Phenotypic and Genomic Properties of Brachybacterium vulturis sp. nov. and Brachybacterium avium sp. nov.

Two strains, VM2412T and VR2415T, were isolated from the feces of an Andean condor (Vultur gryphus) living in Seoul Grand Park, Gyeonggi-do, South Korea. Cells of both strains were observed to be Gram-stain positive, non-motile, aerobic, catalase positive and oxidase negative. Growth was found to occur at 10-30°C, showing optimum growth at 30°C. The strains could tolerate up to 15% (w/v) NaCl concentration and grow at pH 6-9. The strains shared 99.3% 16S rRNA gene sequence similarity to each other but were identified as two distinct species based on 89.0-89.2% ANIb, 90.3% ANIm, 89.7% OrthoANI and 38.0% dDDH values calculated using whole genome sequences. Among species with validly published names, Brachybacterium ginsengisoli DCY80T shared high 16S rRNA gene sequence similarities with strains VM2412T (98.7%) and VR2415T (98.4%) and close genetic relatedness with strains VM2412T (83.3–83.5% ANIb, 87.0% ANIm, 84.3% OrthoANI and 27.8% dDDH) and VR2415T (82.8–83.2% ANIb, 86.7% ANIm, 83.9% OrthoANI and 27.2% dDDH). The major fatty acid of the two strains was identified as anteiso-C15:0 and the polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, presumptively phosphatidylethanolamine and three unidentified glycolipids. Strain VR2415T also produced an unidentified phospholipid. The cell walls of the two strains contained meso-diaminopimelic acid as diagnostic diamino acid and the whole cell sugars were ribose, glucose, and galactose. The strains contained MK-7 as their predominant menaquinone. The genomes of strains VM2412T, VR2415T, and B. ginsengisoli DCY80T were sequenced in this study. The genomic G+C contents of strains VM2412T and VR2415T were determined to be 70.8 and 70.4 mol%, respectively. A genome-based phylogenetic tree constructed using an up-to-date bacterial core gene set (UBCG) showed that the strains formed a clade with members of the genus Brachybacterium, supporting their taxonomic classification into the genus Brachybacterium. Based on phenotypic and genotypic analyses in this study, strains VM2412T and VR2415T are considered to represent two novel species of the genus Brachybacterium and the names Brachybacterium vulturis sp. nov. and Brachybacterium avium sp. nov. are proposed for strains VM2412T (=KCTC 39996T = JCM 32142T) and VR2415T (=KCTC 39997T = JCM 32143T), respectively.

Strains of the genus Dermabacter have been classified as Centers of Disease Control and Prevention (CDC) group 3 and group 5 coryneform bacteria (Funke et al., 1994) and have been isolated from diverse clinical specimens such as cerebral abscess (Bavbek et al., 1998), blood cultures (Gómez-Garcés et al., 2001), peritoneal fluid (Radtke et al., 2001), bone deposits from an osteomyelitis patient (Van Bosterhaut et al., 2002) and fatal septicemia (Lee et al., 2011). Therefore, they are considered opportunistic pathogens. Strains of the genus Devriesea are involved in the pathogenesis of dermatitis, cheilitis and septicemia in lizards as facultative pathogenic bacteria (Hellebuyck et al., 2009) and strains of the genus Helcobacillus were isolated from human cutaneous discharge samples, showing the potential for toxicity (Renvoise et al., 2009). However, members within the genus Brachybacterium have not been reported to be pathogenic before now. They have been isolated from environmental samples such as coastal sand, sediment, plants, soil and seawater and from foods including milk products, cheese and salt-fermented seafood. The type strain of B. muris was isolated from murine liver (Buczolits et al., 2003).
Isolation of strains of Brachybacterium species from wideranging sources demonstrates that they have adapted to diverse environmental conditions using characteristics obtained in the process of diverging from other genera. Genotypic analysis based on whole genome sequences can indicate which genes are involved in the divergence and can contribute to more accurate taxonomic classification. In present study, two strains were isolated and found to belong to the genus Brachybacterium based on 16S rRNA gene sequences. The whole genome sequences were obtained for the newly isolated strains and from B. ginsengisoli DCY80 T as a closely related species. Although whole genome sequences of some type strains of Brachybacterium species are available in GenBank/EMBL/DDBJ databases, the genome sequences have not been used previously for taxonomic analysis or subjected to comparative genomic studies. Therefore, we used the available genome sequences of Brachybacterium species along with those of Dermabacter and Devriesea species to investigate the taxonomic status of the newly isolated strains within the genus Brachybacterium and to identify intra-and inter-genus differences based on genotypic characteristics.

Bacterial Strain Isolation
Strains VM2412 T and VR2415 T were isolated from fecal sample of an Andean condor living in Seoul Grand Park, Gyeonggi-do, South Korea (37 • 25 39.7 N 127 • 01 01.2 E). Strains VM2412 T and VR2415 T were isolated using a marine agar (MA) plate and a Reasoner's 2A (R2A) medium, respectively, which were inoculated with 10 −4 diluted fecal sample and incubated at 20 • C. Tryptic soy broth (TSB) and tryptic soy agar (TSA) were used for culture at 30 • C.

16S rRNA Gene Sequence Analysis
Genome-derived 16S rRNA gene sequences of strains VM2412 T and VR2415 T were 1,513 bp. The sequences were compared with those of other type strains using EzBioCloud server (Yoon et al., 2017). The 16S rRNA gene sequences of the two strains and type strains of closely related taxa were aligned using BioEdit (Hall, 2011). Phylogenetic trees were constructed by neighborjoining (NJ) (Saitou and Nei, 1987), maximum likelihood (ML) (Felsenstein, 1981) and maximum parsimony (MP) (Kluge and Farris, 1969) algorithms in the MEGA6 program (Tamura et al., 2013).

Phenotypic Characterization
Based on the 16S rRNA gene sequences, the strains were observed to share high sequence similarities (>98%) with B. ginsengisoli DCY80 T and B. faecium DSM 4810 T . Therefore, these type strains were obtained from the Korean Collection for Type Cultures (B. ginsengisoli KCTC 29226 T ) and the Japan Collection of Microorganisms (B. faecium JCM 11609 T ) and used for comparison of phenotypic characteristics with the isolated strains.
The two isolates were cultured on TSA [4% (w/v) NaCl, pH 7] and incubated at 30 • C for optimal growth. After 48 h, cell morphology was identified using a light microscope (ECLIPSE 50i; Nikon) and colony morphology was determined macroscopically. Gram-staining was conducted with a Gramstaining kit (bioMérieux) according to the manufacturer's instructions. Cell motility was identified by bacterial migration from an inoculation line in semi-solid agar with 0.4% (w/v) agar (Tittsler and Sandholzer, 1936). Bacterial survival under anaerobic conditions was determined by checking bacterial growth after incubation for 1 week in an anaerobic chamber with 90% N 2 , 5% CO 2 and 5% H 2 .
Catalase activity was identified by bubble formation after adding drops of 3% (v/v) hydrogen peroxide solution and oxidase activity was confirmed by color transition to deep purple or blue after adding drops of 1% (w/v) tetramethyl-p-phenylenediamine solution (bioMérieux). Bacterial enzyme activities were examined using API ZYM and API 20 NE test strips (bioMérieux). API 20 NE test strips were also used to check the ability of bacteria to assimilate carbohydrates. Bacterial ability to ferment various carbohydrates was tested with API 50 CH test strips (bioMérieux) using API 50 CHB/E media.
For analyses of fatty acids, cell wall components, polar lipids and menaquinone, culture was performed on TSA [4% (w/v) NaCl, pH 7] at 30 • C for 48 h. Bacterial fatty acid methyl esters were extracted by saponification and methylation of bacterial cells according to the protocol of the Sherlock Microbial Identification Systems (MIDI, 1999). The composition of fatty acid methyl esters was determined by gas chromatography using a calibration standard (Microbial ID). For analysis of cell wall amino acids, peptidoglycan was purified by the method of Schumann (2011). The purified peptidoglycan was hydrolyzed using 4M HCl and the hydrolysate was spotted onto a cellulose thin layer chromatography (TLC) plate with alanine, lysine, glutamic acid, glycine, aspartic acid, and meso-diaminopimelic acid as reference amino acids. One-dimensional TLC was performed using methanol-water-6M HCl-pyridine (80:26:4:10, v/v) as solvent. The plate was air-dried and sprayed with ninhydrin reagent. Spots were visualized by heating at 100 • C for 5 min. For analysis of cell wall sugars, bacterial cells were hydrolyzed according to the protocol of Cummins and Harris (1956). The whole cell hydrolysate was spotted onto a cellulose TLC plate with ribose, arabinose, glucose, galactose, rhamnose, xylose and mannose for spot comparison. The solvent used for onedimensional TLC development was n-butanol-water-pyridinetoluene (10:6:6:1, v/v). After drying, the plate was sprayed with aniline phthalate reagent and heated at 120 • C for 5 min for spot detection. Polar lipids were extracted from bacterial cells and dissolved in chloroform-methanol (2:1, v/v) solution (Minnikin et al., 1984). The composition of polar lipids was determined by two-dimensional TLC using chloroform-methanol-water (65:24:4, v/v) for one dimension and chloroform-acetic acidmethanol-water (80:15:12:4, v/v) for second dimension. Total lipids were detected by spraying with 10% (v/v) ethanolic molybdophosphoric acid, then heating at 180 • C for 15 min. Ninhydrin and α-naphthol reagents were used for detection of aminolipids and glycolipids, respectively. Spots were visualized by heating at 110 • C for 15 min. Bacterial menaquinone was extracted from bacterial cells using chloroform-methanol (2:1, v/v) solution (Collins and Jones, 1981). The composition of bacterial menaquinones was determined by high-performance liquid chromatography (HPLC) using methanol-isopropanol (2:1, v/v) for elution.

Whole Genome Sequence Analysis
Genomic DNA was extracted using a MG Genomic DNA Purification kit (MGmed), checked by electrophoresis in 1% agarose gel and quantified with a NanoDrop spectrophotometer (Thermo Scientific) and a Qubit fluorometer (Thermo Scientific).
To generate a 20 kb library, genomic DNA was sheared with g-TUBE (Covaris) and purified using AMPure PB magnetic beads (Beckman Coulter). A total library was prepared using a DNA Template Prep Kit 1.0 (Pacific Biosciences). DNA/Polymerase Binding Kit P6 (Pacific Biosciences) was used for binding DNA template libraries to DNA polymerase P6. Whole genome sequencing was performed using the PacBio RS II sequencing platform. DNA Sequencing Kit 4.0 (Pacific Biosciences) and PacBio RS II single-molecule real-time (SMRT) Cells 8 Pac (Pacific Biosciences) were used for SMRT sequencing on the PacBio system. Genomes were assembled by RS HGAP Assembly v3.0 and polished with Quiver. Complete genome sequences were annotated using the Rapid Annotation using Subsystem Technology (RAST). Genomic relatedness was evaluated by different algorithms for genome-to-genome comparison. Average nucleotide identity (ANI) based on BLAST (ANIb) and MUMmer (ANIm) values were calculated in the JSpeciesWS online server (Richter et al., 2016). Digital DNA-DNA hybridization (dDDH) values were computed with Genome-to-Genome Distance Calculator (GGDC) version 2.1 (Meier- Kolthoff et al., 2013). OrthoANI values were determined using the Orthologous Average Nucleotide Identity Tool (OAT) . Genome-based phylogenetic tree was constructed using an up-to-date bacterial core gene set (UBCG) consisting of 92 genes (Na et al., 2018).

16S rRNA Phylogeny
Genome-derived 16S rRNA gene sequences of strains VM2412 T and VR2415 T were found to share high sequence similarities with those of B. ginsengisoli DCY80 T (98.7 and 98.4%, respectively) and B. faecium DSM 4810 T (98.3% for both strains). The two strains formed a monophyletic clade with B. ginsengisoli DCY80 T in the phylogenetic tree based on 16S rRNA gene sequences (Figure 1). The clade containing type strains of Brachybacterium species was distinct from those composed of the members of other genera within the family Dermabacteraceae. Furthermore, the four genera in the family Dermabacteraceae formed a clade distinct from representative members of the family Micrococcaceae. These results suggest that the 16S rRNA tree accurately reflects the phylogenetic relationships.

Phenotypic Characteristics
The two strains were found to grow at 10-30 • C, with optimum growth at 30 • C. They could tolerate salinity up to 15% (w/v) and grow at pH 6-9. Their cells were observed to be coccoid, Gramstain positive, non-motile and aerobic. Colonies were observed to be 0.5-1 mm in diameter, ivory-colored, circular and low-convex with entire margins after incubation for 48 h on TSA. Strains VM2412 T and VR2415 T were found to be catalase positive and oxidase negative. Further biochemical characteristics of strains VM2412 T , VR2415 T , B. ginsengisoli KCTC 29226 T and B. faecium JCM 11609 T were investigated using API kits.
Strain VM2412 T was found to have anteiso-C 15:0 (48.9%), anteiso-C 17:0 (22.3%) and iso-C 16:0 (13.5%) as major fatty acids (>10%). By contrast, strain VR2415 T was found to have anteiso-C 15:0 (57.7%) and iso-C 16:0 (13.7%) as predominant fatty acids (>10%). The fatty acid compositions of strains VM2412 T , VR2415 T and closely related taxa are given in Values <1% in all strains are not shown. −, Not detected. All data from this study. * Summed features are groups of two fatty acids that could not be separated by gas chromatography using the MIDI system. Summed feature 4 consists of iso-C 17:1 I and/or anteiso-C 17:1 B. Summed feature 8 consists of C 18:1 ω7c and/or C 18:1 ω6c. Table 2. The major fatty acid of all strains was identified as anteiso-C 15:0 , accounting for 42.9-57.7% of the total composition. The peptidoglycan of the two strains was found to contain alanine, glutamic acid, glycine and meso-diaminopimelic acid. Strain VR2415 T also contained aspartic acid within the peptidoglycan. Ribose, glucose, and galactose were found to be present as whole cell sugars. Polar lipids included phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE, presumptively) and three unidentified glycolipids (Supplementary Figures 1A,B). In addition, strain VR2415 T produced an unidentified phospholipid. Both strains were found to have MK-7 as their predominant menaquinone.
Along with the closely related taxa B. ginsengisoli DCY80 T and B. faecium NCIB 9860 T , phenotypic characteristics of the type strains of B. squillarum and B. nesterenkovii were investigated because they were used for genotypic analyses using their available genome sequences. All type strains of Brachybacterium, Devriesea and Dermabacter spp. are non-motile and contain meso-diaminopimelic acid within their cell wall. Differential characteristics between the type strains of the three genera are presented in Table 3. PE was tentatively detected only in strains VM2412 T and VR2415 T .

Comparative Genomic Analysis
Genome annotation was performed using the RAST annotation system. The following are comparisons of the genomic features derived from the genomes of strains VM2412 T and VR2415 T . Common features shared by all nine strains used in the comparative genomic study are mentioned at the end of each section. Differences among characteristics derived from genome sequences of the members of the three genera are given in Table 6.

Defense Against Foreign DNA
Gene editing using CRISPR/Cas and restriction-modification systems is used for protection from foreign DNA. Strain VM2412 T has pre-crRNA processing-related genes cse1-4, 5e and cas1, 3 (Gesner et al., 2011). Genes encoding the type III restriction-modification system are present in both strains, but the type I restriction-modification system is present only in strain VM2412 T .

Motility
Genes belonging to the motility subsystem were not detected in the genomes of the two strains, consistent with the result of bacterial motility test using semi-solid agar. Similarly, the other strains analyzed do not encode genes that involved in the motility subsystem. Indeed, non-motility is one of the common features of the members of the family Dermabacteraceae (Stackebrandt, 2014).

Peptidoglycan Biosynthesis
Both strains contain gene murE, which catalyzes the addition of meso-diaminopimelic acid into peptidoglycan. This confirms the result of cell wall analysis using one-dimensional TLC. All the other strains analyzed also encode gene murE within their genomes. Members of the family Dermabacteraceae are known to have A4γ-type peptidoglycan, which contains mesodiaminopimelic acid (Stackebrandt, 2014).

Glycerophospholipid Metabolism
The enzyme for DPG synthesis is encoded in the genomes of the two strains. In both strains, enzymes for the biosynthesis of PG including phosphatidate cytidylyltransferase and CDPdiacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase  are encoded but phosphatidylglycerophosphate is absent. Genes involved in the synthesis of phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylethanolamine (PE) are not present in the two strains while PE was detected by two-dimensional TLC. This latter identification needs to be confirmed; it is an atypical polar lipid in the majority of Brachybacterium species although PE has been detected in B. aquaticum (Kaur et al., 2016). All nine strains analyzed have genes for the synthesis of DPG and PG. The genome of D. agamarum IMP2 T includes the cls gene encoding cardiolipin (DPG) synthetase and the pcs gene encoding PC synthetase but DPG and PC were not detected in a previous characterization (Martel et al., 2008). This indicates potential discrepancies between genome-derived features and those obtained from some chemotaxonomic methods that will await detailed re-evaluation.

Sulfur Metabolism
In relation to metabolic pathways of sulfur compounds, strain VM2412 T possesses aryl sulfatase. However, none of the nine strains analyzed have genes for sulfur oxidation.

Anaerobic Respiration
Ferredoxin reductase, one of the anaerobic respiratory reductases, is present in the two strains. Furthermore, strain VM2412 T has arsenate reductase. In spite of the presence of anaerobic respiratory reductases, the strains are not able to grow under anaerobic conditions. Both strains possess respiratory nitrate reductase, which converts nitrate into nitrite. The ability to reduce nitrate was also detected using an API test kit. However, denitrification-related enzymes including nitrite reductase, nitric oxide reductase and nitrous oxide reductase are not present in any of the nine genomes analyzed. Nitrogenase involved in nitrogen fixation is also not encoded in the genomes of these nine strains.

Stress Response
Both strains contain enzymes involved in the biosynthesis of betaine from choline, including choline dehydrogenase (betA) and betaine aldehyde dehydrogenase (betB). Genes for ectoine biosynthesis (ectB, ectA, and ectC) are also present in the two strains. Osmolytes such as betaine and ectoine can protect cells against osmotic stress. Oxidative stress is suppressed by catalase, ferroxidase and manganese-dependent superoxide dismutase in the two strains. A synthetase for glutathione, known to be an important antioxidant, is encoded in the genome of strain VM2412 T . Catalase and manganese-dependent superoxide dismutase-encoding genes are also present in the other strains analyzed.

Resistance to Antibiotics
Both strains encode β-lactamase that may provide resistance to β-lactam antibiotics. Strain VR2415 T has a gene encoding VanW, a vancomycin B-type resistance protein. All the other strains analyzed here also contain genes encoding β-lactamase.

CONCLUSION
This study indicates that strains VM2412 T and VR2415 T represent two novel species of the genus Brachybacterium based on the results of phenotypic and genotypic analyses. Phylogenetic trees constructed using 16S rRNA gene sequences and core gene sets from whole genome sequences reveal the phylogenetic relationships between the isolates and other type strains of the genus Brachybacterium. In addition, this is the first study to reveal the taxogenomic relationships between members of the genus Brachybacterium. The following are descriptions of the newly isolated strains. The Digital Protologue database 1 TaxoNumbers for strains VM2412 T and VR2415 T are TA00602 and TA00603.
The type strain is VM2412 T (=KCTC 39996 T = JCM 32142 T ), isolated from a fecal sample of an Andean condor (Vultur gryphus) in Seoul Grand Park, Gyeonggi-do, South Korea.
Description of Brachybacterium avium sp. nov.
The type strain is VR2415 T (=KCTC 39997 T = JCM 32143 T ), isolated from a fecal sample of an Andean condor (Vultur gryphus) in Seoul Grand Park, Gyeonggi-do, South Korea.