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Domibacillus Tundrae Sp The 21st International Symposium on Polar Science May 19-20, 2015 Domibacillus tundrae sp. nov., isolated from active layer soil of tussock tundra in Council, Alaska Hye Ryeon Gyeong, Kiwoon Baek, Chung Yeon Hwang, Key Hun Park, Hye Min Kim, HongKum Lee and Yoo Kyung Lee* E-mail to [email protected] Korea Polar Research Institute, Republic of Korea [email protected] ABSTRACT A novel Gram-positive, spore-forming, aerobic, motile and rod-shaped bacterium designated as strain PAMC 80007T was isolated from active layer soil sample of moist acidic tundra in Council, Alaska. Optimal growth of strain PAMC 80007T was observed at 30 ºC, pH 7.0 and in the presence of 2 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain PAMC 80007T belonged to the genus Domibacillus. This strain was closely related to Domibacillus enclensis (98.3 %), D. robiginosus (98.3 %) and D. indicus (97.2 %). Genomic DNA G+C content was 43.5 mol % and genomic relatedness analyses based on the average nucleotide identity and the genome-to-genome distance showed that strain PAMC 80007T is clearly distinguished from the closely related Domibacillus species. The major fatty acids (>5 %) were iso-C15:0 (24.7 %), C16:1 ω11c (16.8 %), anteiso-C15:0 (16.5 %), C16:0 (15.6 %) and anteiso-C17:0 (8.7 %). The major respiratory isoprenoid quinines MK-6 and MK-7, and the polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipid, phospholipid and two unidentified lipids. The major whole-cell sugar was ribose with minor quantity of glucose and meso-diaminopimelic acid (type A1γ) was present in the cell-wall peptidoglycan. Results from polyphasic study suggested that strain PAMC80007T is a novel species of the genus Domibacillus for which the name Domibacillus tundrae sp. nov. is proposed. INTRODUCTION METHODS The genus Domibacillus was a member of the family Bacillaceae and first proposed with a description of Domibacillus robiginosus (Seiler et al., 2013). Domibacillus robiginosus was isolated from pharmaceutical clean room in eastern Germany and its morphological, chemotaxonomic and phylogenetic characteristics were different from the closely related members. Taxonomically Domibacillus belongs to phylum Firmicutes, class Bacilli, order Bacillales and family Bacillaceae (Parte, 2014). At the time of writing, three species, D. Domibacillus tundrae PAMC 80007T (KM657429) robiginosus (Seiler et al., 2013), D. indicus (Sharma et al., 2014) and D. enclensis Active layer soil collected from moist acidic tussock tundra in Council, Alaska (64.50765° N, 163.42696° W) (Sonalkar et al., 2014) with validly published names are included in the genus Domibacillus. Members of genus Domibacillus are Gram-positive, spore-forming, oxidative, and aerobic rods. MK-6 and MK-7 are the dominant quinones and the cell wall An aliquot of the resuspended soil sample was spread on TSA and incubated at 20 ºC for 10 days. peptidoglycan contains meso-diaminopimelic acid (type A1γ). The major whole cell sugars are glucose and ribose. In this current study, we proposed that strain PAMC 80007T represents a novel species of the genus Domibacillus based on its differences in 16S rRNA gene similarity analysis and polyphasic analysis on microorganism phenotypic and genotypic characteristics. RESULTS 78 Domibacillus tundrae PAMC 80007T (KM657429) 98 Domibacillus enclensis NIO-1016T (JF893466) 94 T 56 Domibacillus robiginosus WS 4628 (HE577175) Domibacillus indicus DSM 28032T (KF732820) Characteristics 1 2 3 4 Fatty acid 1 2 3 4 Bacillus badius ATCC 14574T (X77790) T † ‡ § L1 Bacillus thermotolerans SgZ-8 (JX261934) Spore position C, S T C, S C, S 59 T C14:0 2.8 1.0 1.6 3.9 Bacillus drentensis LMG 21831 (AJ542506) L2 78 Bacillus vireti LMG 21834T (AJ542509) Temperature range for DPG 99 † ‡ § Bacillus soli LMG 21838T (AJ542513) 10-42 25-45 13-45 10-40 C16:0 15.6 6.8 6.2 11.7 79 growth (°C) Bacillus bataviensis LMG 21833T (AJ542508) PG T C ω11c 16.8 8.3 10.5 17.2 Bacillus circulans ATCC 4513 (AY724690) Motility + + + - 16:1 PL 71 Bacillus gottheilii WCC 4585T (FN995266) T † ‡ § Bacillus kochii WCC 4582 (FN995265) NaCl tolerance (%, w/v) 8 12 8.5 6 C18:0 2.3 0.9 0.7 4.8 T PGL Bacillus eiseniae A1-2 (HM035089) 74 T Oxidase + -† -‡ -§ Bacillus purgationiresistens DS22 (FR666703) C18:1 ω9c 1.2 - - 1.1 Bacillus idriensis SMC 4352-2T (AY904033) T Hydrolysis of: 100 Bacillus cibi JG-30 (AY550276) 57 100 T iso-C14:0 0.5 - - 1.8 Bacillus indicus Sd/3 (AJ583158) Starch + - + + Bacillus kribbensis BT080T (DQ280367) Bacillus lentus IAM 12466T (D16272) Tween 20 - - + + iso-C15:0 24.7 34.0 27.0 18.3 Bacillus sporothermodurans M215T (U49079) Tween 80 + + - + iso-C - - 0.6 1.7 99 Bacillus ginsengihumi Gsoil 114T (AB245378) 16:0 57 Bacillus shackletonii LMG 18435T (AJ250318) Production of : Bacillus fordii R-7190T (AY443039) iso-C17:0 4.5 12.1 7.1 2.2 Bacillus graminis YC6957T (GU322908) Acid phosphatase, α- T - + + + 78 Bacillus panacisoli CJ32 (JQ806742) glucosidase, β-glucosidase iso-C17:1 ω10c 2.1 4.7 2.7 - 89 Jeotgalibacillus alimentarius YKJ-13T (AF281158) Jeotgalibacillus campisalis SF-57T (AY190535) 100 Trypsin - - + - anteiso-C15:0 16.5 13.5 24.6 25.0 Chryseomicrobium imtechense MW 10T (GQ927308) Chryseomicrobium amylolyticum JC16T (FM209428) 57 99 T α-Galactosidase, β- anteiso-C17:0 8.7 12.2 14.2 7.4 Sporosarcina luteola Y1 (AB473560) - + + - 85 Sporosarcina thermotolerans CCUG 53480T (FN298445) galactosidase 100 Sporosarcina saromensis HG645T (AB243859) C16:1 ω7c alcohol - - - 2.0 95 Acid production from : Sporosarcina koreensis F73T (DQ073393) Planomicrobium chinense DX3-12T (AJ697862) Glycerol, D-Melezitose + + - - Summed features Planomicrobium glaciei 423T (EU036220) 63 Planococcus salinarum ISL-16T (FJ765415) Inositol - + - + 3 (C16:1 ω7c and/or C16:1 ω 6c) 0.9 1.5 1.8 - Planomicrobium flavidum ISL-41T (FJ265708) 0.5 μm 61 Planococcus rifietoensis M8T (AJ493659) Amidon - - + + 100 4 (C17:1 iso and/or C17:1 anteiso) 3.8 4.3 2.5 3.0 Planococcus columbae PgEx11T (AJ966515) Glycogen + - + + 64 Planococcus plakortidis AS/ASP6(II)T (JF775504) Planococcus maitriensis S1T (AJ544622) D-Arabitol, xylitol + - - - Planococcus maritimus TF-9T (AF500007) Table 2. Cellular fatty acid composition of strain PAMC 80007T and members of the genus Paenibacillus polymyxa ATCC 842T (AFOX01000032) Potassium 5-ketogluconate - - + - Antibiotic susceptibility Domibacillus 0.02 Nalidixic acid (30 µg) + - - - Fig. 1. Two-dimensional thin-layer chromatogram of total polar lipid profile of strain PAMC 80007T. Fig. 3. Neighbor-joining tree showing the phylogenetic positions T T of Domibacillus tundrae PAMC 80007 and related species DNA G+C content (mol %) 43.5 46.5 42.8 44.6 Fig. 2. Transmission electron micrograph of cells of strain PAMC 80007 . Bar, 0.5 µm on the basis of 16S rRNA gene sequence. Only bootstrap MK-6, MK-6, Major Menaquinone MK-6‡ MK-6§ values above 50 % are shown (1000 resamplings) at the MK-7 MK-7† branching points. Solid circles indicate that the corresponding nodes were also obtained in both the T T Table 1. Differential characteristics between strain PAMC 80007 and type strains of other Domibacillus species Strains: 1, Domibacillus tundrae sp. nov. PAMC 80007 ; 2, maximum-likelihood and the minimum-evolution trees, D. enclensis DSM 25145T; 3, D. robiginosus DSM 25058T; 4, D. indicus DSM 28032T. while open circles in the latter tree only. Bar, 0.02 † Data from Sonalkar et al. (2014); ‡ Data from Seiler et al. (2013); § Data from Sharma et al. (2014) nucleotide substitution per site. CONCLUSION ACKNOWLEDGEMENTS This study was supported by the National Research Foundation of T Strain PAMC 80007 formed a robust clade with the three type strains of the genus Domibacillus. Korea Grant funded by the Korean Government (MSIP) (NRF-2011- The chemotaxonomic profiles of strain PAMC 80007T were generally similar to those of the genus 0021067) (PN14082, KOPRI) and by Korea Polar Research Institute Domibacillus. However, phenotypic characteristics, including hydrolysis of some macromolecules, (PE15080). and enzyme activities differentiated strain PAMC 80007T from other Domibacillus species. Therefore, strain PAMC 80007T represents a novel species of the genus Domibacillus, for which REFERENCES [1] Minnikin, D. E. et al. (1984) J Microbiol Methods 2, 233–241. the name Domibacillus tundrae sp. nov. is proposed. [2] Seiler, H., Wenning, M. & Scherer, S. (2013) Int J Syst Evol Microbiol 63, 2054–2061. [3] Sharma, A. et al. (2014) Int J Syst Evol Microbiol 64, 3010–3015. [4] Sonalkar, V. V. et al. (2014) Int J Syst Evol Microbiol 64, 4098–4102..
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