DOI: 10.15468/ym3v63
title: Arachidicoccus soli sp. nov., a bacterium isolated from soil

description: This dataset contains the digitized treatments in Plazi based on the original journal article Lee, Shin Ae, Kim, Tae-Wan, Sang, Mee-Kyung, Song, Jaekyeong, Kwon, Soon-Wo, Weon, Hang-Yeon (2021): Arachidicoccus soli sp. nov., a bacterium isolated from soil. International Journal of Systematic and Evolutionary Microbiology (004566) 71 (1): 1-6, DOI: 10.1099/ijsem.0.004566, URL: http://dx.doi.org/10.1099/ijsem.0.004566AbstractA Gram-stain-negative, aerobic, non-motile and rod-shaped bacterium, designated KIS 59-12 T, was isolated from a soil sample collected on Hodo island, Boryeong, Republic of Korea. The strain grew at 10–33 ° C, pH 6.0–7.5 and with 0–4% NaCl (w/v). Results of phylogenetic analysis based on 16 S rRNA gene sequences showed that strain KIS 59-12 T was in the same clade as Arachidicoccus rhizosphaerae Vu-144 T and Arachidicoccus ginsenosidivorans Gsoil 809 T with 97.5 and 97.2% sequence similarity, respectively. Comparative genome analysis between strain KIS 59-12 T and A. rhizosphaerae Vu-144 T showed that average nucleotide identity value was 69.4% and the digital DNA–DNA hybridization value was 19.1%. The major respiratory quinone was menaquinone-7. The major polar lipids were phosphatidylethanolamine and an unknown polar lipid. The predominant cellular fatty acids were iso-C 15:0, iso-C 15: 1 G and iso-C 17:0 3- OH, which supported the affiliation of strain KIS 59-12 T with the genus Arachidicoccus. The major polyamines were homospermidine and putrescine. The genomic DNA G + C content was 36.4mol%. On the basis of phylogenetic, physiological and chemotaxonomic characteristics, strain KIS 59-12 T represents a novel species of the genus Arachidicoccus, for which the name Arachidicoccus soli sp. nov. is proposed. The type strain of Arachidicoccus soli is KIS 59-12 T (= KACC 17340 T = NBRC 113161 T).The genus Arachidicoccus was proposed by Madhaiyan et al. [1] as a member of the family Chitinophagaceae. Arachidicoccus rhizosphaerae Vu-144 T, the first-described Arachidicoccus species, was isolated from cowpea rhizosphere and showed plant-growth-promoting properties including indole-3-acetic acid production, 1-aminocyclopropane-1-carboxylate deaminase activity and phosphate solubilization [1]. Arachidicoccus ginsenosidivorans Gsoli 809 T isolated from ginseng field soil was the second proposed species of Arachidicoccus [2]. Complete genome sequence and experimental analyses revealed that a novel species, ‘ Arachidicoccus ginsenosidimutans’ BS20 T, produces glycoside hydrolases converting major ginsenosides into minor ginsenosides [3]. At the time of manuscript preparation, only these three species have been described in this genus (www.bacterio.net/arachidicoccus.html). The members of Archidicoccus are Gram-stain-negative, non-motile, aerobic and non-spore-forming. The major respiratory quinone is menaquinone-7 (MK-7) and the major fatty acids are iso-C 15:0, iso-C 15:1 G and iso-C 17:0 3- OH [2]. In this study, we isolated a novel bacterial strain, KIS 59-12 T, from forest soil collected from Hodo island and its taxonomic characteristics were investigated using a polyphasic approach.To study the diversity and structure of the microbiome in various types of soil, forest soil samples were collected from Hodo island, Boryeong, Chungcheongnam-do Province, Republic of Korea (36° 18′ 01″ N, 126° 15′ 49″ E). The soil samples were thoroughly suspended with 0.85% NaCl (w/v) and the suspension following serial dilution was spread on Reasoner's 2 A (R 2 A) agar medium (LAB M). After 2weeks of incubation at 28 ° C, a novel bacterium designated KIS 59-12 T was isolated and selected for taxonomic characterization. A single colony was purified by subculture and preserved in 15% (v/v) glycerol solution at −80 ° C.The genome sequencing of strain KIS 59-12 T was performed using Pacific Biosciences (PacBio) RSII with the singlemolecule real-time (SMRT) sequencing platform at Microgen Inc. (Seoul, Republic of Korea). All generated reads were assembled de novo using RS HGAP Assembly (version 3.0) software. The complete genome sequence of strain KIS 59-12 T was submitted to the GenBank database (www.ncbi.nlm.nih. gov/) and annotated using the NCBI Prokaryotic Genome Annotation Pipeline [4] and the Rapid Annotation Subsystem Technology (RAST) server [5]. The genome of strain KIS 59-12 T was found to consist of a circular chromosome with 4099663 bp and the DNA G + C content was 36.4 mol%. The sequencing depth coverage of genome assembly was 249-fold. The genome included 3352 protein-coding genes, six rRNAs and 42 tRNAs (Table S1, available in the online version of this article).Author affiliations: 1 Agricultural Microbiology Division, National Institute of Agricultural Sciences, Rural Development Administration, Wanju-gun, Jeollabuk-do, 55365, Republic of Korea.*Correspondence: Hang-Yeon Weon,why@korea.krKeywords: Arachidicoccus; novel species; polyphasic taxonomy; strain KIS 59-12 T.Abbreviations: ANI, average nucleotide identity; dDDH, digital DNA-DNA hybridization; MK, menaquinone.The GenBank/ EMBL / DDBJ accession numbers for the 16 S rRNA gene sequence and the genome sequence of strain KIS 59-12 T are MH 933978 and CP 032489, respectively.Four supplementary figures and two supplementary tables are available with the online version of this article.Fig. 1. Neighbour-joining tree based on a comparative analysis of 16S rRNA gene sequences, showing the phylogenetic relationships of strain KIS59-12 T and other type strains in the family Chitinophagaceae. Filled circles indicate that the corresponding nodes were also recovered in trees generated with maximum-parsimony and maximum-likelihood algorithms. Percentage bootstrap values (from 1000 replications) greater than 70% are shown at nodes. Bar, 0.02 substitutions per nucleotide position. Saprospira grandis LewinT was used as an outgroup.Phylogenetic analysis was performed based on 16 S rRNA gene sequences. The full-length 16 S rRNA gene sequence of strain KIS 59-12 T (1516 bp) obtained from the annotated genome sequence and those of type strains in the family Chitinophagaceae were aligned using the online tool SINA Alignment Service version 1.2.11 (www.arb-silva.de/aligner) [6]. The aligned sequences were exported to Molecular Evolutionary Genetics Analysis (MEGA) program version 7 [7] and phylogenetic trees were reconstructed using the maximum-likelihood (ML), neighbour-joining (NJ) and maximum-parsimony (MP) algorithms with 1000 bootstrap iterations. The genetic distances for the NJ and ML analysis were calculated using Kimura’s two-parameter model [8]. The sequence similarities between strain KIS 59-12 T and the closely related type strains were calculated using the EzBioCloud server (www.ezbiocloud.net/identify) [9]. The 16 S rRNA gene sequence analysis revealed that strain KIS 59-12 T showed the highest similarity to A. rhizosphaerae Vu-144 T (97.5%), A. ginsenosidivorans Gsoil 809 T (97.2%) and ‘ A. ginsenosidimutans ’ BS20 T (94.7%) and had low sequence similarities (below 93.0%) to other members of the family ‘ Chitinophagaceae ’ in the phylum Bacteroidetes. Phylogenetic analysis based on the NJ tree revealed that strain KIS 59-12 T formed a clade with the type strains belonging to the genus Arachidicoccus, which was also supported by the MP and ML trees (Figs 1, S1 and S 2).Multilocus sequence analysis was performed with genome sequences of strain KIS 59-12 T and the closely related strains available from the NCBI database using an up-to-date bacterial core gene set (UBCG) [10]. The phylogenomic trees reconstructed by the UBCG pipeline showed that the type strains of the genus Arachidicoccus were in the same cluster as strain KIS 59-12 T. Therefore, A. rhizosphaerae KACC 19049 T, A. ginsenosidivorans KACC 19399 T and ‘ A. ginsenosidimutans’ KACC 18624 T were used as reference strains that belonged to the same clade based on 16 S rRNA gene and genome sequence analyses.The genome sequences of the reference type strains were obtained from the EzBioCloud database (www.ezbiocloud. net) [9]. Average nucleotide identity (ANI) scores and digital DNA–DNA hybridization (dDDH) values between KIS 59- 12 T and the reference type strains were calculated using the OrthoANI algorithm with the ANI calculator (www.ezbiocloud.net/toos/ani) [9] and the Genome-to-Genome Distance Calculation (GGDC) version 2.1 (http://ggdc.dsmz.de/distcalc2.php) [11], respectively. The ANI values between strain KIS 59-12 T and A. rhizosphaerae Vu-144 T, A. ginsenosidivorans Gsoli 809 T and ‘ A. ginsenosidimutans ’ BS20 T were 68.6, 69.0 and 71.9%, and the dDDH values between them were 19.1, 20.2 and 18.7%, respectively (Table S1). As the accepted threshold values for species boundaries in terms of ANI and dDDH are 95–96% and 70% [12], respectively, strain KIS 59- 12 T represents a distinct species of the genus Arachidicoccus.We further annotated functional genes in the genomes of strain KIS 59-12 T. The RAST server predicted 3631 proteincoding sequences (CDSs) for strain KIS 59-12 T, of which 788 CDSs (22%) were in the subsystem and 2843 CDSs (78%) were not in the subsystem. The assigned genes involved in amino acids and derivatives (201) were the most abundant followed by carbohydrate metabolism (176), protein metabolism (149) and cofactors, vitamins, prosthetic groups and pigments (125) (Table S 2). Type species of Arachidicoccus including strain KIS 59-12 T might be resistant to antibiotics and toxic compounds with several modes of actions, such as copper homeostasis and beta-lactamase. Strain KIS 59-12 T also possessed genes associated with multidrug-resistance efflux pumps, which were not detected in the other closely related type species (Table S 2). Strain KIS 59-12 T and A. ginsenosidimutans KACC 18624 T contained potential genes related to conjugative transposon but those were not found in the other closely related type species (Table S 2). We identified functional genes associated with phosphorus and nitrogen cycling in soil. Strain KIS 59-12 T possessed five copies of the glucose-1-dehydrogenase (gcd) gene responsible for gluconic acid production, which solubilizes mineral phosphates, thus increasing biologically available phosphate for plants [13]. The genome of KIS 59-12 T contained genes encoding nitrite reductase large and small subunits (nirB and nirD) that are involved in reduction of nitrite to ammonium in the nitrogen cycle [14]. This novel strain isolated from soil might potentially a play role in the biogeochemical process of terrestrial ecosystems.The cell morphology and motility of strain KIS 59-12 T were examined by oil-immersion phase-contrast microscopy (Axioplan 2, Zeiss) with cells cultured for 2 days on R 2 A agar at 28 ° C. Gram staining was performed using the Difco Gram-staining kit (Difco), according to the manufacturer’s instructions. Motility was observed by light microscopy. The temperature range for growth was tested at 4, 10, 15, 20, 25, 30, 33, 37 and 40° C. To investigate tolerance to NaCl, R 2 A broth was prepared with NaCl concentrations adjusted to 0–5.0%, w/v (at intervals of 1.0%). The pH range for growth was assessed after 10 days of incubation in R 2 A broth at pH 4.0–10.0, with increments of one pH unit. The pH of the R 2 A broth was adjusted using citrate/phosphate buffer (pH 3.0–7.0), Tris/hydrochloride buffer (pH 8.0–9.0) and carbonate/bicarbonate buffer (pH 10.0–11.0). After sterilization (121 ° C, 15 min), the pH of the R 2 A broth was checked and adjusted if necessary. Cells of strain KIS 59-12 T were aerobic, Gram-stain-negative, non-motile rods (0.5–0.6 µm wide and 1.3–10µm long) (Fig. S 4). Colonies grown on R 2 A agar medium were light yellow, round and convex (0.65– 1.75 mm in diameter). The growth ranges of temperature, pH and NaCl were 10–33 ° C, pH 6.0–7.5 and 0–4% (w/v) NaCl, respectively.Biochemical characteristics and enzyme activities of strain KIS 59-12 T were compared with its reference strains, A. rhizosphaerae KACC 19049 T and A. ginsenosidivorans KACC 19399 T. Catalase activity was detected by dispersing colonies in 3% (v/v) hydrogen peroxide and checking for bubble formation. The oxidase activity was determined by using the Oxidase Reagent (bioMérieux). Hydrolyses of carboxymethyl cellulose (CM-cellulose, 0.1%, w/v), casein (skimmed milk, 10% w/v), hypoxanthine (0.5%, w/v), tyrosine (0.5%, w/v), Tween 80 (1.0 %, v/v), starch (1.0%, w/v) and xanthine (0.5%, w/v) were examined using R 2 A agar medium. DNase activity was determined using DNase test agar (Difco). Growth under anaerobic conditions was tested by incubating R 2 A agar plates in AnaeroGen sachets (Oxoid) for 7 days at 28 ° C. Additional enzyme activities and biochemical features were determined with API ZYM, API 20NE and API 32GN kits (bioMérieux) following the manufacturer’s instructions. The API 20NE and API 32GN tests were recorded after 5 days (A. rhizosphaerae KACC 19049 T) and 10 days (A. ginsenosidivorans KACC 19399 T) at 37 ° C and the API ZYM test results were read after 5 h of incubation at 37 ° C. Detailed biochemical characteristics distinguishing strain KIS 59-12 T from A. rhizosphaerae KACC 19049 T and A. ginsenosidivorans KACC 19399 T are listed in Table 1.To analyse chemotaxonomic characteristics, cells of strain KIS 59-12 T, A. rhizosphaerae KACC 19049 T and A. ginsenosidivorans KACC 19399 T were grown on R 2 A agar for 2 days at 28 ° C until the exponential phase. The cellular fatty acids were extracted, methylated and separated by gas chromatography (model 6890, Hewlett Packard) according to the protocol of the Sherlock Microbial Identification System (MIDI, version 5.0) [15]. Fatty acid methyl esters were identified and quantified using the TSBA 5.0 database of the MIDI. Polar lipids were extracted and separated by twodimensional silica-gel TLC on silica gel 60 plates (Merck) of the cell extract. After the two-dimensional TLC, the following reagents were sprayed for the detection of polar lipids: ninhydrin for free amino groups and molybdenum blue for phosphorus-containing lipids. Menaquinones and phospholipids were extracted and purified according to the protocol of Minnikin et al. [16]. Polyamines were extracted and analysed according to the method of Schenkel et al. [17]. The major fatty acids (&gt;10.0 %) of strain KIS 59-12 T were composed of iso-C 15:0 (42.49%), iso-C 17:0 3- OH (18.33%) and iso-C 15:1 G (16.03%). The fatty acid profile of strain KIS 59-12 T was similar to members of the genus Arachidicoccus with small differences in qualitative and quantitative composition. The complete fatty acid profile is provided in Table 2. Polar lipids of strain KIS 59-12 T were phosphatidylethanolamine, four unknown aminolipids and three unknown lipids (Fig. S 4). The major polyamines of strain KIS 59-12 T were homospermidine and putrescine. Homospermidine is a representative polyamine of the genus Arachidicoccus [2] and putrescine is a differentiated feature with other type species. Consistent with data from A. rhizosphaerae KACC 19049 T and A. ginsenosidivorans KACC 19399 T, the major respiratory quinone was MK-7.Table 1. Phenotypic characteristics that differentiate strain KIS59-12 T from the type strains of related Arachidicoccus speciesStrains: 1, KIS59-12 T; 2, Arachidicoccus rhizosphaerae KACC 19049 T; 3, Arachidicoccus ginsenosidivorans KACC 19399 T; 4, ‘ Arachidicoccus ginsenosidimutans ’ KACC 18624 T. All strains excluding ‘ A. ginsenosidimutans ’ KACC 18624 T were grown on R2A agar at 28°C for 2 or 3days until reaching the exponential growth phase.All data were obtained in this study unless indicated. +, Positive; −, negative; W, weak.Characteristic 1 2 3 4 Isolation source Soil Rhizosphere soil Ginseng field soil Compost c Cell morphology Rod Coccoid rod Rod Rod c Temperature range for growth (°C) 10–33 20–37 a 10–37 b 15–37 c pH range for growth 6.0–7.5 6.0–8.0 a 5.0–7.0 b 6.0–9.0 c NaCl range for growth (%) 0–4 0-2 a 0-4 b 0.5–1 c Assimilation of: D-Glucose + − + + c Sucrose − + + + c Glycogen − − + + c Salicin − + + + c Melibiose − + + + c Enzyme acitivity: Oxidase − + + + c Catalase − + + + c Arginine dihydrolase − − + – c Esterase (C4) + + − + c Esterase lipase (C8) − − − + c Valine arylamidase + W − + c β - Glucuronidase + − − + c α -Glucosidase W − − + c β -Glucosidase − − − + c α - Mannosidase − − − + c α -Fucosidase W W − + cIn conclusion, strain KIS 59-12 T clustered with A. rhizosphaerae Vu-144 T and A. ginsenosidivorans Gsoil 809 T in the phylogenetic trees based on 16 S rRNA gene sequences. The members of Archidicoccus and strain KIS 59-12 T are Gram-stain-negative, non-motile, aerobic and non-sporeforming. The major respiratory quinone of strain KIS 59-12 T was menaquinone-7 (MK-7) and the major fatty acids were iso-C 15:0, iso-C 15:1 G and iso-C 17:0 3- OH, which is similar to the members of the genus Archidicoccus. However, ANI and dDDH values between strain KIS 59-12 T and A. rhizosphaerae Vu-144 T, which is the closest type strain in the phylogenetic trees, were less than 95–96% and 70%, respectively. The strain consists of different numbers of unknown aminolipids and lipids compared to the reference strains. Several differentiating physiological traits, including temperature and NaCl range for growth, assimilation of five carbon sources and eight enzyme activities, were also observed. The phylogenetic, phenotypic, genomic and chemotaxonomic data clearly indicate that strain KIS 59-12 T represents a novel species in the genus Arachidicoccus, for which we propose the name Arachidicoccus soli sp. nov.