NRC Publications Archive Archives des publications du CNRC Tumebacillus permanentifrigoris gen. nov., sp. nov., an aerobic, spore- forming bacterium isolated from Canadian high Arctic permafrost Steven, Blaire; Chen, Min Qun; Greer, Charles W.; Whyte, Lyle G.; Niederberger, Thomas D. This publication could be one of several versions: author’s original, accepted manuscript or the publisher’s version. / La version de cette publication peut être l’une des suivantes : la version prépublication de l’auteur, la version acceptée du manuscrit ou la version de l’éditeur. For the publisher’s version, please access the DOI link below./ Pour consulter la version de l’éditeur, utilisez le lien DOI ci-dessous. Publisher’s version / Version de l'éditeur: https://doi.org/10.1099/ijs.0.65101-0 International Journal of Systematic and Evolutionary Microbiology (IJSEM), 58, 6, pp. 1497-1501, 2008 NRC Publications Record / Notice d'Archives des publications de CNRC: https://nrc-publications.canada.ca/eng/view/object/?id=938ea1ac-2624-408b-a6f2-583b02ccbaf5 https://publications-cnrc.canada.ca/fra/voir/objet/?id=938ea1ac-2624-408b-a6f2-583b02ccbaf5 Access and use of this website and the material on it are subject to the Terms and Conditions set forth at https://nrc-publications.canada.ca/eng/copyright READ THESE TERMS AND CONDITIONS CAREFULLY BEFORE USING THIS WEBSITE. L’accès à ce site Web et l’utilisation de son contenu sont assujettis aux conditions présentées dans le site https://publications-cnrc.canada.ca/fra/droits LISEZ CES CONDITIONS ATTENTIVEMENT AVANT D’UTILISER CE SITE WEB. Questions? Contact the NRC Publications Archive team at [email protected]. If you wish to email the authors directly, please see the first page of the publication for their contact information. Vous avez des questions? Nous pouvons vous aider. Pour communiquer directement avec un auteur, consultez la première page de la revue dans laquelle son article a été publié afin de trouver ses coordonnées. Si vous n’arrivez pas à les repérer, communiquez avec nous à [email protected]. International Journal of Systematic and Evolutionary Microbiology (2008), 58, 1497–1501 DOI 10.1099/ijs.0.65101-0 Tumebacillus permanentifrigoris gen. nov., sp. nov., an aerobic, spore-forming bacterium isolated from Canadian high Arctic permafrost Blaire Steven,1 Min Qun Chen,1 Charles W. Greer,2 Lyle G. Whyte1 and Thomas D. Niederberger1 Correspondence 1Department of Natural Resource Sciences, McGill University, Ste-Anne de Bellevue, Quebec, Lyle G. Whyte Canada [email protected] 2Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada A Gram-positive, aerobic, rod-shaped bacterium (strain Eur1 9.5T) was isolated from a 9-m-deep permafrost sample from the Canadian high Arctic. Strain Eur1 9.5T could not be cultivated in liquid medium and grew over the temperature range 5–37 6C; no growth was observed at 42 6C and only slow growth was observed at 5 6C following 1 month of incubation. Eur1 9.5T grew over the pH range 5.5–8.9 and tolerated NaCl concentrations of 0–0.5 % (w/v). Eur1 9.5T grew heterotrophically on complex carbon substrates and chemolithoautotrophically on inorganic sulfur compounds, as demonstrated by growth on sodium thiosulfate and sulfite as sole electron donors. T Eur1 9.5 contained iso-C15 : 0 as the major cellular fatty acid and menaquinone 7 (MK-7) as the major respiratory quinone. The cell-wall peptidoglycan was of type A1c. The DNA G+C content was 53.1 mol%. The 16S rRNA gene sequence of strain Eur1 9.5T was only distantly related (¡87 % sequence similarity over 1407 bp) to any recognized bacterial species. Based on physiological and phylogenetic analyses, strain Eur1 9.5T is suggested to represent a novel species of a new genus, for which the name Tumebacillus permanentifrigoris gen. nov., sp. nov. is proposed. The type strain of Tumebacillus permanentifrigoris is Eur1 9.5T (5DSM 18773T 5JCM 14557T). A novel Gram-positive bacterium, designated strain Eur1 9.5T formed yellow colonies after about 2 days incubation 9.5T, was isolated during the course of a study on the at room temperature. Cells inoculated into liquid R2A microbial diversity in a 9-m-deep permafrost sample from medium produced an insoluble yellow precipitate that Eureka (79u 599 410 N85u 489 480 W), Ellesmere Island, appeared to be free of bacterial cells based on microscopic Nunavut, Canada (Steven et al., 2007). Comparative 16S investigations (61000 magnification) of recovered precip- rRNA gene sequence analysis indicated that strain Eur1 itate material. Likewise, strain Eur1 9.5T did not grow in 9.5T formed an independent branch in the order Bacillales, liquid modified sulfur media (composition described and data from a polyphasic study was used to define in below) supplemented with 20 mM sodium thiosulfate or 2 detail the taxonomic position of this novel isolate. 2.0 g glucose l 1, LB broth (Becton Dickinson) or trypti- T case soy broth (Becton Dickinson). Phase-contrast micro- Strain Eur1 9.5 was isolated by using standard dilution T scopy revealed that cells of strain Eur1 9.5 were non- plate techniques on Difco R2A agar (Becton Dickinson) motile rods with ellipsoidal spores formed in large terminal and, unless otherwise stated, cells were grown on R2A agar sporangia (Fig. 1a). Microscopic examinations of Gram- (pH 7.0) incubated at room temperature (approximately T u T stained cells of strain Eur1 9.5 revealed Gram-positive cell 22–25 C). Strain Eur1 9.5 proved to be recalcitrant to walls; older cultures occasionally appeared to be Gram- cultivation in liquid media and therefore all experiments negative. Rod-shaped vegetative cells were 3–3.5 mmin were performed on agar media. On R2A agar, strain Eur1 length and approximately 0.5 mm in width. Spores were approximately 1 mm in width and 2 mm in length based on Abbreviation: TEM, transmission electron microscopy. estimates from transmission electron microscopy (TEM) The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene images (Fig. 1b); TEM was undertaken on cells grown on sequence of Eur1 9.5T is DQ444975. R2A medium. Cells, ultrathin sections and copper TEM A table detailing the cellular fatty acid composition of strain Eur1 9.5T is grids were prepared as described by Vali et al. (2004). The available as supplementary material with the online version of this paper. grids were stained with 4 % aqueous uranyl acetate and 65101 G 2008 IUMS Printed in Great Britain 1497 B. Steven and others Fig. 1. Micrographs of cells of strain Eur1 9.5T. (a) Phase-contrast micrograph showing vegetative cells and cells with large terminal sporangia; (b) TEM comparison between a vegetative cell and a spore (bottom left). Bars, 1 mm. Reynold lead citrate and observed with a JEOL JEM- [modified sulfur medium (per litre distilled water): 3.0 g 2000FX TEM at 80 kV. KH2PO4, 0.5 g MgSO4 .7H2O, 0.3 g (NH4)2SO4, 0.25 g CaCl .2H O, 0.02 g FeCl .6H O, 15.0 g agar, adjusted to Chromosomal DNA was prepared from cells of strain Eur1 2 2 3 2 T pH 7.0 with HCl; Atlas (1993)]. Carbon sources were 9.5 by using the Gram-positive protocol of the DNeasy 2 added at a final concentration of 2.0 g l 1. Acid production Tissue kit (Qiagen) and was used as a template to amplify from carbohydrates was determined by using API 50CH the nearly full-length 16S rRNA gene by using primers 27F ´ and 1492R (Lane, 1991). The PCR mixtures and conditions test strips (bioMerieux) and carbon source utilization was also tested by using Biolog GP microplates. Strain Eur1 were described by Steven et al. (2007). Sequencing of the T PCR product resulted in a sequence of 1407 bp that was 9.5 formed white colonies on basal media with varying used for phylogenetic comparisons. Alignment of the 16S times of appearance depending on the carbon source or T electron donor provided. It grew well on galactose, starch, rRNA gene sequence of strain Eur1 9.5 with BLAST (Altschul et al., 1990) and RDP-II release 9 Classifier tryptone, cellobiose, lactose, trehalose, mannitol, maltose, software (Cole et al., 2007) indicated that strain Eur1 9.5T glucose and Casamino acids but showed relatively weak growth on glycerol, fructose, sodium lactate and yeast formed a monophyletic clade affiliated with the order T Bacillales (data not shown). The closest relative to strain extract. Xylose did not support growth of strain Eur1 9.5 . Eur1 9.5T in the GenBank database was an uncharacterized The novel strain did not grow in the presence of the member of the Bacillales (Gsoil 1105; 94 % 16S rRNA gene electron donors NaNO2 (1 and 5 mM) or cysteine sequence similarity) and the closest matching recognized hydrochloride (1 and 5 mM). Both Na2SO3 (5 mM) and Na2S2O3 (5 and 20 mM) supported growth, indicating that relative was the type strain of Alicyclobacillus contaminans T (87 % sequence similarity; Goto et al., 2007). Phylogenetic strain Eur1 9.5 was capable of facultative sulfur oxidation. relationships of some of the 16S rRNA gene sequences that No positive results were detected by using either API 50CH were most closely related to strain Eur1 9.5T were test strips or Biolog microplates, presumably due to the T determined by aligning 16S rRNA gene sequences with inability of strain Eur1 9.5 to grow in liquid media. the CLUSTAL W program and phylogenetic inference To investigate tolerance to NaCl, modified sulfur medium 2 packages in MacVector 7.2 (Accelrys). Evolutionary supplemented with 2.0 g sodium thiosulfate l 1 was distances between sequences were estimated by using the prepared as above and NaCl concentrations were varied Jukes–Cantor model (Jukes & Cantor, 1969) and a from 0 to 2 % (w/v) at intervals of 0.5 %.