competition.

The draft genome sequence of strains DR4-4T is publicly available on DDBJ/ENA/GenBank with accession number JADWYS000000000. The genomic G+C content of strain DR4-4T was found to be 67.5%, falling within the range (62.7–68.7%) for Caenimonas species [2]. The phylogenomic tree indicated that strain DR4-4T clustered closely with C. soli S4T (Fig. 2). Strain DR4-4T showed the highest AAI value with the type species of the genus Caenimonas (74.37%), followed by Ramlibacter (73.02%), Variovorax (66.56%), Limnohabitans (65.04%), Curvibacter (64.81%), and Rhodoferax (63.02%) (Table S1). These results suggested that DR4-4T should be regarded as a member within the genus Caenimonas [39]. The ANI and dDDH values for strain DR4-4T with its closely related type strains were below 79.46% and 22.3%, respectively (Table S2). Such values are much lower than the species boundaries of 95% for ANI and 70% for dDDH [40-43], suggesting the novel status of strain DR4-4T within the genus Caenimonas.

Figure 2. Phylogenomic tree constructed using Type (Strain) Genome Server (TYGS) showing the position of strain DR4-4T among the species of the family Comamonadaceae. GenBank accession numbers are given in parentheses. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values > 60% from 100 replications with an average branch support of 85.1%. The tree was rooted at the midpoint.

Phenotypic, Physiological, and Biochemical Characteristics

Strain DR4-4T cells were Gram-stain-negative, non-motile, rod-shaped (0.3–0.5 μm × 2.5–4.5 μm) (Fig. S5), and positive for oxidase and catalase. The strain was unable to hydrolyze skim milk, Tween 20, Tween 80, carboxyl methylcellulose, and starch. Growth was observed on NA and R2A but not on TSA and LB media. Colonies formed on R2A were white, smooth, and convex with entire margins. The strain exhibited abundant growth at 30°C, pH 7.0, and in the absence of sodium chloride. DR4-4T was susceptible to all tested antibiotics except cloxacillin. Strain DR4-4T and reference strains shared many common phenotypic properties, such as non-motile, colony color, the activity of oxidase, and alkaline phosphatase. However, it could be differentiated from the closely related Caenimonas species by several biochemical characteristics such as hydrolysis of gelatin, producing enzyme lipase (C14), cysteine arylamidase, and trypsin (Table 1). Detailed comparison of phenotypic features of strain DR4-4T with those of the closely related species is listed in Table 1 and Table S3.

Table 1 . Differential characteristics of strain DR4-4T and type strains of closely related species..

Characteristic12345
Catalase++-++
Hydrolysis of Tween 20-+---
Growth range
pH7.0-7.56.0-9.05.0-8.0#5.0-10.0††6.0-8.0**
Temperature (°C)10-3710-3510-40#10-37††15-30**
Enzymatic activity (API ZYM)
Lipase (C14)+----
Leucine arylamidase++-++
Cystine arylamidase+----
Trypsin+----
Other biochemical tests (API 20NE)
Reduction of nitrate to nitrite--+-+
Hydrolysis of:
Esculin---++
Gelatin+----
Polar lipidsPE,PG, DPGPE,PG, DPG,ALPE,PG, DPG, 3AL#PE,2PL, AL,L††PE,PG,PC, DPG,3APL**
DNA G+C content67.5%63.5%*68.7mol%#65.1%††67.5%**

Strains: 1, DR4-4T; 2, C. koreensis KCTC 12616T ( data was obtained from Ryu et al., (2008) [1]; *data was obtained from wholegenome sequences data under accession number WJBU00000000); 3, C. terrae KACC 13365T (#data was obtained from Kim et al., [2]); 4, C. soli KCTC 72742T(††data was obtained from Dahal et al., [4]); 5, ‘Caenimonas sedimenti’ KCTC 72473T(**data was obtained from Xu et al., [5]). PE, phosphatidylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; AL, unidentified aminolipid; APL, unidentified aminophospholipid; PL, unidentified phospholipids; PC, phosphatidylcholine; L, unidentified polar lipid. +, positive; -, negative; w, weakly positive reaction; NA, not available..



Chemotaxonomic Characteristics

In line with the description of the genus Caenimonas, the fatty acid profile of strain DR4-4T was dominated (≥10% of the total fatty acids) by C16:0 (33.7%), cyclo C17:0 (21.1%), summed feature 3 (C16:1ω7c and/or C16:1ω6c)(22.0%) and summed feature 8 (C18:1ω7c and/or C18:1ω6c) (10.0%). Nevertheless, the ratios of some components such as C14:0, C15:1 ω6c, C17:0, and C18:1 ω7c 11-methyl are a key chemotaxonomic difference between DR4-4T and references strains (Table 2). Strain DR4-4T is distinct from Variovorax species with respect to the presence of iso-C17:0 3-OH and C18:1 ω9c and the absence of C12:0 and C18:0 (Table S4). The fatty acid profile of Curvibacter fontanus AQ9T differs from those of DR4-4T by the presence of C15:0 as the predominant fatty acid (Table S4). The cellular fatty acid compositions of strain DR4-4T and its close neighbor taxon are mentioned in Table 2 and Table S4. The polar lipid composition of strain DR4-4T encompassed phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylglycerol (Fig. S6 and Table 1). Strain DR4-4T is differentiated from C. terrae SGM1-15T, C. koreensis EMB320T, and C. soli S4T by the absence of unidentified aminolipid. Additionally, phosphatidylcholine and three unidentified aminophospholipids were found in C. sedimenti HX-9-20T but absent in strain DR4-4T. The respiratory quinone was Q-8, which is consistent with its affiliation as a member belonging to the genus Caenimonas. The polyamine profile of strain DR4-4 T was composed of putrescine 61.01%, cadaverine 33.86%, and spermidine 5.13% (Fig. S7), which is in accordance with the characteristic of members Betaproteobacteria [44]. Taken together, the data from phylogenetic, genomic, phenotypic and chemotaxonomic analyses supported that strain DR4-4T should be considered as a novel species of the genus Caenimonas, for which the name C. aquaedulcis sp. nov. is proposed.

Table 2 . Fatty acid contents (%) of strain DR4-4T and type strains of closely related species..

Fatty Acids1234 5§
C8:0 3-OH---4.4-
C10:0----2.7
C10:0 3-OH4.63.72.83.42.6
iso-C11:03-OH---1.3-
C14:0TR1.1TR3.74.2
anteiso-C14:0---1.3-
C15:1 ω6cTR3.3---
C16:033.722.035.622.924.3
C17:1 ω6c-3.4---
cyclo C17:021.1-27.25.45.6
C17:01.23.6-1.0-
iso-C17:0 3-OH2.0----
C18:0---1.0-
C18:1 ω9c1.0TR---
C18:1 ω7c 11-methyl-2.4---
Summed Feature 3*22.044.519.829.146.6
Summed Feature 7**2.32.2---
Summed Feature 810.010.312.014.510.6
Summed Feature 9††---1.4-

Strains: 1, DR4-4T; 2, C. koreensis KCTC 12616T; 3, C. terrae KACC 13365T; 4, C. soli KCTC 72742T (‡Data was obtained from Dahal et al., [4]); 5, ‘Caenimonas sedimenti’ KCTC 72473T (§Data was obtained from Xu et al., [5]). Values are percentages of the total fatty acids. All data were obtained from this study, except where indicated otherwise. Major components (≥10.0%) are highlighted in bold. TR, Trace amount (<1.0%); −, not detected. * Summed Feature 3 comprises of C16:1ω7c and/or C16:1ω6c; ** Summed Feature 7 comprises C19:1 ω6c, C19:0 cyclo and/or an unknown fatty acid with an equivalent chain length of 18.846. † Summed Feature 8 comprises of C18:1ω7c and/or C18:1ω6c. Summed Feature 9†† comprises iso-C17:1ω9c and/or 10-methyl C16:0. Summed features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total..



Description of Caenimonas aquaedulcis sp. nov.

C. aquaedulcis (a.quae.dul’cis. L. fem. n. aqua water; L. masc. adj. dulcis sweet; N.L. gen. n. aquaedulcis of fresh water).

Cells are Gram-stain-negative, rod-shaped, approximately 2.5–4.5 μm in length and 0.3–0.5 μm in width. Oxidase and catalase-positive. Colonies grown on R2A after 3 days of incubation are white, smooth, and convex with entire margins. Growth is observed on R2A and NA at 10-37°C (optimally at 30°C) and at pH 7.0-7.5 (optimally at pH 7.0). It does not require NaCl for growth but can tolerate up to 0.5%. Negative for hydrolyzing skim milk, Tween 80, Tween 20, carboxymethyl cellulose, and starch. In the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, cystine arylamidase, trypsin, acid phosphatase, and naphthol-AS-BI-phosphohydrolase are positive but valine arylamidase, α–chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase are negative. According to API 20NE test, the cells are positive for hydrolysis of esculin and gelatine but negative for reduction of nitrate to nitrite, reduction of nitrate to nitrogen, indole production, glucose acidification, arginine dihydrolase, urease, assimilation of D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. In the API 50CH system, a weakly positive reaction is observed for aesculin but not for the other substrates. The major cellular fatty acids are C16:0, cyclo C17:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c), and summed feature 8 (C18:1ω7c and/or C18:1ω6c). The polar lipids are phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylglycerol. The respiratory quinone is ubiquinone Q-8. The polyamines are putrescine, cadaverine, and spermidine. The type strain, DR4-4T (=KCTC 82470T =JCM 34453T), was isolated from Daechung Reservoir. The DNA G+C content of the type strain is 67.5%. The GenBank/EMBL/DDBJ accession number of the 16S rRNA gene sequence and the whole genome sequence of type strain are OL587860 and JADWYS000000000, respectively.

Supplemental Materials

Acknowledgments

We thank Professor Aharon Oren for his expert advice concerning the genus and species epithet and Latin etymology. This study was supported by National Research Foundation of Korea (2019R1A2C2007038 and 2021R1A2C1005151) and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program.

Conflicts of Interest

The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Neighbor-joining phylogenetic tree based on the 16S rRNA gene sequences showing the relationship of strain DR4-4T to other members of the family Comamonadaceae. Burkholderia cepacia ATCC 25416T (GenBank accession No. NR114491) was used as an outgroup. Bootstrap values (≥50%) based on 1000 replications were indicated at branch nodes. Filled circles at nodes indicate that the corresponding nodes were recovered in the trees reconstructed with three algorithms (NJ, ME, and ML methods) whereas the nodes with empty circles were recovered by two algorithms. Bar, 0.01 nucleotide substitutions per nucleotide position.
Journal of Microbiology and Biotechnology 2022; 32: 575-581https://doi.org/10.4014/jmb.2201.01023

Fig 2.

Figure 2.Phylogenomic tree constructed using Type (Strain) Genome Server (TYGS) showing the position of strain DR4-4T among the species of the family Comamonadaceae. GenBank accession numbers are given in parentheses. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values > 60% from 100 replications with an average branch support of 85.1%. The tree was rooted at the midpoint.
Journal of Microbiology and Biotechnology 2022; 32: 575-581https://doi.org/10.4014/jmb.2201.01023

Table 1 . Differential characteristics of strain DR4-4T and type strains of closely related species..

Characteristic12345
Catalase++-++
Hydrolysis of Tween 20-+---
Growth range
pH7.0-7.56.0-9.05.0-8.0#5.0-10.0††6.0-8.0**
Temperature (°C)10-3710-3510-40#10-37††15-30**
Enzymatic activity (API ZYM)
Lipase (C14)+----
Leucine arylamidase++-++
Cystine arylamidase+----
Trypsin+----
Other biochemical tests (API 20NE)
Reduction of nitrate to nitrite--+-+
Hydrolysis of:
Esculin---++
Gelatin+----
Polar lipidsPE,PG, DPGPE,PG, DPG,ALPE,PG, DPG, 3AL#PE,2PL, AL,L††PE,PG,PC, DPG,3APL**
DNA G+C content67.5%63.5%*68.7mol%#65.1%††67.5%**

Strains: 1, DR4-4T; 2, C. koreensis KCTC 12616T ( data was obtained from Ryu et al., (2008) [1]; *data was obtained from wholegenome sequences data under accession number WJBU00000000); 3, C. terrae KACC 13365T (#data was obtained from Kim et al., [2]); 4, C. soli KCTC 72742T(††data was obtained from Dahal et al., [4]); 5, ‘Caenimonas sedimenti’ KCTC 72473T(**data was obtained from Xu et al., [5]). PE, phosphatidylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; AL, unidentified aminolipid; APL, unidentified aminophospholipid; PL, unidentified phospholipids; PC, phosphatidylcholine; L, unidentified polar lipid. +, positive; -, negative; w, weakly positive reaction; NA, not available..


Table 2 . Fatty acid contents (%) of strain DR4-4T and type strains of closely related species..

Fatty Acids1234 5§
C8:0 3-OH---4.4-
C10:0----2.7
C10:0 3-OH4.63.72.83.42.6
iso-C11:03-OH---1.3-
C14:0TR1.1TR3.74.2
anteiso-C14:0---1.3-
C15:1 ω6cTR3.3---
C16:033.722.035.622.924.3
C17:1 ω6c-3.4---
cyclo C17:021.1-27.25.45.6
C17:01.23.6-1.0-
iso-C17:0 3-OH2.0----
C18:0---1.0-
C18:1 ω9c1.0TR---
C18:1 ω7c 11-methyl-2.4---
Summed Feature 3*22.044.519.829.146.6
Summed Feature 7**2.32.2---
Summed Feature 810.010.312.014.510.6
Summed Feature 9††---1.4-

Strains: 1, DR4-4T; 2, C. koreensis KCTC 12616T; 3, C. terrae KACC 13365T; 4, C. soli KCTC 72742T (‡Data was obtained from Dahal et al., [4]); 5, ‘Caenimonas sedimenti’ KCTC 72473T (§Data was obtained from Xu et al., [5]). Values are percentages of the total fatty acids. All data were obtained from this study, except where indicated otherwise. Major components (≥10.0%) are highlighted in bold. TR, Trace amount (<1.0%); −, not detected. * Summed Feature 3 comprises of C16:1ω7c and/or C16:1ω6c; ** Summed Feature 7 comprises C19:1 ω6c, C19:0 cyclo and/or an unknown fatty acid with an equivalent chain length of 18.846. † Summed Feature 8 comprises of C18:1ω7c and/or C18:1ω6c. Summed Feature 9†† comprises iso-C17:1ω9c and/or 10-methyl C16:0. Summed features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total..


References

  1. Ryu SH, Lee DS, Park M, Wang Q, Jang HH, Park W, et al. 2008. Caenimonas koreensis gen. nov., sp. nov., isolated from activated sludge. Int. J. Syst. Evol. Microbiol. 58: 1064-1068.
    Pubmed CrossRef
  2. Kim SJ, Weon HY, Kim YS, Moon JY, Seok SJ, Hong SB, et al. 2012. Caenimonas terrae sp. nov., isolated from a soil sample in Korea, and emended description of the genus Caenimonas Ryu et al. 2008. J. Microbiol. 50: 864-868.
    Pubmed CrossRef
  3. Parte AC. 2018. LPSN - List of prokaryotic names with standing in nomenclature (Bacterio. net), 20 years on. Int. J. Syst. Evol. Microbiol. 68: 1825-1829.
    Pubmed CrossRef
  4. Dahal RH, Lee H, Chaudhary DK, Kim DY, Son J, Kim J, et al. 2021. Caenimonas soli sp. nov., isolated from soil. Arch. Microbiol. 203: 1123-1129.
    Pubmed CrossRef
  5. Xu J, Sheng M, Yang Z, Qiu J, Zhang J, Zhang L, He J. 2020. Caenimonas sedimenti sp. nov., isolated from sediment of the wastewater outlet of an agricultural chemical plant. Curr. Microbiol. 77: 3767-3772.
    Pubmed CrossRef
  6. Xu L, Han Y, Yi M, Yi H, Guo E, Zhang A. 2019. Shift of millet rhizosphere bacterial community during the maturation of parent soil revealed by 16S rDNA high-throughput sequencing. Appl. Soil Ecol. 135: 157-165.
    CrossRef
  7. Tindall BJ, Rosselló-Móra R, Busse HJ, Ludwig W, Kämpfer P. 2010. Notes on the characterization of prokaryote strains for taxonomic purposes. Int. J. Syst. Evol. Microbiol. 60: 249-266.
    Pubmed CrossRef
  8. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173: 697-703.
    Pubmed KoreaMed CrossRef
  9. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J. 2017. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int. J. Syst. Evol. Microbiol. 67: 1613-1617.
    Pubmed KoreaMed CrossRef
  10. Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425.
    Pubmed CrossRef
  11. Felsenstein J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol. 17: 368-376.
    Pubmed CrossRef
  12. Nei M, Kumar S, Takahashi K. 1998. The optimization principle in phylogenetic analysis tends to give incorrect topologies when the number of nucleotides or amino acids used is small. Proc. Natl. Acad. Sci. USA 95: 12390-12397.
    Pubmed KoreaMed CrossRef
  13. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35: 1547-1549.
    Pubmed KoreaMed CrossRef
  14. Kimura M. 1983. The neutral theory of molecular evolution. Cambridge University Press, Cambridge.
    CrossRef
  15. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30: 2114-2120.
    Pubmed KoreaMed CrossRef
  16. Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. 2015. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31: 3210-3212.
    Pubmed CrossRef
  17. Aziz RK, Bartels D, Best A, DeJongh M, Disz T, Edwards RA, et al. 2008. The RAST server: rapid annotations using subsystems technology. BMC Genomics 9: 75.
    Pubmed KoreaMed CrossRef
  18. Blin K, Shaw S, Steinke K, Villebro R, Ziemert N, Lee SY, et al. 2019. AntiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res. 47: W81-W87.
    Pubmed KoreaMed CrossRef
  19. Davis JJ, Wattam AR, Aziz RK, Brettin T, Butler R, Butler RM, et al. 2020. The PATRIC bioinformatics resource center: expanding data and analysis capabilities. Nucleic Acids Res. 48: D606-D612.
    Pubmed KoreaMed CrossRef
  20. Meier-Kolthoff JP, Göker M. 2019. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat. Commun. 10: 2182.
    Pubmed KoreaMed CrossRef
  21. Yoon SH, Ha SM, Lim J, Kwon S, Chun J. 2017. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek 110: 1281-1286.
    Pubmed CrossRef
  22. Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. 2013. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14: 60.
    Pubmed KoreaMed CrossRef
  23. Smibert R, Krieg NR. 1994. Phenotypic characterization, pp. 607-654. In: Gerhardt P, Murray R, Wood W, Krieg N (eds), Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington DC, USA.
    CrossRef
  24. Bauer AW, Kirby WM, Sherris JC, Turck M. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45: 493-496.
    Pubmed CrossRef
  25. Sasser M. Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101.
  26. Minnikin DE, O'Donnell AG, Goodfellow M, Alderson G, Athalye M, Schaal A, et al. 1984. An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J. Microbiol. Methods 2: 233-241.
    CrossRef
  27. Tindall BJ, Sikorski J, Smibert RA, Krieg NR. 2007. Phenotypic characterization and the principles of comparative systematics, pp. 330-393. In: Reddy CA, Beveridge TJ, Breznak JA, Marzluf G, Schmidt TM, Snyder LR (eds), Methods for General and Molecular Microbiology, 3rd Ed. American Society for Microbiology, Washington DC, USA.
    CrossRef
  28. Kates M. 1972. Techniques of lipidology. Elsevier, New York, USA.
    CrossRef
  29. Oren A, Duker S, Ritter S. 1996. The polar lipid composition of Walsby's square bacterium. FEMS Microbiol. Lett. 138: 135-140.
    CrossRef
  30. Tamaoka J. 1986. Analysis of bacterial menaquinone mixtures by reverse-phase high-performance liquid chromatography. Methods Enzymol. 123: 251-256.
    Pubmed CrossRef
  31. Busse J, Auling G. 1988. Polyamine pattern as a chemotaxonomic marker within the Proteobacteria. Syst. Appl. Microbiol. 11: 1-8.
    CrossRef
  32. Sharrar AM, Crits-Christoph A, Méheust R, Diamond S, Starr EP, Banfield JF. 2020. Bacterial secondary metabolite biosynthetic potential in soil varies with phylum, depth, and vegetation type. mBio 11: e00416-20.
    Pubmed KoreaMed CrossRef
  33. Hibbing ME, Fuqua C, Parsek MR, Peterson SB. 2010. Bacterial competition: surviving and thriving in the microbial jungle. Nat. Rev. Microbiol. 8: 15-25.
    Pubmed KoreaMed CrossRef
  34. Helfrich EJN, Lin GM, Voigt CA, Clardy J. 2019. Bacterial terpene biosynthesis: challenges and opportunities for pathway engineering. Beilstein J. Org. Chem. 15: 2889-2906.
    Pubmed KoreaMed CrossRef
  35. Agrawal S, Acharya D, Adholeya A, Barrow CJ, Deshmukh SK. 2017. Nonribosomal peptides from marine microbes and their antimicrobial and anticancer potential. Front. Pharmacol. 8: 828.
    Pubmed KoreaMed CrossRef
  36. Zhang J, Du L, Liu F, Xu F, Hu B, Venturi V, et al. 2014. Involvement of both PKS and NRPS in antibacterial activity in Lysobacter enzymogenes OH11. FEMS Microbiol. Lett. 355: 170-176.
    Pubmed KoreaMed CrossRef
  37. Schöner TA, Gassel S, Osawa A, Tobias NJ, Okuno Y, Sakakibara Y, et al. 2016. Aryl polyenes, a highly abundant class of bacterial natural products, are functionally related to antioxidative carotenoids. Chembiochem 17: 247-253.
    Pubmed CrossRef
  38. Hegemann JD, Zimmermann M, Xie X, Marahiel MA. 2015. Lasso peptides: an intriguing class of bacterial natural products. Acc. Chem. Res. 48: 1909-1919.
    Pubmed CrossRef
  39. Ding YP, Khan IU, Li MM, Xian WD, Liu L, Zhou EM, et al. 2019. Calidifontimicrobium sediminis gen. nov., sp. nov., a new member of the family Comamonadaceae. Int. J. Syst. Evol. Microbiol. 69: 434-440.
    Pubmed CrossRef
  40. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR, da Costa MS, et al. 2018. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int. J. Syst. Evol. Microbiol. 68: 461-466.
    Pubmed CrossRef
  41. Kim M, Oh HS, Park SC, Chun J. 2014. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int. J. Syst. Evol. Microbiol. 64: 346-351.
    Pubmed CrossRef
  42. Auch AF, von Jan M, Klenk HP, Göker M. 2010. Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand. Genomic Sci. 2: 117-134.
    Pubmed KoreaMed CrossRef
  43. Richter M, Rosselló-Móra R. 2009. Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl. Acad. Sci. USA 106: 19126-19131.
    Pubmed KoreaMed CrossRef
  44. Busse HJ. 2011, pp. 239-259. Polyamines. Rainey F, Oren A (eds). Academic Press, Methods in Microbiology.
    CrossRef