Ramlibacter Alkalitolerans Sp. Nov., Alkali-Tolerant Bacterium Isolated from Soil of Ginseng

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 maximum-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.

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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

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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). rized in Table 1 (Table S1).

Table 1. Differential characteristics of strain CJ661T and related type strains of the genus Ramlibacter Strains: 1, strain CJ661T; 2, R. ginsenosidimutans KCTC 22276T; 3, R. henchirensis DSM 14656T; 4, R. tataouinensis DSM 14655T. Data are from this study unless indicated otherwise. Utilization of the following carbon sources are positive for strain CJ661T and negative for all other reference strains; tween 80, N-acetyl-D-glucosamine, L-arabinose, D-arabitol, cellobiose, D-fructose, L-fucose, gentiobiose, maltose, D-mannitol, D-mannose, b- methyl-D-glucoside, D-psicose, L-rhamnose, D-sorbitol, sucrose, turanose, pyruvic acid methyl ester, cis-aconitic acid, citric acid, D-galatonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, p-hydroxyphenylacetic acid, a-ketoglutaric acid, D-saccharic acid, succinamic acid, D- alanine, L-alanine, L-alanylglycine, L-asparagine, L-aspartic acid, L-proglutamic acid, D-serine, L-serine, urocanic acid, inosine, thymidine-5’-monophosphate, glycerol, a-D-glucose-1-phosphate, D-glucose-6-phosphate and potassium gluconate. +, positive; –, negative.

Characteristic 1 2 3 4

Reduction of nitrate to nitrite + À + + Hydrolysis Starch ÀÀÀ + Gelatin + À + À Aesculin + ÀÀÀ Enzyme activity Alkaline phosphatase ÀÀÀ + Esterase lipase (C8) + À + + Leucine arylamidase ÀÀÀ + Valine arylamidase + + + À Crystine arylamidase + ÀÀÀ Naphtol-AS-BI-phosphohydrolase + + ÀÀ a-Galactosidase + ÀÀÀ Carbon source utilization Tween 40 + À + À Succinic acid monomethyl ester À + + À D-Tagatose, L-leucine À + ÀÀ Acetic acid, g-hydroxybutyric acid ÀÀÀ + b-Hydroxybutyric acid, D,L-lactic acid, propionic acid + ÀÀ + N-Acetyl-glucosamine, trisodium citrate, phenylacetic acid + À + + D-Arabitol, gentiobiose, a-ketobutyric acid, a-ketovaleric acid, potassium gluconate ÀÀ + À Dextrin, adonitol, D-galatose, a-D-glucose, lactose, lactulose, L-glutamic acid, potassium-5-ketogluconate + + ÀÀ Raffinose, trehalose, L-proline, D,L-carnitine, glycyl-L-aspartic acid, potassium-2-ketogluconate, g-aminobutyric acid ÀÀ + + DNA G+C content (mol%) 65.4 68.1*a 66.6*b 69.6*b

*Data taken from: a, [2]; b, [1].

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Table 2. Fatty acid contents (%) of strain CJ661T and related type a relatively higher amount of summed feature 3 and the strains of the genus Ramlibacter absence of summed feature 5 were differentiated from the Strains: 1, strain CJ661T; 2, R. ginsenosidimutans KCTC 22276T; related type strains. Isoprenoid quinone was extracted as 3, R. henchirensis DSM 14656T; 4, R. tataouinensis DSM 14655T. Data described by Minnikin et al. [21] and identified by HPLC – are from this study. , not detected. analysis [22]. The major isoprenoid quinone was ubiqui- Fatty acid 1 2 3 4 none (Q-8). Polar lipids were determined by two-dimen- sional silica gel TLC analysis [23, 24]. Total lipids and Straight-chain specific functional group lipids were detected using molyb- – – C10 : 0 0.4 0.5 dophosphoric acid, molybdenum blue spray, ninhydrin – C12 : 0 3.1 1.5 9.9 reagents and a-naphthol. The predominant polar lipids ––– C13 : 0 0.5 were phosphatidylethanolamine (PE), diphosphatidylgly- C14 : 0 4.4 4.5 1.6 1.7 cerol (DPG) and phosphatidylglycerol (PG). Several uniden-

C16 : 0 32.1 38.1 35.6 26.2 tified polar lipids including aminolipids were also detected –– C17 : 0 1.5 1.4 (Fig. S2). – C18 : 0 0.6 0.7 1.3 As indicated by phylogenetic analysis based on 16S rRNA – C19 : 0 1.2 0.6 1.3 gene sequence, strain CJ661T belongs to the genus Ramli- Branched bacter and shows a distinct phyletic branch. Phenotypic and anteiso-C15 : 0 0.7 0.5 0.6 0.6 chemotaxonomic characteristics including carbon source –– iso-C17 : 0 1.0 0.6 utilization, enzyme activity, DNA G+C content, isoprenoid – anteiso-C17 : 0 0.3 0.6 0.6 quinone, polar lipid, and fatty acid results were concordant Cyclic with the traits listed in the genus description. On the basis C17 : 0 cyclo 1.0 35.8 22.9 20.0 of the polyphasic taxonomy analysis, several differential ! ––– C19 : 0 8c cyclo 0.4 characteristics and DNA–DNA relatedness to reference Unsaturated strains (<70 %) clearly differentiated strain CJ661T from the ! ––– T C14 : 1 5c 1.1 closely related type strains. Thus, strain CJ661 represents a ! – C15 : 1 6c 0.5 1.1 0.9 novel species of the genus Ramlibacter, for which the name – C16 : 1 !5c 0.5 0.5 0.3 Ramlibacter alkalitolerans sp. nov. is proposed. – C17 : 1 !7c 0.6 0.9 0.6

iso-C17 : 1 !5c 0.9 0.6 1.5 0.9 DESCRIPTION OF RAMLIBACTER ! –– anteiso-C17 : 1 9c 0.9 0.6 ALKALITOLERANS SP. NOV. Hydroxy Ramlibacter alkalitolerans (al.ka.li.to¢le.rans. N.L. n. alkali C10 : 0 3-OH 4.4 4.3 2.6 2.8 – alkali; L. part. adj. tolerans tolerating; N.L. part. adj. alkalito- C12 : 0 3-OH 1.4 0.7 0.9 lerans alkali-tolerating). Summed feature* 2 ––– 1.0 Cells are Gram-staining-negative, aerobic, non-motile and 3 44.4 6.2 15.9 15.4 rod-shaped (2 µm long, 1 µm wide). Colonies on R2A agar 5 – 1.3 2.2 1.8 are circular and white colour but become red-brown with

7.3 – 6.8 8.4 aging. Orange fluorescence typical for the Nile red complex 8 with PHA appears. Growth occurs at 25-37 C (optimum *Summed features represent groups of two or three fatty acids that 30 C), at pH 5.0–10.0 (optimum pH 7.0) and at 0 % con- could not be separated by GLC with MIDI system. Summed feature 2 centration of NaCl. Casein, aesculin and gelatin are hydro- comprises C 3-OH and/or iso-C I. Summed feature 3 com- 14 : 0 16 : 1 lyzed, but cellulose and starch are not. Nitrate is reduced to prises C16 : 1 !6c and/or C16 : 1 !7c. Summed feature 5 comprises ante- D L iso-C18 : 0 and/or C18 : 2 !6,9c. Summed feature 8 comprises C18 : 1 !7c nitrite, but nitrogen gas is not produced. -Glucose, -arabi- and/or C18 : 1 !6c. nose, D-mannose, D-mannitol, N-acetylglucosamine, maltose, potassium gluconate, malic acid, trisodium citrate and phenylacetic acid are assimilated, but capric acid and adipic acid are not. Enzyme activities for esterase (C4), esterase For chemotaxonomic characteristics, cells were cultivated lipase (C8), lipase (C14), valine arylamidase, crystine aryla- on optimal media at 30 C and harvested at exponential midase, naphtol-AS-BI-phosphohydrolase and a-galactosi- growth phases. Fatty acid methyl ester analysis was carried dase are positive, but negative for arginine dihydrolase, out according to the instruction of the standard Microbial urease, alkaline phosphatase, leucine, trypsin, a-chymotryp- Identification System (MIDI) version 6.1 and RTSBA6 6.10 sin, acid phosphatase, b-galactosidase, b-glucuronidase, database (Microbial ID). The major fatty acids of strain a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, T ! CJ661 were summed feature 3 (C16 : 1 6c and/or C16 : 1 a-mannosidase and b-fucosidase. Acid is produced from L- ! ! 7c), C16 : 0 and summed feature 8 (C18 : 1 7c and/or C18 : 1 arabinose, D-ribose, D-xylose, D-adonitol, D-galactose, D-glu- T !6c). As described in Table 2, strain CJ661 and the closely cose, D-fructose, D-mannose, L-rhamnose, aesculin ferric related type strains had similar fatty acid components, while citrate and potassium 5-ketogluconate but not from

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