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1 Dipartimento di Biologia Evolutiva, Università degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy
2 Dipartimento di Biotecnologie Agrarie, Sezione di Microbiologia, Università degli Studi di Firenze, Piazzale delle Cascine 24, 50144 Firenze, Italy
Correspondence
Laura Marri
marri{at}unisi.it
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and the description has subsequently been emended based on newly obtained data (Goto et al., 2003; Karavaiko et al., 2005). At the time of writing, the genus comprises 17 recognized species: Alicyclobacillus acidocaldarius, A. acidoterrestris (Wisotzkey et al., 1992), A. hesperidum (Albuquerque et al., 2000), A. acidiphilus (Matsubara et al., 2002), A. sendaiensis (Tsuruoka et al., 2003), A. vulcanalis (Simbahan et al., 2004), A. tolerans, A. disulfidooxidans (Karavaiko et al., 2005), A. cycloheptanicus (Deinhard et al., 1987), A. herbarius (Goto et al., 2002), A. pomorum (Goto et al., 2003), A. contaminans, A. fastidiosus, A. kakegawensis, A. macrosporangiidus, A. sacchari and A. shizuokensis (Goto et al., 2007). Moreover, three Japanese strains and two isolated from the island of São Miguel in the Azores were denoted as representing two genomic species of Alicyclobacillus based on 16S rRNA gene sequence analysis and DNA–DNA hybridization assays (Hiraishi et al., 1997; Albuquerque et al., 2000). In addition, a novel subspecies of A. acidocaldarius, subsp. ritmannii, was isolated from a geothermal site in Antarctica (Nicolaus et al., 1998). Although continental Antarctica is largely ice-bound and supports only sparse growth of terrestrial life, it possesses a small number of sites where the ground is warmed by geothermal sources and is supplied with moisture from the condensation of steam emissions (Bargagli et al., 2004). Novel thermophilic species ascribed to the genera Bacillus, Brevibacillus, Aneurinibacillus and Anoxybacillus have recently been isolated from Antarctic geothermal grounds (Nicolaus et al., 1996; Logan et al., 2000; Allan et al., 2005; Poli et al., 2006).
In the present study, we describe a novel thermophilic bacterium isolated from warm soils collected in the immediate vicinity of a steam vent on the north-west slope of Mount Melbourne, northern Victoria Land, continental Antarctica (7 ° 20.994' S 16 ° 39.792' E, 2498 m altitude) (Bargagli et al., 2004). Brevibacillus levickii is the only novel species previously recovered from the north-west slope of Mt Melbourne despite repeated soil sampling on separate occasions followed by enrichment and isolation at different times in the laboratory (Allan et al., 2005). In February 2002, four soil samples were collected along a thermal gradient ranging from 25 to 43 °C in the vicinity of a small area (100 cm2) colonized by shoots of the moss Pohlia nutans (Bargagli et al., 2004). Analytical determinations of chemical properties of soil water extracts showed pH values ranging from 5.46 to 5.71 and relatively higher concentrations of sodium, iron and manganese when compared with chemical characteristics of other geothermal sites in Victoria Land. In particular, the iron concentration ranged from 0.94 to 1.22 µg g–1 (Bargagli et al., 2004). After incubation in DSMZ medium 259 (l–1: 3.5 g Na2HPO4 . 12H2O, 1.0 g K2HPO4, 0.03 g MgSO4 . 7H2O, 0.5 g NH4Cl, 4.0 g yeast extract, pH 6.5) at 55 °C, all four samples yielded positive cultures. Subcultivation, carried out on tryptone soy agar (TSA; Oxoid) plus 250 µM Fe2SO4 . 7H2O (TSA-Fe) at 55 °C, allowed the isolation of four thermophilic strains, designated MP1, MP2, MP3 and MP4T (Bargagli et al., 2004). Cultures were further propagated on FeP liquid medium [l–1: 5.0 g tryptone (Difco), 0.5 g D-glucose, 0.1 g (NH4)2SO4, 0.1 g FeSO4 . 7H2O], BAM basal salts medium (Deinhard et al., 1987) and/or DSMZ medium 259, all amended with 250 µM Fe2SO4 . 7H2O. In order to establish the requirement for mineral substrates for growth, DSMZ medium 259, containing 0.01 or 0.1 g yeast extract l–1, was supplemented with 250 µM Fe2SO4 . 7H2O, FeCl3, CaCl2 . 2H2O, MgSO4 . 7H2O or Na2O3S, or 0.15 % elemental sulfur (S0). Growth in liquid media was monitored by measuring the optical density at 570 nm (Ultrospec 2100 UV-VIS spectrophotometer; Amersham Biosciences). The temperature range for growth was determined by incubating the four isolates at temperatures ranging from 25 to 70 °C. The pH range for growth was determined in FeP medium and modified DSMZ medium 259 with pH adjusted by using 1 M H2SO4. All pH measurements were made at room temperature. Anaerobic growth was tested in triplicate by spreading each strain on FeP agar (1.5 % Bacto agar; Difco). Plates were placed in a GENbag Anaer chamber (bioMérieux) and incubated for 1 week (O2 <0.1 % after 2–5 h). For studies of heat resistance, 1-week-old colonies were resuspended in fresh liquid medium and exposed to temperatures of 80 or 100 °C for 10 min. Aliquots were then plated on FeP agar and survival was measured based on the number of colonies formed after 24–48 h incubation at 55 °C. After growth on TSA-Fe, colony morphology was observed under a stereomicroscope. Vegetative cells and sporangial morphologies were observed visually by bright-field light microscopy and phase-contrast microscopy, respectively. Gram determination was carried out either by staining (Gram stain kit; Medical Chemical) or a chemical method (Powers, 1995). Catalase and oxidase activities were determined according to Smibert & Krieg (1981). Sensitivity of the four isolates to ampicillin, chloramphenicol, tetracycline, gentamicin, penicillin G, bacitracin, erythromycin, kanamycin and streptomycin was tested on FeP agar. Acidification was examined with the API 50 CH test system (bioMérieux) essentially as described by Albuquerque et al. (2000) at the optimum growth temperature. Acidification of the medium was monitored every 2 days for 10 days. All tests were repeated in triplicate. The ability to oxidize different carbon sources was examined by using Biolog GN and GP microplates. Cell suspensions, after growth on TSA-Fe, were prepared by following the manufacturer's instructions and then used to inoculate the wells of Biolog microtitre plates. Microplates were incubated in a humidified chamber at 55 °C, and readings of the rate of colour development were taken after 24, 48 and 72 h (Viti & Giovannetti, 2005). Results of the phenotypic characterization tests are given in the species description and in Table 1.
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). For the present study, more complete sequences of the 16S rRNA gene of strains MP1, MP2 and MP3 were determined. By comparing 16S rRNA gene sequences between positions 36 and 1490 (Escherichia coli numbering system), strains MP1, MP3 and MP4T showed 100 % identity to each other, while strain MP2 differed from the other three at one nucleotide position. The four 16S rRNA gene sequences were then used to retrieve closely matching type sequences at the Ribosomal Database Project (Cole et al., 2003). Phylogenetic analysis was performed by using the software package TREECON for Windows (Van de Peer & De Wachter, 1997) after multiple alignment of the data by CLUSTAL_X (Thompson et al., 1997). Distances were converted to evolutionary distances according to the Jukes and Cantor method, and clustering with the neighbour-joining method was determined by using bootstrap values based on 1000 replications (Fig. 1). The 16S rRNA gene sequence of strain MP4T was a continuous stretch of 1505 bp. After a neighbour-joining analysis, 16S rRNA gene sequence similarity calculations indicated that the closest relatives of strain MP4T were the type strains of A. pomorum (91.0 %), A. sacchari (90.9 %), A. hesperidum and A. macrosporangiidus (90.8 %), A. acidoterrestris (90.7 %) and A. cycloheptanicus (90.6 %). Lower sequence similarities (<90.5 %) were found with the type strains of all other recognized species of the genus.
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. Analysis of the fatty acids at the conditions applied revealed that strain MP4T was dominated by iso-branched fatty acids iso-C15 : 0 (45.56 %) and iso-C17 : 0 (35.81 %). In addition, smaller amounts of anteiso-branched fatty acids were found (anteiso-C15 : 0, 6.4 %; anteiso-C17 : 0, 3.36 %). The DNA base composition for MP4T, determined according to the method of Mesbah et al. (1989), was 55.1 mol% G+C, which is within the range reported for recognized Alicyclobacillus species (48.7–62.7 mol%) (Goto et al., 2003; Karavaiko et al., 2005). On the basis of the data presented, we conclude that strains MP1, MP2, MP3 and MP4T represent a single novel species of the genus Alicyclobacillus, for which the name Alicyclobacillus pohliae sp. nov. is proposed.
Description of Alicyclobacillus pohliae sp. nov.
Alicyclobacillus pohliae (poh'li.ae. N.L. gen. fem. n. pohliae of Pohlia, a genus of moss colonizing the area where the type strain was isolated).
Aerobic, Gram-positive, spore-forming rods measuring 1.5–2.5x0.4–0.6 µm. Round spores lie terminally in swollen sporangia. Oxidase- and catalase-negative. Does not grow in the presence of 5 % NaCl. Colonies are cream-coloured, entire and convex and grow to 1.5–2 mm in diameter after 48 h at 55 °C in TSA-Fe. Iso-branched fatty acids iso-C15 : 0 (45.56 %) and iso-C17 : 0 (35.81 %) are the major cellular fatty acids. Facultatively anaerobic. Optimal growth occurs aerobically at 55 °C (range 42–60 °C) and at pH 5.5 (range pH 4.5–7.5). Growth on yeast extract (0.01 %) occurs in the presence of ferrous iron (Fe2+) at micromolar concentrations. Sensitive to (ml–1) ampicillin (25 µg), chloramphenicol (10 µg), tetracycline (50 µg), gentamicin (30 µg), penicillin G (25 µg), bacitracin (10 µg), erythromycin (5 µg), kanamycin (30 µg) and streptomycin (25 µg). Acid is produced from D- and L-arabinose, ribose, D- and L-xylose, D-galactose, glucose, D-fructose, mannose, L-sorbose, rhamnose, inositol, N-acetylglucosamine, aesculin, D-cellobiose, maltose, lactose, melibiose, sucrose, glycogen, gentiobiose, D-turanose, D-lyxose, D-tagatose, D- and L-fucose and 2- and 5-ketogluconate. Oxidizes D-xylose, D-ribose, 
-ketovaleric acid, L-proline and pyruvic acid methyl ester. The DNA G+C content is 55.1 mol%.
The type strain, MP4T (=CIP 109385T =NCIMB 14276T), was isolated from geothermal soil on the north-west slope of Mount Melbourne on the Antarctic continent. Strains MP1, MP2 and MP3, isolated from the same vicinity, are also included in the species.
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ACKNOWLEDGEMENTS |
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