TAXONOMIC DESCRIPTION Lee and Cha, Int J Syst Evol Microbiol 2017;67:4619–4623 DOI 10.1099/ijsem.0.002342 Ramlibacter alkalitolerans sp. nov., alkali-tolerant bacterium isolated from soil of ginseng Do-Hoon Lee and Chang-Jun Cha* Abstract A novel bacterial strain, designated CJ661T, was isolated from soil of ginseng in Anseong, South Korea. Cells of strain CJ661T were white-coloured, Gram-staining-negative, non-motile, aerobic and rod-shaped. Strain CJ661T grew optimally at 30 C and pH 7.0. The analysis of 16S rRNA gene sequence of strain CJ661T showed that it belongs to the genus Ramlibacter within the family Comamonadaceae and was most closely related to Ramlibacter ginsenosidimutans KCTC 22276T (98.1 %), followed by Ramlibacter henchirensis DSM 14656T (97.1 %). DNA–DNA relatedness levels of strain CJ661T were 40.6 % to R. ginsenosidimutans KCTC 22276T and 25.0 % to R. henchirensis DSM 14656T. The major isoprenoid quinone was ubiquinone (Q-8). The predominant polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The T major cellular fatty acids of strain CJ661 were summed feature 3 (C16 : 1 !6c and/or C16 : 1 !7c), C16 : 0 and summed feature 8 (C18 : 1 !7c and/or C18 : 1 !6c). The G+C content of the genomic DNA was 65.4 mol%. On the basis polyphasic taxonomic data, strain CJ661T represents a novel species in the genus Ramlibacter, for which name Ramlibacter alkalitolerans sp. nov. is proposed; the type strain is CJ661T (=KACC 19305T=JCM 32081T). The genus Ramlibacter was introduced by Heulin et al. [1], (Qiagen). The 16S rRNA gene sequence was determined at and belongs to the family Comamonadaceae in the class Solgent (Daejeon, Korea) using the BigDye Terminator Cycle Betaproteobacteria. Four species in this genus have been Sequencing Ready Reaction kit (Applied Biosystems) and an reported at the time of writing, which include Ramlibacter automated DNA analyzer (PRISM 3730XL; Applied Biosys- ginsenosidimutans KCTC 22276T [2], R. henchirensis DSM tems). The 16S rRNA gene sequence of strain CJ661T was 14656T, R. tataouinensis DSM 14655T [1], and R. solisilvae aligned with those of related type strains from the EzTaxon-e [3]. Members of the genus Ramlibacter are Gram-staining- server (http://www.ezbiocloud.net/) [5] using the multiple negative, aerobic and rod-shaped, and contain ubiquinone sequence alignment MUSCLE 3.8 [6]. Nucleotide substitution (Q-8) as a major respiratory quinone and a high G+C con- model test was based on the Akaike information criterion ntent of 66.6–69.9 mol%. using jModelTest [7]. Phylogenetic trees based on maxi- mum-likelihood (ML) [8] and neighbour-joining (NJ) [9] Strain CJ661T was isolated from ginseng soil in Anesong methods were reconstructed using the package phangorn in (37 59¢ 57.503† N, 126 59¢ 58.9052† E), South Korea using R [10]. Tree visualization was done in MEGA7.0 [11]. Accord- R2A agar (BD) at 30 C for 3 days and preserved with glyc- ingly, evolutionary distances were calculated using the Gen- erol suspension (30 %, v/v). In this study, we describe the eral Time Reversible model with non-uniformity of taxonomic characterization of strain CJ661T, including the evolutionary rates among sites by using a discrete Gamma most closely related strains, R. ginsenosidimutans KCTC distribution (+G) with five rate categories and by assuming 22276T, R. henchirensis DSM 14656T and R. tataouinensis that a certain fraction of sites are evolutionarily invariable DSM 14655T as reference strains in parallel tests. (+I). Nearest-Neighbour-Interchange was used for heuristic Genomic DNA of strain CJ661T and the reference strains searches and an initial tree was obtained automatically by were extracted using the DNeasy Blood and Tissue kit (Qia- using Neighbour-Join and BioNJ algorithms. All positions gen) following to the manufacturer’s protocols. Amplifica- containing gaps and missing data were eliminated from the tion of 16S rRNA genes by PCR was performed using the dataset (complete deletion option). The NJ phylogeny was universal bacterial primers 27F, 785F, 805R and 1492R [4]. reconstructed by using Jukes–Cantor model [12]. Both tree The PCR amplicon was purified using a gel extraction kit topologies were evaluated based on bootstrap analysis of Author affiliation: Department of Systems Biotechnology, Chung-Ang University, Anseong 456-756, Republic of Korea. *Correspondence: Chang-Jun Cha, [email protected] Keywords: Ramlibacter alkalitolerans; Comamonadaceae; Proteobacteria; alkali-tolerant. Abbreviation: GLC, Gas Liquid Chromatography. The GenBank accession number for the 16S rRNA gene sequence of the strain CJ661T is KF740333. Two supplementary figures and one supplementary table are available with the online Supplementary Material. 002342 ã 2017 IUMS Downloaded from www.microbiologyresearch.org by IP: 165.194.103.154619 On: Mon, 29 Apr 2019 08:31:28 Lee and Cha, Int J Syst Evol Microbiol 2017;67:4619–4623 100 Rhodoferax saidenbachensis ED16T (AWQR01000064) Ǹ Albidiferax ferrireducens T118T (CP000267) 94 Caenimonas koreensis EMB320T (DQ349098) 90 Ǹ Caenimonas terrae SGM1-15T (GU181268) 96 Variovorax dokdonensis DS-43T (DQ178978) 100 Variovorax defluvii 2C1-bT (HQ385753) Ǹ T 96 Variovorax soli GH9-3 (DQ432053) 87 T 90 Ǹ91 Variovorax boronicumulans BAM-48 (AB300597) Ǹ 87 T Ǹ Variovorax paradoxus IAM 12373 (D88006) Ǹ Ǹ Variovorax guangxiensis GXGD002T (JF495126) 91 84 55 Variovorax ginsengisoli Gsoil 3165T (AB245358) Xylophilus ampelinus ATCC 33914T (AF078758) 97 Curvibacter delicatus LMG 4328T (AF078756) Ǹ Curvibacter fontanus AQ9T (AB120963) Curvibacter lanceolatus ATCC 14669T (AB021390) Ǹ 52 100 Curvibacter gracilis 7-1T (AB109889) 92 Ramlibacter henchirensis TMB834T (AF439400) Ǹ 94 T Ǹ Ramlibacter tataouinensis TTB310 (CP000245) Ramlibacter solisilvae 5-10T (KC569791) 69 Ǹ 100 Ramlibacter alkalitolerans KACC 19305 T (KF740333) Ǹ 0.020 97 Ramlibacter ginsenosidimutans BXN5-27T (EU423304) 68 Pseudorhodoferax soli TBEA3T (EU825700) 70 Ǹ T Ǹ Pseudorhodoferax caeni SB1 (AJ606333) 97 Pseudorhodoferax aquiterrae NAFc-7T (GU721026) 66 Comamonas humi GAU11T (AB907700) Ǹ 75 Pseudacidovorax intermedius CC-21T (EF469609) 95 Hydrogenophaga caeni EMB71T (DQ372983) Ǹ 100 Hydrogenophaga defluvii BSB 9.5T (AJ585993) Acidovorax caeni R-24608T (AM084006) Ǹ 84 Acidovorax wautersii NF 1078T (JQ946365) Hylemonella gracilis ATCC 19624T (AEGR01000103) Aquabacterium commune B8T (AF035054) Fig. 1. Maximum-likelihood phylogenetic tree of strain CJ661T and related type strains based on 16S rRNA gene sequences. Filled circles indicate that the corresponding nodes were recovered in both trees generated by neighbour-joining and maximum-likelihood methods. Numbers at the nodes are levels of bootstrap value (%) based on 1000 replicated datasets; only values above 50 % are shown. Aquabacterium commune B8T (AF035054) was used as an outgroup. Bar, 0.02 substitutions per nucleotide position. 1000 datasets [13]. The complete 16S rRNA gene sequence of strain CJ661T was 40.6 % (CJ661T as probe) and 35.2 % (R. strain CJ661T showed 98.1, 97.1, and 96.8 % similarity to ginsenosidimutans KCTC 22276T as probe) to R. ginsenosidi- R. ginsenosidimutans KCTC 22276T, R. henchirensis DSM mutans KCTC 22276T, and 25.0 % (CJ661T as probe) and 14656T and R. tataouinensis DSM 14655T, respectively 28.5 % (R. henchirensis DSM 14656T as probe) to R. henchir- (Fig. 1). ensis DSM 14656T. These values are well below the 70 % threshold recommended for genomic species delineation by DNA–DNA hybridization experiments were carried out flu- Wayne et al. [15], clearly suggesting that strain CJ661T repre- orometrically [14] using the DIG-High Prime DNA sents a novel species of the genus Ramlibacter. The DNA G Labelling and Detection Starter kit according to the manufac- +C content of strain CJ661T was determined by HPLC analy- turer’s instructions (Roche). DNA–DNA relatedness of sis [16] with a reverse phase column (Capcell Pak C18 UG Downloaded from www.microbiologyresearch.org by IP: 165.194.103.154620 On: Mon, 29 Apr 2019 08:31:28 Lee and Cha, Int J Syst Evol Microbiol 2017;67:4619–4623 120; Shisheido). The G+C content of strain CJ661T was Anaerobic growth was determined after 2 weeks of cultiva- 65.4 mol%. tion at 30 C on R2A agar using the GasPak EZ Anaerobe T Pouch System (BD). Oxidase and catalase activities were Cellular morphology of strain CJ661 was observed by determined in 1 % (w/v) tetramethyl phenylene-diamine transmission electron microscopy (JEM 1010; JEOL) using cells grown for 3 days at 30 C on R2A agar (Fig. S1, avail- reagent (bioMerieux) and 3 % (v/v) hydrogen peroxide solu- able in the online Supplementary Material). Gram reaction tion, respectively. Hydrolysis of starch, cellulose, casein and was carried out using a Gram staining kit following the DNA was tested using soluble starch 1.0 % (w/v), CMC (car- manufacturer’s instructions (Sigma-Aldrich). Gliding motil- bon methyl cellulose) 0.2 % (w/v), skimmed milk 3 % (w/v) ity was tested by production of colonies that had spread to and DNase test agar (BD), respectively. Production of intra- the edge [17]. Growth was observed on R2A agar (BD) and cellular PHA (polyhydroxyalkanoate) granules was exam- nutrient agar (NA, Difco). Growth was determined at 4, 10, ined on R2A agar supplemented with the Nile red dye 15, 20, 25, 30, 37 and 40 C and pH 4.0–12.0 (1.0 pH unit under ultraviolet light [20]. Carbon utilization and enzyme intervals) in R2A broth for up to 7 days. The different pH activities were carried out by API 20NE, API ZYM, API 50CH kits (bioMerieux) and GN2 MicroPlate system media were prepared using appropriate buffers: C6H8O7/C6 – ’ H5O7Na3 for pH 5.0, Na2HPO4/NaH2PO4 for pH 6.0 8.0, (Biolog) according to the manufacturer s instructions. Bio- – T Na2CO3/NaHCO3 for pH 9.0 10.0 and Na2HPO4/NaOH chemical and physiological characteristics of strain CJ661 for pH 11.0–12.0 [18, 19]. Salt tolerance was tested in R2A that differentiated it from related type strains are summa- broth supplemented with 0–4 % (w/v) NaCl (1 % intervals).