Isolation and maintenance
In May 2018, an environmental bacterial strain, designated CJ43T, was isolated from fresh water at Gangwon-do, South Korea (N37.483472, E128.657556), using the method described by Lee et al. (2020). Briefly, environmental antibiotic-resistant bacteria were isolated by the dilution-plating method on Mueller-Hinton agar (BD) containing 8 mg/L gentamicin (Sigma-Aldrich). Colonies were individually streaked on Mueller-Hinton agar at 30 ℃, and the obtained pure culture was preserved at -80 ℃ with glycerol suspension (30%, w/v).
Phylogenetic analysis
The 16S rRNA gene of strain CJ43T was amplified by PCR using the universal primers 27F, and 1492R for bacteria (Frank et al. 2008). The PCR product was purified, and sequenced at Biofact (Daejeon, South Korea) using the sequencing primers 27F, 1492R, 785F, and 805R (Baker et al. 2003). The 16S rRNA gene sequence of strain CJ43T was aligned using the multiple sequence alignment program clustal W (Thompson et al. 1994) with those of related type strains obtained from the EzBioCloud database (www.ezbiocloud.net) (Yoon et al. 2017). Phylogenetic analysis was conducted using mega X version 10.1.7 (Kumar et al. 2018). Evolutionary distances were calculated for the neighbor-joining (NJ) tree (Saitou and Nei 1987) using the Jukes-Cantor model (Jukes and Cantor 1969). The best-fit substitution model for the maximum-likelihood (ML) tree was determined to be Kimura two-parameter model (Kimura 1980) by model test option of mega X version 10.1.7 (Kumar et al. 2018). The maximum-parsimony (MP) tree was obtained using the subtree pruning and regrafting algorithm (Nei and Kumar 2000). The topology of NJ, ML, and MP trees were evaluated based on the bootstrap resampling method with 1000 replications (Felsenstein 1985). Three closely related type strains, identified from 16S rRNA phylogenetic tree, were used as reference strains for phenotypic tests and chemotaxonomic analysis.
Physiological characterisation
Strain CJ43T was examined to find out optimal media for growth on different media such as R2A agar (BD), tryptic soy agar (TSA, BD), Luria-Bertani (LB) agar (BD), nutrient agar (NA, BD), and marine agar (MA, BD) at 30 ℃. The optimum growth temperature was determined at 4, 15, 20, 25, 30, and 37 ℃ on R2A agar. The pH range for growth was tested using different pH buffers in R2A broth (MB cell), which were 0.1 M citrate buffer for pH 5, 0.2 M phosphate buffer for pH 6-8 and 0.1 M bicarbonate-carbonate buffer for pH 9. Salt tolerance was tested in R2A broth supplemented with 0-3% (w/v) NaCl (at 1% intervals). Cellular morphology of strain CJ43T was observed by transmission electron microscopy (JEM 1010; JEOL) using three-day cultured cells on R2A agar at 30 ℃. Gliding motility was tested by stab culture in semi-solid R2A medium [0.4% (w/v) agar]. Anaerobic growth was determined after two weeks of cultivation at 30 ℃ on R2A agar using the GasPak Anaerobe Pouch System (BD).Gram-stain was carried out using a gram staining kit according to the manufacturer’s protocols (Sigma-Aldrich). Oxidase activity was assessed using oxidase reagent (bioMérieux), and catalase activity was evaluated by bubble production in a 3% (v/v) H2O2 solution on several fresh colonies. Hydrolysis of starch, cellulose, casein, and deoxyribonucleic acid were tested using 1% (w/v) soluble starch, 0.2% (w/v) carboxymethyl cellulose, 3% (w/v) skimmed milk, and deoxyribonuclease (DNase) test agar (BD), respectively, after seven days of incubation, following methods described by Reichenbach (2006). The reactions in API 20NE, API ZYM, and API 50CH strips (bioMérieux) were incubated at 30 ℃ and assessed following the producer's prescriptions.
Chemotaxonomic analysis
The respiratory quinone and polar lipids were extracted and analysed from freeze-dried cells following methods described by Minnikin et al. (1984). Isoprenoid quinone was extracted using hexane:methanol (1:2, v/v) in darkness, evaporated using a vacuum rotary evaporator, separated by thin-layer chromatography (TLC) plate using petroleum ether:acetone (95:5, v/v), and detected by UV absorbance at 254 nm. The purified quinones were detected by high performance liquid chromatography (Collins and Jones 1981). The polar lipids of strain CJ43T were analysed by two-dimensional TLC using chloroform:methanol:water (65:25:4, v/v/v) for the first-dimension solvent and chloroform:methanol:acetic acid:water (80:12:15:4, v/v/v/v) for the second-dimension solvent. Appropriate detection reagents were used to identify the spots; 5% (w/v) phosphomolybdic acid, 0.25% (w/v) ninhydrin, molybdenum blue spray reagent (Sigma-Aldrich), and 15% (w/v) a-naphthol reagent were used to detect total polar lipids, amino lipids, phospholipids, and glycolipids, respectively (Costa et al. 2011). To carry out whole cell fatty acid methyl esters analysis, strains CJ43T, and the related type strains were grown on R2A at 30 ℃ and harvested at the mid-exponential phase. Cells were saponified, methylated, and extracted following the instruction of the standard Microbial Identification system version 6.1. The fatty acids were analysed by gas chromatography (HP 6890 Series GC System; Hewlett Packard), and identified using the RTSBA6 6.10 database of the Microbial Identification system (Sasser 1990).
Whole-genome sequencing and genomic analyses
The genomic DNA of strain CJ43T was extracted using DNeasy Powersoil Kit (Qiagen) according to the instructions. Thereafter, the whole-genome sequencing was performed using the PacBio RS II (Pacific Biosciences) Single Molecule Real-Time (SMRT) platform with 20 kb SMRTbellTM template library. 71240 sequencing reads were obtained and assembled de novo using the Hierarchical Genome Assembly Process implemented in the PacBio SMRT analysis software version 2.3.0. The up-to-date bacterial core-gene (UBCG) set and the UBCG pipeline version 3 (Na et al. 2018) were used for the reconstruction of phylogenomic tree. 92 UBCGs were concatenated for alignments and the phylogenomic tree was inferred by the approximate ML method using FastTree (version 2.1.3) (Price et al. 2010). The Genome-to-Genome Distance Calculator 3.0 (http://ggdc.dsmz.de/distcalc2.php) (Meier-Kolthoffet al. 2014) was used to assess digitalDNA-DNA hybridization (dDDH). Average nucleotide identity (ANI) values between genomic sequences of strain CJ43T, and its closest phylogenomic neighbors were calculated using the OrthoANI tools (Lee et al. 2016). Reference genome sequences of type strains were downloaded from the EzBioCloud database (www.ezbiocloud.net) (Yoon et al. 2017). Annotation was performed using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline software revision 4.11 (Tatusova et al. 2016), and NCBI reference sequences (Haft et al. 2018). PRODIGAL version 2.6.3 (Hyatt et al. 2010) was used for finding protein-coding sequences (CDSs). Functional annotations were played against various databases, including Clusters of Orthologous Groups (COGs) categories based on the COG database (Tatusov et al. 2000), SEED subsystem (Overbeek et al. 2014), Kyoto Encyclopedia of Genes and Genomes pathway database (Kanehisa and Goto 2000). The antibiotic resistance genes were determined using the comprehensive antibiotic resistance database (CARD) (Jia et al. 2017).
Antimicrobial susceptibility testing
The antibiotic susceptibility of strain CJ43T was evaluated by the disk diffusion assay using antibiotic impregnated disks (Liofilchem, Italy) with amoxicillin (10 mg), cephalexin (30 mg), chloramphenicol (30 mg), ciprofloxacin (5 mg), clindamycin (2 mg), colistin sulfate (10 mg), fosfomycin (200 mg), gentamicin (10 mg), linezolid (10 mg), meropenem (10 mg), rifampicin (5 mg), streptomycin (10 mg), sulfamethoxazole (50 mg), tetracycline (30 mg), and trimethoprim (5 mg). Susceptibilities were recorded as susceptible (S) or resistant (R) based on breakpoints for Escherichia coli according to the EUCAST QC breakpoint table (version 11.0) (www.eucast.org/clinical_breakpoints/). E. coli ATCC 25922 was used as a quality control strain.