TAXONOMIC DESCRIPTION Zhang et al., Int J Syst Evol Microbiol 2018;68:47–51 DOI 10.1099/ijsem.0.002426 Arthrobacter paludis sp. nov., isolated from a marsh Qi Zhang,1 Mihee Oh,1 Jong-Hwa Kim,1 Rungravee Kanjanasuntree,1 Maytiya Konkit,1 Ampaitip Sukhoom,2 Duangporn Kantachote2 and Wonyong Kim1,* Abstract A novel Gram-stain-positive, strictly aerobic, non-endospore-forming bacterium, designated CAU 9143T, was isolated from a hydric soil sample collected from Seogmo Island in the Republic of Korea. Strain CAU 9143T grew optimally at 30 C, at pH 7.0 and in the presence of 1 % (w/v) NaCl. The phylogenetic trees based on 16S rRNA gene sequences revealed that strain CAU 9143T belonged to the genus Arthrobacter and was closely related to Arthrobacter ginkgonis SYP-A7299T (97.1 % T similarity). Strain CAU 9143 contained menaquinone MK-9 (H2) as the major respiratory quinone and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, two glycolipids and two unidentified phospholipids as the major polar lipids. The whole-cell sugars were glucose and galactose. The peptidoglycan type was A4a (L-Lys–D-Glu2) and the major cellular fatty T acid was anteiso-C15 : 0. The DNA G+C content was 64.4 mol% and the level of DNA–DNA relatedness between CAU 9143 and the most closely related strain, A. ginkgonis SYP-A7299T, was 22.3 %. Based on phenotypic, chemotaxonomic and genetic data, strain CAU 9143T represents a novel species of the genus Arthrobacter, for which the name Arthrobacter paludis sp. nov. is proposed. The type strain is CAU 9143T (=KCTC 13958T,=CECT 8917T). The genus Arthrobacter, a member of the family Micrococ- by subculture and maintained at À70 ˚C in marine broth caceae, was established by Conn and Dimmick [1] with the 2216 (MB; Difco) supplemented with 25 % (v/v) glycerol. description of Arthrobacter globiformis as the type species of Strain CAU 9143T has been deposited in the Korean Collec- the genus. Currently, the genus comprises 49 recognized tion for Type Cultures (KCTC; Jeongeup, Republic of Korea) species with validly published names (www.bacterio.net/ and the Spanish Type Culture Collection (CECT; Valencia, arthrobacter.html). The genus Arthrobacter consists of aero- Spain) as KCTC 13958T and CECT 8917T, respectively. The bic, rod–coccus-shaped, Gram-stain-positive, catalase- type strains of the most closely related species, Arthrobacter positive bacteria that are characterized by the presence of ginkgonis SYP-A7299T (=KCTC 39 592T), Arthrobacter ory- A3a and A4a, respectively, as the major peptidoglycan and zae KV-651T (=KCTC 29628T) and Arthrobacter halodurans – T T MK-9 (H2) or MK-8/MK-9 as the major menaquinones [2 JSM 078085 (=KCTC 19430 ) were obtained from the 4]. Members of the genus Arthrobacter have been isolated Korean Collection for Type Cultures (KCTC; Jeongeup, from various environments, such as sediment [5], soil envi- Republic of Korea) and used as reference strains. ronment [6–11], pond [12], sea water [2], Antarctica [13] Genomic DNA of strain CAU 9143T was extracted and and rhizosphere of Ginkgo biloba L. [3]. A bacterial strain, T purified using a genomic DNA extraction kit (Intron). The designated CAU 9143 , was isolated from a hydric soil sam- T the 16S rRNA gene of strain CAU 9143 was amplified by ple beside reservoir in Seogmo Island (37 40¢ 54.84† N, PCR using the universal primers 8F/1525R following estab- 126 22¢ 15.14† E) in the Republic of Korea. In this study, a lished procedures [15]. The amplicon of the 16S rRNA bacterial strain, CAU 9143T, was characterized that was gene was sequenced directly using a 3730 automatic DNA phylogenetically closely related to the genus Arthrobacter. sequencer (Applied Biosystems). Multiple alignments and Isolation was performed according to Gordon and Mihm calculation of sequence similarity levels with 16 recognized [14] using marine agar 2216 (MA; Difco). The soil sample members of the genus Arthrobacter and representative was crushed, serially diluted and incubated under aerobic members of the family Micrococcaceae were carried out by T conditions at 30 ˚C for 7 days. Strain CAU 9143 was purified using the CLUSTAL_X 2.1 software [16] and EzTaxon-e Author affiliations: 1Department of Microbiology, Chung-Ang University College of Medicine, Seoul 06974, Republic of Korea; 2Department of Microbiology, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand. *Correspondence: Wonyong Kim, [email protected] Keywords: Arthrobacter paludis; actinobacteria; marsh. Abbreviations: CECT, Spanish Type Culture Collection; DPG, diphosphatidylglycerol; FAMEs, fatty acid methyl esters; GL, two glycolipids; KCTC, Korean Collection for Type Cultures; PG, phosphatidylglycerol; PI, phosphatidylinositol; PL, two unknown phospholipids. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CAU 9143T is JN176140. Three supplementary figures are available with the online version of this article. 002426 ã 2018 IUMS Downloaded from www.microbiologyresearch.org by IP: 165.194.103.2547 On: Thu, 04 Apr 2019 07:33:31 Zhang et al., Int J Syst Evol Microbiol 2018;68:47–51 (www.ezbiocloud.net/). Phylogenetic trees were generated reference sequences in the GenBank database (access Sep- by using three algorithms: neighbour-joining [17], maxi- tember 2017). Phylogenetic analysis indicated that the strain mum-likelihood [18] and maximum-parsimony [19]. The fell into the genus Arthrobacter. The neighbour-joining distance matrix was produced on the basis of the Jukes– tree is shown in Fig. 1. The trees reconstructed by the Cantor model [20]. The tree topology was evaluated by the maximum-likelihood and maximum-parsimony algorithms bootstrap resampling method [21] with 1000 replicates of showed a similar topology (Fig. S1, available in the online the neighbour-joining dataset with the SEQBOOT and version of this article). Pairwise analysis showed that A. T T CONSENSE programs from the PHYLIP 3.66 package. The ginkgonis SYP-A7299 (97.1 %), A. oryzae NRRL B-24478 T G+C content of the genomic DNA was determined using (96.7 %) and A. halodurans JSM 078085 (96.7 %) were the T high-performance liquid chromatography (HPLC) by using most closely related species to strain CAU 9143 . The T the method of Tamaoka and Komagata [22]. The extent of G+C content of the DNA of strain CAU 9143 was 64.4 mol DNA–DNA relatedness between CAU 9143T and the most %, which was close to the values observed for other species T of the genus Arthrobacter. The DNA–DNA relatedness closely related strain, A. ginkgonis SYP-A7299 , was deter- T mined by following Goris et al. [23]. between CAU 9143 and the most closely phylogenetic neighbour, A. ginkgonis SYP-A7299T, was 22.3 %. The value T The 16S rRNA gene sequence of strain CAU 9143 is below the 70 % cut-off point suggested by Wayne et al. (1497 bp) was determined and compared with the available [24] for the determination of genomic species, supporting 98 Citricoccus muralis 4-0 T (AJ344143) 76 Citricoccus alkalitolerans YIM 70010 T (AY376164) 75 Citricoccus nitrophenolicus PNP1 T (GU797177) 98 T 0.01 Citricoccus zhacaiensis FS24 (EU305672) 71 Citricoccus yambaruensis PS9T (AB473562) Micrococcus terreus V3M1 T (FJ423763) 98 Paeniglutamicibacter psychrophenolicus AG31 T (AJ616763) 82 Paeniglutamicibacter kerguelensis KGN15 T (AJ606062) Paeniglutamicibacter cryotolerans LI3T (GQ406812) 89 Glutamicibacter protophormiae DSM 20168 T (X80745) Glutamicibacter soli SYB2T (EF660748) 98 Glutamicibacter mysorens LMG 16219 T (AJ639831) T 90 Glutamicibacter arilaitensisRe117 (FQ311875) Glutamicibacter nicotianae DSM 20123 T (X80739) Glutamicibacter halophytocola KLBMP 5180 T (JX993762) Arthrobacter rhombi CCUG 38813 T (Y15885) T 100 Zhihengliuella halotolerans YIM 70185 (DQ372937) Zhihengliuella salsuginis NBRC 109062 T (AB778264) 95 Arthrobacter nanjingensis A33 T (KF479547) Arthrobacter woluwensis CUL 1808 T (X93353) Arthrobacter enclensis NIO-1008 T (JF421614) Arthrobacter cupressi D48 T (HQ657321) Arthrobacter pascens DSM 20545 T (X80740) 96 Arthrobacter humicola KV-653 T (AB279890) 91 Arthrobacter halodurans JSM 078085 T (EU583729) 99 Arthrobacter oryzae KV-651 T (AB279889) Arthrobacter paludis CAU9143T (JN176140) Arthrobacter ginkgonis SYP-A7299 T (KP128918) 83 Arthrobacter deserti YIM CS25 T (KT946776) Arthrobacter subterraneus CH7 T (DQ097525) 100 Arthrobacter tumbae DSM 16406 T (AJ315069) Cellulomonas composti TR7-06 T (AB166887) Fig. 1. Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences showing the relationships between strain CAU 9143T and the type strains of recognized Arthrobacter species. Dots indicate that the corresponding nodes were also recov- ered in the trees generated with maximum-likelihood and maximum-parsimony algorithms. The numbers at the nodes indicate levels of bootstrap support based on a neighbour-joining analysis of 1000 resampled datasets; only values >70 % are given. Bar, 0.01 substi- tutions per nucleotide position. Cellulomonas composti TR7-06T (AB166887) is used as an outgroup. Downloaded from www.microbiologyresearch.org by IP: 165.194.103.2548 On: Thu, 04 Apr 2019 07:33:31 Zhang et al., Int J Syst Evol Microbiol 2018;68:47–51 the proposal that strain CAU 9143T represents a separate Table 1. Differential properties of strain CAU 9143T and the type species. strains of the most closely related Arthrobacter species T T T Strains: 1, CAU 9143 ; 2, A. ginkgonis SYP-A7299 ; 3, A. oryzae KV- Strain CAU 9143 was cultivated routinely on an MA plate T T 651 ; 4, A. halodurans