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Tumebacillus Soli Sp. Nov., Isolated from Non- Rhizosphere Soil Jong-Hwa Kim and Wonyong Kim

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Tumebacillus Soli Sp. Nov., Isolated from Non- Rhizosphere Soil Jong-Hwa Kim and Wonyong Kim International Journal of Systematic and Evolutionary Microbiology (2016), 66, 2192–2197 DOI 10.1099/ijsem.0.001009 Tumebacillus soli sp. nov., isolated from non- rhizosphere soil Jong-Hwa Kim and Wonyong Kim Correspondence Department of Microbiology, Chung-Ang University College of Medicine, Seoul, Republic of Korea Wonyong Kim [email protected] A Gram-stain-positive, strictly aerobic, motile, spore-forming, rod-shaped bacterial strain, CAU 11108T, was isolated from soil in Danghangpo, Republic of Korea, and its taxonomic position was investigated using a polyphasic approach. The bacterium grew optimally at 37 C, at pH 8, and in the presence of 1 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence similarity revealed that strain CAU 11108T formed a distinct lineage within the genus Tumebacillus and was most closely related to Tumebacillus luteolus U13T (98.2 %). The strain contained menaquinone-7 (MK-7) as the major respiratory quinone and iso-C15 : 0 and anteiso- C15 : 0 as the major fatty acids. The DNA G+C content was 54.6 mol%. On the basis of phenotypic differentiation, phylogenetic and chemotaxonomic data, strain CAU 11108T represents a novel species of the genus Tumebacillus, for which the name Tumebacillus soli sp. nov. is proposed. The type strain is CAU 11108T (=KCTC 33141T=CECT 8918T). The genus Tumebacillus, a member of the family Alicyclo- saline solution. Sample dilutions were plated on R2A agar À bacillaceae, was first created by Steven et al. (2008) with (BD Difco) supplemented with cycloheximide (50 mg l 1) À the description of Tumebacillus permanentifrigoris as the and nalidixic acid (20 mg l 1). One hundred microlitres of type species of the genus. The genus Tumebacillus com- the diluted samples (10–2 and 10–6) were spread on R2A prises Gram-stain-positive, motile, rod-shaped bacteria agar plates and incubated at 30 ˚C for 7 days under aerobic that are characterized chemotaxonomically by the presence conditions. Colonies were randomly selected through sub- T of MK-7 as the major isoprenoid quinone and iso-C15 : 0 as culture and strain CAU 11108 was purified from a single the predominant cellular fatty acid. At the time of writing, colony from an R2A agar plate. Strain CAU 11108T has this genus consists of four species with validly published been deposited in the Korean Collection for Type Cultures names, T. permanentifrigoris (Steven et al., 2008), T. gin- (KCTC; Taejon, Korea) and Colección Española de Cultivos sengisoli (Baek et al., 2011), T. flagellatus (Wang et al., Tipo (CECT; Paterna, Spain). 2013) and T. algifaecis (Wu et al., 2015). They have been The type strains of four species of the genus Tumebacillus isolated from diverse environments such as Arctic perma- were used as reference strains in fatty acid analyses. T. per- frost, soil, wastewater and decomposing algal scum. In the manentifrigoris JCM 14557T, T. ginsengisoli KCTC 13942T, course of screening bacteria from environmental samples, T T Tumebacillus. flagellatus DSM 25748 , T. algifaecis NBRC a novel bacterial strain, designated CAU 11108 , was iso- T T T lated from a non-rhizosphere soil sample (pH 8.0 and 108765 , Tumebacillus luteolus U13 (JCM 19866 ) and salinity 1.5 %) taken at a depth 10 cm in Danghangpo Tumebacillus luteolus U14 (JCM 19867) were obtained from (35 03¢ 17.28† N, 128 23¢ 42.82† E) in the Republic of the Japan Culture Collection of Microorganisms (JCM; Korea. The purpose of present study was to establish the Osaka, Japan), the KCTC, the Deutsche Sammlung von taxonomic position of this bacterial strain using a polypha- Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, sic characterization that included chemotaxonomic, phe- Germany) and the Biological Resource Center, NITE notypic and genotypic properties. (NBRC; Chiba, Japan). T Isolation was performed using the dilution plating tech- Genomic DNA of strain CAU 11108 was extracted by the nique. The crushed soil sample was diluted with sterilized method of Marmur (1961). PCR amplification of 16S rRNA genes was accomplished using 27F and 1525R primers (Lane, 1991) with conditions according to Nam et al. Abbreviation: TEM, transmission electron microscopy. (2004). The amplified 16S rRNA gene was sequenced The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene directly using a BigDye Terminator Cycle Sequencing kit sequence of strain CAU 11108T is JX233496. (Applied Biosystems) and a 3730 automatic DNA sequencer One supplementary figure is available with the online Supplementary (Applied Biosystems). The 16S rRNA gene sequence of the Material. strain was determined and compared with available 2192 001009 ã 2016 IUMS Printed in Great Britain Tumebacillus soli sp. nov. Tumebacillus ginsengisoli Gsoil 1105 T (AB245375) 0.1 Tumebacillus permanentifrigoris Eur1 9.5 T (DQ444975) 100 Tumebacillus flagellatus GST4 T (JQ421297) 100 Tumebacillus algifaecis THMBR28 T (JX110710) Tumebacillus luteolus U14 (KP050359) 98 Tumebacillus luteolus U13 T (KP050358) 88 100 Tumebacillus soli CAU 11108 T (JX233496) Effusibacillus consociatus CCUG 53762 T (HE613268) 100 Effusibacillus lacus skLN1 T (AB828155) 100 Effusibacillus pohliae MP4 T (AJ564766) Alicyclobacillus aeris ZJ-6 T (FM179383) Alicyclobacillus fastidiosus S-TAB T (AB264021) 100 100 Alicyclobacillus acidoterrestris ATCC 49025 T (AURB01000138) Alicyclobacillus cycloheptanicus DSM 4006 T (AB042059) 100 Alicyclobacillus tolerans K1 T (AF137502) Alicyclobacillus pomorum 3A T (AB089840) 100 Alicyclobacillus contaminans 3-A191 T (AB264026) Thermoflavimicrobium dichotomicum KCTC 3667 T (AF138733) Tuberibacillus calidus 607 T (AB231786) Halobacillus profundi IS-Hb4 T (AB189298) Virgibacillus kekensis YIM kkny16 T (AY121439) Bacillus thermophilus SgZ-9 (JX274437) Anoxybacillus voinovskiensis TH13 T (AB110008) 74 Planococcus maritimus TF-9 T (AF500007) Planococcus plakortidis AS/ASP6 (II) T (JF775504) Actinomyces bovis NCTC 11535 T (X81061) Fig. 1. Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences showing the relationships between strain CAU 11108T and representative strains of recognized species of the genus Tumebacillus. Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and least-squares algo- rithms. Numbers at nodes indicate levels of bootstrap support based on a neighbour-joining analysis of 1000 resampled data- sets; only values >70 % are given. Bar, 0.1 substitutions per nucleotide position. Actinomyces bovis NCTC 11535T (X81061) is used as an outgroup organism. reference sequences in the GenBank database (Benson et al., fluorometric microplate method (Ezaki et al., 1989), as 2013), accessed June 2015. Multiple sequence alignments modified by Goris et al. (1998). The DNA G+C content with sequences of a broad selection of members of the (mol%) of the genomic DNA was determined using HPLC genus Tumebacillus and calculation of sequence similarity by the method of Tamaoka & Komagata, 1984. levels were carried out by using EzTaxon-e (Kim et al, The nearly complete 16S rRNA gene sequence of strain 2012; http://www.ezbiocloud.net/eztaxon) and CLUSTAL -X CAU 11108T (1522 bp) was sequenced and compared with 2.1 (Larkin et al., 2007). Evolutionary distance matrices four reference sequences of type strains of the genus were generated by the neighbour-joining method described Tumebacillus in the GenBank database (accessed August by Jukes & Cantor (1969). Phylogenetic trees were recon- 2015). Phylogenetic analysis indicated that strain CAU structed using the neighbour-joining (Saitou & Nei, 1987), 11108T fell into the genus Tumebacillus (Fig. 1). Strain least-squares (Fitch & Margoliash, 1967) and maximum- CAU 11108T exhibited the highest similarity of 16S rRNA likelihood (Felsenstein, 1981) algorithms in the PHYLIP gene sequence to T. luteolus U13T (98.2 %), T. luteolus package (Felsenstein, 1989). Tree topology was evaluated by U14 (97.9 %), T. ginsengisoli Gsoil 1105T (95.3 %), T. flag- the bootstrap resampling method (Felsenstein, 1985) with ellatus GST4T (95.0 %), T. algifaecis THMBR28T (95.0 %) 1000 replicates of the neighbour-joining dataset with the and T. permanentifrigoris Eur1 9.5T (93.1 %). The DNA– SEQBOOT and CONSENSE programs from the PHYLIP package. DNA relatedness between CAU 11108T and the most The extent of DNA–DNA relatedness between CAU 11108T closely related strains, T. luteolus U13T and T. luteolus and the most closely related phylogenetic neighbours, T. U14, was 23 % and 25 %, respectively. These values are luteolus U13T and T. luteolus U14, was determined using the well below the 70 % cut-off point recommended by Moore http://ijs.microbiologyresearch.org 2193 J.-H. Kim and W. Kim Table 1. Differential properties of strain CAU 11108T and the type strains of the most closely related species of the genus Tumebacillus species Strains: 1, CAU 11108T; 2, T. permanentifrigoris JCM 14557T; 3, T. ginsengisoli KCTC 13942T; 4, T. flagellatus DSM 25748T; 5, T. algifaecis NBRC T 108765 . +, Positive; À, negative; W, weakly positive. All strains were positive for hydrolysis of casein, and assimilation of cellobiose and lactose. All strains were sensitive to ampicillin, chloramphenicol, kanamycin and streptomycin. Data were obtained in this study unless indicated. Characteristic 1 2 3 4 5 Colony colour Cream Yellow* White* Light yellow* White† Motility + À* À* +* +† Flagella + À* À* +* +† Temperature range for growth ( C) 25–45 5–37* 20–42* 20–42* 20-45† Optimum temperature for growth 37 25–30* 30* 37* 30† pH range for growth 5.5–8.5 5.5–9.0* 5.0–8.5* 4.5–8.5* 5.0–9.5† NaCl tolerance (%) 0–2 0–0.5* 0–1* 0–1* 0–1† Optimum 1 0* 0* 0.5* 0.5† Oxidase + À* +* +* +† Hydrolysis of: Gelatin + À* +* +* +† Starch À +* +* +* À† Citrate + w* À* À* À† Response to antibiotics Erythromycin (15 µg) S S* R* S* S Penicillin (10 U) S R* S* S* S† Acid production from: D-Fructose À + À + À L-Arabinose ÀÀ + À + D-Xylose ÀÀ + + À D-Galactose À + + + + D-Glucose À + + + + D-Mannitol À + + + À D-Sorbitol ÀÀ + + À D-Maltose À + À + + D-Trehalose À + + + À DNA G+C content (mol%) 54.6 53.1* 55.6* 53.7* 57.6† *Data from Wang et al.
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