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International Journal of Systematic and Evolutionary Microbiology (2009), 59, 2977–2986 Janibacter hoylei sp. nov., Bacillus isronensissp. nov. and Bacillus aryabhattai sp. nov., isolatedfrom cryotubes used for collecting air from theupper atmosphere S. Shivaji,1 Preeti Chaturvedi,1 Zareena Begum,1 Pavan Kumar Pindi,1R. Manorama,1 D. Ananth Padmanaban,2 Yogesh S. Shouche,3Shrikant Pawar,3 Parag Vaishampayan,3 C. B. S. Dutt,4 G. N. Datta,4R. K. Manchanda,5 U. R. Rao,4 P. M. Bhargava6 and J. V. Narlikar7 1Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India 2Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Sector 39A, 3Microbial Culture Collection, National Centre for Cell Science, Pune University Campus, 4ISRO Headquarters, Department of Space, Bangalore 560 023, India 5Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India 6Anveshna, 12-13-100, Lane No. 1, Street No. 3, Tarnaka, Hyderabad 500 017, India 7Inter-University Centre for Astronomy and Astrophysics, Ganeshkhind, Post Bag 4, Pune 411 007, Three novel bacterial strains, PVAS-1T, B3W22T and B8W22T, were isolated from cryotubesused to collect air samples at altitudes of between 27 and 41 km. Based on phenotypiccharacteristics, chemotaxonomic features, DNA–DNA hybridization with the nearest phylogeneticneighbours and phylogenetic analysis based on partial 16S rRNA gene sequences (PVAS-1T,1196 nt; B3W22T, 1541 nt; B8W22T, 1533 nt), the three strains were identified as representingnovel species, and the names proposed are Janibacter hoylei sp. nov. (type strain PVAS-1T5MTCC 8307T 5DSM 21601T 5CCUG 56714T), Bacillus isronensis sp. nov. (type strainB3W22T 5MTCC 7902T 5JCM 13838T) and Bacillus aryabhattai sp. nov. (type strain B8W22T5MTCC 7755T 5JCM 13839T).
Evidence for the occurrence of micro-organisms in the atmosphere (Bruch, 1967; Greene et al., 1964; Lysenko, 1979; upper atmosphere at altitudes of 17–85 km (Bruch, 1967; Rogers & Meier, 1936; Harris et al., 2002). In these earlier Greene et al., 1964; Lysenko, 1979), obtained using a reports, the emphasis was on detection of micro-organisms meteorological rocket (Imshenetsky et al., 1978; Lysenko, rather than their identification. Wainwright et al. (2003) 1979), a specially designed direct-flow sampler (Greene reported the presence of two bacterial species (Bacillus simplex et al., 1964; Rogers & Meier, 1936) and cryosamplers sent and Staphylococcus pasteurii) and a fungus (Engyotontium up on a balloon (Harris et al., 2002; Wainwright et al., album), whereas Shivaji et al. (2006) identified four novel 2003; Shivaji et al., 2006), has established unequivocally species of Bacillus, Bacillus aerius, B. aerophilus, B. strato- that life forms are present in the upper atmosphere. To sphericus and B. altitudinis, from cryogenic tubes used to date, only a few studies have been published on the collect air samples at altitudes of 24, 28 and 41 km.
quantity and nature of the micro-organisms in the upper In this paper, a polyphasic taxonomic approach was usedto characterize 12 bacterial strains isolated from cryotubes The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene that were used to collect air at altitudes of between 27 and sequences of strains PVAS-1T, B3W22T and B8W22T are DQ317608,EF114311 and EF114313.
41 km, during a balloon flight. Three of the 12 bacterialisolates, strains PVAS-1T, B3W22T and B8W22T, represent Polar lipid profiles of the novel strains and related type strains andresults of UV-sensitivity experiments are available as supplementary three novel species of the genera Bacillus (B3W22T and material with the online version of this paper.
Detection of viable bacteria from air collected inthe cryotubes The balloon with the cryosampler payload used forcollecting high-altitude air samples (altitude 20–41.4 km) All the work related to detection of microbes in air samples was launched on 20 April 2005 from the National Scientific from the cryotubes was carried out in a room with very Balloon Facility of the Tata Institute of Fundamental little human traffic and inside a laminar flow hood Research at Hyderabad, India. The cryogenic sampler, available exclusively for the handling of the probes. The comprising a 16-cryoprobe assembly, is similar to one used probes were transferred to the laminar flow hood one at a previously (Wainwright et al., 2003; Shivaji et al., 2006). All time and, prior to the transfer, the surface of the cryotubes 16 cryosampling tubes were autoclaved at 120 uC for 4 h and was cleaned and sterilized with alcohol. All instruments integrated into the payload in a tissue-culture laboratory used to unscrew the outlet valve of the cryotube such as which was UV-sterilized overnight. The cryotubes were also spanners and screwdrivers were surface-sterilized with checked for sterility after the assembly. For this purpose, alcohol and flame-heated prior to use. The outlet valve of three cryotubes were disconnected from the assembly, the cryotube was then connected to a series of two opened in a laminar flow hood, rinsed with sterile phosphate Millipore filtration units using sterile tubing and the air buffer (0.1 M, pH 7.2) and plated on Luria–Bertani (LB) was sequentially filtered through a 0.45 mm filter (Millipore agar and incubated at 37 uC. No colonies had appeared after cat. no. HAWP 04700) and a 0.22 mm filter (Millipore cat.
1 month, indicating that the cryotubes were sterile.
no. GSWP 04700) under aseptic conditions in the laminar Therefore, the isolates reported in Table 1 should have flow hood. The 0.45 mm and 0.22 mm filters were then used originated from the air samples collected at the given for detection of bacteria. For this purpose, each filter was altitudes. Cryotubes 1, 3, 5, 7, 9, 12, 13 and 15 were cut into quarters and one of the quarters was transferred to examined at the Centre for Cellular and Molecular Biology, a nutrient agar plate (0.5 % peptone, 0.3 % beef extract, Hyderabad, while cryotubes 2, 4, 6, 8, 10 and 14 were 0.5 % NaCl and 1.5 % agar, w/v) and incubated at 15 uC.
similarly studied at the National Centre for Cell Sciences, After about 10 days, if no growth was observed, the filter Pune. Care was taken that the two laboratories followed was transferred to an LB agar plate (1 % tryptone, 0.5 % similar protocols, and there was frequent interaction and yeast extract, 1.0 % NaCl and 2.0 % agar, w/v; pH 7.2) or discussion between the two groups to ensure homogeneity an oligotrophic LB agar plate (1/10 LB) and incubated at 15 uC for a further 15 days. Subsequently, if no colonies Table 1. Bacteria isolated from cryotubes used for collecting air at altitudes of 20–41.4 km With the exception of strain PVAS-1T, which was isolated from an air sample, all the strains were isolated from washes of the cryotubes.
International Journal of Systematic and Evolutionary Microbiology 59 Novel Janibacter and Bacillus species from the stratosphere appeared, the filters were incubated on LB agar at 25 uC for minimal salts agar, blood agar and Sabouraud agar, and up to a month. It should be noted that, when hydrophobic incubated under the conditions described above. The plates 0.45 mm (Millipore cat. no. HVHP 04700) and 0.22 mm were observed for the appearance of colonies over a period (cat. no. GVHP 04700) filters were used for filtering the air, a thin layer of medium of the same composition was From the 15 cryotube wash samples, 12 bacterial colonies applied to the surface of the plates before the filters were were detected (Table 1). Nine (PVAS-2, -3, -4, -5, -6, -8 placed on the surface of the medium.
and -10 and B5W22-1 and B5W22-2) showed more than Two of the remaining quarters of the filter were transferred 98 % 16S rRNA gene sequence similarity to reported to minimal salts agar. This medium contained (per 100 ml) species (Table 1). Therefore, further attempts were not 20 ml of a 3.39 % (w/v) sodium dihydrogen phosphate made to characterize them to the species level.
solution, 20 ml 1.5 % (w/v) potassium phosphate, 20 ml0.25 % (w/v) NaCl, 20 ml 0.5 % (w/v) ammonium chloride and 18 ml Millipore water. The final concentration of agar was 2 %. The medium was also supplemented with 2 mlsterile 20 % glucose and 0.2 ml sterile 0.1 M MgSO4 Morphological, growth and biochemical studies of viable solution; the pH was adjusted to 7. This medium was colonies were performed using standard methods (Holding used for the cultivation of organisms which require low & Collee, 1971; Smibert & Krieg, 1994). LB agar was used nutrients. Blood agar [2.3 % (w/v) peptone, 0.1 % (w/v) for growth and maintenance and for the determination of corn starch, 0.5 % (w/v) NaCl and 1.5 % (w/v) agar, to phenotypic and chemotaxonomic characteristics of all which 5 % (v/v) defibrinated blood was added (after strains except PVAS-2 and -3. Strains PVAS-2 and -3 were autoclaving and cooling the medium)] was used for the grown on a medium containing 0.25 % (w/v) K2HPO4, cultivation of organisms which require rich media. Plates 0.225 % (w/v) NaH2PO4, 0.05 % (w/v) (NH4)2SO4, 0.02 % of minimal salt agar and blood agar were incubated at (w/v) MgSO4 . 7H2O and 0.1 % (v/v) methanol. The shape, 30 uC. One quarter of the filter was also incubated at 30 uC size and motility of the strains were ascertained using a on Sabouraud agar (1.0 % enzymic digest of casein, 4 % Leitz Diaplan phase-contrast microscope with an oil- glucose and 1.5 % agar, w/v) to facilitate fungal growth immersion objective (6100). The sensitivity of the cultures to antibiotics was determined by using antibiotic discs(HiMedia). Utilization of various carbon compounds as Of the 15 cryotubes from which air was sampled, only a sole carbon sources was tested in mineral liquid medium single novel bacterial colony was detected, in cryotube 11, supplemented with 0.2 % filter-sterilized carbon source as corresponding to air collected at an altitude of 40–41.4 km described previously (Shivaji et al., 2006). Fatty acid and (Table 1). When placed on minimal salts agar, the filter lipid composition (Sato & Murata, 1988; Kiran et al., paper from cryotube 11 showed a single cream-coloured 2004), DNA G+C content (Shivaji et al., 1992, 2005) and colony after 24 days of incubation at 30 uC, and it was DNA–DNA hybridization (Tourova & Antonov, 1987; Shivaji et al., 1992) were determined according to standard During the filtration process, plates containing fungal and procedures. Analyses of polar lipids, respiratory quinones bacterial media were left open in the laminar flow hood and meso-diaminopimelic for PVAS-1T were carried out by throughout the operation to check the sterility of the hood.
the Identification Service of the DSMZ (Braunschweig, These plates were incubated at 25 uC (for fungi) or 37 uC Germany). Isoprenoid quinones were also extracted (bacteria) for 7 days prior to use, to confirm the sterility of according to the method of Collins et al. (1977), separated the plates. Colonies were not detected on any of these by HPLC and identified as described previously (Reddy plates, indicating that the sampling was done under sterile et al., 2003). Peptidoglycan was prepared and analysed according to the method described by Komagata & Suzuki(1987).
Detection of viable bacteria from washes of Detailed phenotypic and chemotaxonomic characteristics (listed in Tables 2, 3 and 4 and in the species descriptions)were compared with those of Bacillus silvestris DSM It seemed possible that some bacteria might have remained 12223T, Bacillus megaterium MTCC 428T, Janibacter at the bottom of the tubes or attached to their polished anophelis CCUG 49715T, Janibacter terrae DSM 13876T, walls and thus escaped detection. To check this possibility, Janibacter melonis DSM 16063T, Janibacter limosus DSM all 15 empty cryotubes were injected with 200 ml sterile 11140T and Janibacter corallicola DSM 18906T.
phosphate buffer (0.1 M, pH 7.2) and agitated at 22 uC for6 h in a shaker. The liquid was then removed using sterile Cells of strain PVAS-1T are Gram-positive, coccoid, non- tubing and a syringe and filtered through a 0.22 mm endospore-forming and non-motile and occur singly or in Millipore filter (47 mm diameter). These filters were each clumps. Some biochemical characteristics are listed in cut aseptically into four sectors and each sector was placed Table 2. meso-Diaminopimelic acid is the diagnostic on a plate containing one of four different media, LB agar, diamino acid in the cell-wall peptidoglycan and iso-C16 : 0 Table 2. Physiological properties that differentiate strain C16 : 1D9c as the predominant fatty acids (Tables 3 and 4).
PVAS-1T and the type strains of other species of the genus The polar lipid profile of strain B3W22T contains (PE), phosphatidylserine (PS) and one unidentified lipid, Strains: 1, Janibacter hoylei sp. nov. PVAS-1T (data from this study); whereas that of strain B8W22T has only PG and PE. MK-6, 2, Janibacter anophelis DSM 18333T; 3, Janibacter terrae DSM 13876T; MK-7 and MK-8 are the menaquinones in strain B3W22T 4, Janibacter limosus DSM 11140T (unless indicated, data in columns whereas, in B8W22T, only MK-7 was present (Table 3).
2–4 from Ka¨mpfer et al., 2006); 5, Janibacter melonis DSM 16063T The DNA G+C contents of the two strains are 38– (Yoon et al., 2004); 6, Janibacter corallicola DSM 18906T (Kageyama et 40 mol%. These characteristics indicated that strains al., 2007). All strains were negative for growth at 40 uC, utilization of B3W22T and B8W22T are members of the genus Bacillus N-acetylglutamic acid, haemolysis, motility, methyl red test and H2S production and positive for catalase, starch hydrolysis and gelatinhydrolysis. All strains tolerate up to 6 % NaCl. +, Positive; 2,negative; W, weakly positive; d, delayed reaction; TSBA, tryptic soy broth agar; CASO agar, casein-peptone-soymeal peptone agar.
The 16S rRNA gene was amplified from genomic DNA,purified and sequenced as described previously (Shivaji et al., 2000; Pidiyar et al., 2004). To ascertain the phylogenetic affiliation of the novel strains, the almost-complete 16S rRNA gene sequences of the bacterial isolates were manually corrected and aligned using CLUSTAL_X (Thompson et al., 1994). Phylogenetic trees were constructed based on neighbour-joining (Saitou & Nei, 1987) and maximum- parsimony methods using MEGA 3.1 (Kumar et al., 2004).
Evolutionary distances were determined with Kimura’s two- parameter model (Kimura, 1980). Bootstrap analysis (Felsenstein, 1993) was performed for 1000 replications.
Reference sequences were retrieved from GenBank under the accession numbers indicated on the trees.
Phylogenetic analysis of the 16S rRNA gene sequence of strain PVAS-1T (1196 bp) indicated that its closest relatives were Janibacter anophelis CCUG 49715T (98 % similarity) and Janibacter terrae DSM 13953T (98 %) (Lang et al., 2003). Both the neighbour-joining tree (Fig. 1) and the maximum-parsimony tree (not shown) revealed that strain PVAS-1T clustered most closely with these strains. DNA– DNA relatedness of PVAS-1T with Janibacter terrae DSM 13953T was 29.8 % and with Janibacter anophelis DSM 18333T was 13.7 %, and the reciprocal reactions in both cases gave values of about 9–10 % . Thus, strain PVAS-1T represents a novel species of the genus Janibacter, for whichthe name Janibacter hoylei sp. nov. is proposed.
*Data generated for this study by the Identification Services of the Strains B3W22T and B8W22T are related at the 16S rRNA gene sequence level (91 % similarity), but the sequencesdiffer by more than 2.5 %, indicating that they probablyrepresent different species. BLASTN analysis indicated that (50.4 %), iso-C18 : 0 (5.1 %), 10-methyl C17 : 0 (10.9 %), the nearest phylogenetic neighbour of B3W22T is Bacillus C17 : 1D9c (12.1 %) and C18 : 1D9c (6.0 %) are the predom- silvestris H3-23T (99.5 % similarity), with which it forms a inant fatty acids. Mycolic acids are absent. The predom- clade in the phylogenetic tree, and it is separated from inant isoprenoid quinone present is MK-8(H4). The DNA Caryophanon latum DSM 14151T and Caryophanon tenue G+C content is 72.8 mol%. These characteristics and DSM 14152T, with which it exhibits about 97 and 96 % phylogenetic analysis of the 16S rRNA gene sequence similarity, respectively (Fig. 2). The members of the genus indicated that PVAS-1T is a member of the genus Caryophanon are characterized by the presence of slightly curved to straight trichomes, and are thus clearly differentfrom B3W22T.
Cells of strains B3W22T and B8W22T are Gram-positive,rod-shaped, endospore-forming and catalase-positive, with DNA–DNA hybridization studies indicated that the C15 : 0, C16 : 0, iso-C15 : 0, anteiso-C14 : 0, anteiso-C16 : 0 and relatedness between B3W22T and Bacillus silvestris DSM International Journal of Systematic and Evolutionary Microbiology 59 Novel Janibacter and Bacillus species from the stratosphere gravitophotophoresis (Rohatschek, 1996). Finally, in 1982, Furthermore, these two strains exhibit phenotypic and Hoyle and Wickramasinghe proposed the theory of chemotaxonomic differences (Tables 3 and 4) that imply ‘Panspermia’ (Hoyle & Wickramasinghe, 1986, 1993, that B3W22T represents a novel species; the name proposed Description of Janibacter hoylei sp. nov.
In the phylogenetic tree, strain B8W22T forms a robustclade with its nearest phylogenetic neighbour Bacillus Janibacter hoylei (hoy9le.i. N.L. gen. n. hoylei of Hoyle, megaterium IAM 13418T (Fig. 2), with which it shows named after Sir Fred Hoyle, the famous English astro- 99.7 % similarity at the 16S rRNA gene sequence level.
Despite this high similarity, it is observed that, at the whole Colonies on LB agar are creamish, entire, round and 1– genome level, the DNA–DNA relatedness between B8W22and Bacillus megaterium MTCC 428T is only 35 % 2 mm in diameter. Cells stain Gram-positive and show (reciprocal reaction 43 %), indicating that B8W22T could oxidative metabolism; they are non-motile, non-endo- be assigned to a novel species. Furthermore, B8W22T and spore-forming cocci, 0.4–0.7 mm in diameter, that occur Bacillus megaterium MTCC 428T differ in their phenotypic singly or in clumps. Good growth (visible colonies with a and chemotaxonomic characteristics (Tables 3 and 4), diameter of 1 mm) occurs after 2 days of incubation on implying that B8W22T does indeed represent a novel nutrient agar at 25–30 uC. Grows in LB broth at 20–40 uC species, for which the name proposed is Bacillus aryabhat- and at pH 5–10, with optimum growth at 30 uC and pH 9.
tai sp. nov. Similarity of B8W22T to other Bacillus species Grows in the presence of 5 % (w/v) NaCl in LB broth and at the 16S rRNA gene sequence level was less than 95 %.
exhibits weak growth in LB broth containing 10 % NaCl.
Resistant to UV radiation. Catalase- and oxidase-positive;H2S is not produced. Voges–Proskauer negative, indole- negative and reduces nitrate to nitrite. Results of carbon source utilization tests are shown in Table 2. Does not sensitivity to UV radiation was determined by exposure to produce acid from alcohols or the following sugars: D- a UV lamp (UV-B, 15 W64; Sankyo Denki) as described glucose, D-fructose, D-galactose, lactose, maltose, cello- previously (Shivaji et al., 2006). Cultures of Bacillus biose, D-mannitol, D-mannose, raffinose, L-rhamnose, Micrococcus luteus MTCC 106T, Janibacter limosus DSM Diaminopimelic acid is the diagnostic diamino acid in 11140T and Janibacter terrae DSM 13876T were used as the cell-wall peptidoglycan. The main fatty acids present controls in these experiments. The results indicate that are iso-C16 : 0, 10-methyl C17 : 0, C18 : 1D9c and C17 : 1D9c. The strains B3W22T, B8W22T and PVAS-1T are more resistant predominant isoprenoid quinone present is MK-8(H4). In to UV irradiation than their nearest phylogenetic neigh- whole-cell hydrolysates, ribose and glucose are present, but bours (Supplementary Table S1, available in IJSEM arabinose, galactose and mannose are absent. Mycolic acids are absent. The DNA base composition of the type strain is72.8 mol% G+C.
The type strain is PVAS-1T (5MTCC 8307T 5DSM21601T 5CCUG 56714T), isolated from an air sample The bacterial strains identified are unlikely to be laboratory collected at an altitude of 40–41.4 km using a cryosampler.
contaminants, because no such cultures were handled inthe laboratory. The control plates that were exposed to theair flow of the laminar flow hood during the entire air Description of Bacillus isronensis sp. nov.
Bacillus isronensis ( N.L. masc. adj. isronensis Furthermore, all possible precautions were taken to rule arbitrary name pertaining to ISRO, the acronym of the out contamination of the cryotubes throughout the Indian Space Research Organization, which largely funded assembly and deployment stages of the experiment; it is the studies in which the type strain was isolated).
unlikely that they came from the cryotube assembly facilityand survived the transit through the stratosphere, because Colonies on nutrient agar are white, entire, round and 3– the control cryotubes were completely sterile. It has been 4 mm in diameter. Cells produce round terminal argued that micro-organisms can travel across interplan- endospores and are motile. Grows at 5–37 uC and etary space during routine meteoritic exchanges between pH 6–10. Does not grow at 42 uC or at pH 4 or 11.
the Earth and Mars (Gladman et al., 1996; Mileikowsky Tolerates up to 5.8 % NaCl. Resistant to UV radiation.
et al. 2000; Nicholson et al., 2000). Furthermore, it is Grows on peptone. Positive for oxidase, lipase, gelatinase, possible that micro-organisms reach the stratosphere from starch hydrolysis, caseinase, tryptophan deamination, the Earth as a result of volcanic eruptions, the updraft nitrate reduction and indole production. Produces acid caused by blue lightning strikes (Pasko et al., 2002), from cellobiose and utilizes a number of sugars, amino thunderstorms and forest fires (Fromm et al., 2004) and acids and other carbon compounds as sole carbon sources Table 3. Physiological properties that differentiate strains B3W22T and B8W22T and their nearest phylogenetic neighbours Strains: 1, Bacillus isronensis sp. nov. B3W22T; 2, Bacillus silvestris DSM 12223T; 3, Bacillus aryabhattai sp. nov. B8W22T; 4, Bacillus megateriumMTCC 428T. All four strains have Gram-positive, motile, rod-shaped cells that produce endospores, grow at 20–37 uC and pH 6–10, tolerate 5.8 %NaCl and grow in peptone broth and are positive for catalase and b-galactosidase but do not grow at pH 4 or 11 and are negative for citrateutilization, the methyl red test and H2S production. None of the strains produces acid from sucrose or inulin or uses dextran, citric acid, cellulose,glycogen, thioglycolate, hydroxybutyric acid, salicin, D-glucuronic acid, L-aspartic acid, sodium succinate, valeric acid, L-cysteine, L-tyrosine, L-phenylalanine, L-proline or L-histidine as sole carbon sources. All four strains utilize glycerol, myo-inositol, methyl a-D-galactoside, L-ornithine and L-creatinine as sole carbon sources, are resistant to discs containing colistin (10 mg) and are sensitive to discs containing norflaxacin (10 mg), tobramycin (15 mg), lomefloxacin (30 mg), amikacin (30 mg), roxithromycin (15 mg), ciprofloxacin (30 mg), nitrofurantoin (300 mg), cefoperazone(75 mg), vancomycin (30 mg), lincomycin (15 mg), cephotaxime (30 mg), kanamycin (30 mg), novobiocin (30 mg), chloramphenicol (30 mg),ampicillin (25 mg), tetracycline (30 mg), bacitracin (10 mg), gentamicin G (30 mg), polymyxin B (50 mg), oleandomycin (15 mg), spectinomycin(100 mg), rifampicin (30 mg), erythromycin (15 mg) and carbenicillin (100 mg). +, Positive; 2, negative; V, variable; W, weak; S, sensitive;R, resistant.
Trehalose, maltose, adonitol, erythritol, International Journal of Systematic and Evolutionary Microbiology 59 Novel Janibacter and Bacillus species from the stratosphere L-arabinose, melibiose, sucrose, D-mannitol,raffinose, D-mannose, D-xylose, L-Sorbose, D-fructose, trehalose, L-xylose, lactose, maltose, propionate, starch, methyla-D-mannoside, polyethylene glycol,sodium acetate, potassium acetate,amygdalin, xylitol, erythritol Lactic acid, adonitol, N-acetylglucosamine, L-Serine, L-threonine, L-arginine, L-glutamic Cefazolin (30 mg), co-trimoxazole (25 mg) Streptomycin (25 mg), nalidixic acid (30 mg) *DAP, Diaminopimelic acid.
DDPG, Diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; UL, unknown lipid.
(Table 3). The type strain is sensitive to a number of The type strain is B3W22T (5MTCC 7902T 5JCM antibiotics (Table 3). The fatty acids present are C15 : 0, 13838T), isolated from a cryogenic tube used for collecting C16 : 0, C17 : 0, iso-C13 : 0, iso-C15 : 0, anteiso-C14 : 0, anteiso- an air sample from an altitude of 27–30 km.
C16 : 0, C16 : 1D9c, C16 : 1D11 and C18 : 1D9 (Table 4). Theinterpeptide bridge of the cell-wall peptidoglycan consists Description of Bacillus aryabhattai sp. nov.
of Lys–Glu–Ala. The lipids present are PG, PE, PS and anunidentified lipid. MK-6, MK-7 and MK-8 are the Bacillus aryabhattai (a.ry.a.bhat9ta.i. N.L. gen. n. aryabhat- menaquinones present. The DNA G+C content of the tai of Aryabhatta, named after the renowned Indian Table 4. Total fatty acid compositions of strains B3W22T andB8W22T and related type strains Strains: 1, Bacillus isronensis sp. nov. B3W22T; 2, Bacillus silvestrisDSM 12223T; 3, Bacillus aryabhattai sp. nov. B8W22T; 4, Bacillusmegaterium MTCC 428T. Data are percentages of total fatty acids andwere obtained in this study; strains were grown on nutrient agar (1 %beef extract, 1 % peptone, 0.5 % NaCl and 1.5 % agar; pH 7.2) andincubated overnight at 30 uC.
Colonies on nutrient agar are peach-coloured, entire,round, flat and 5–8 mm in diameter. Cells produce Fig. 2. Neighbour-joining phylogenetic tree reconstructed on the ellipsoidal and central endospores and are motile.
basis of 16S rRNA gene sequences showing the phylogenetic Growth occurs at 10–37 uC and pH 6–10. Does not grow relationships between strains B3W22T and B8W22T and closely at 42 uC or at pH 4 or 11. Tolerates up to 11.6 % NaCl.
related species of the genera Bacillus, Caryophanon and Resistant to UV radiation. Grows on peptone. Positive for Lysinibacillus. The sequence of Microbacterium indicum DSM phosphatase, DNase, tryptophan deamination, ornithine 20030T (GenBank accession no. AJ536198; not shown) was decarboxylase and malonate utilization (Table 3). Produces used as an outgroup. Bootstrap values (expressed as percentages acid from various carbon sources and utilizes a number of of 1000 replications) .50 % are given at nodes. Bar, 2 sugars, amino acids and other carbon compounds as sole substitutions per 100 nucleotide positions.
carbon sources (Table 3). The type strain is sensitive to anumber of antibiotics (Table 3). The fatty acids present areC14 : 0, C15 : 0, C18 : 0, iso-C15 : 0, anteiso-C14 : 0, anteiso-C16 : 0and C16 : 1D9c (Table 4). The interpeptide bridge of the cell-wall peptidoglycan consists of meso-diaminopimelic acid–Ala–Glu. The lipids present are PG and PE. MK-7 is themenaquinone present. The DNA G+C content of the typestrain is 38 mol%.
The type strain is B8W22T (5MTCC 7755T5JCM 13839T), Fig. 1. Neighbour-joining phylogenetic tree reconstructed on the isolated from a cryogenic tube used for collecting an air basis of 16S rRNA gene sequences showing the phylogenetic sample from an altitude of 40–41.4 km.
relationships between strain PVAS-1T and closely related speciesof the genus Janibacter. The sequence of Brevibacterium linensDSM 20425T (GenBank accession no. X77451; not shown) was used as an outgroup. Bootstrap values (expressed as percentages The present study is part of a long-term study partly conceptualized of 1000 replications) .50 % are given at nodes. Bar, 1 and largely funded by the Indian Space Research Organization, substitution per 100 nucleotide positions. The asterisk (*) indicates Department of Space, Government of India. The payload envir- that this was the only branch point that was conserved in the onmental testing and some critical parameter evaluations were carried out at various ISRO Centres (VSSC, LPSC and ISAC). The periodic International Journal of Systematic and Evolutionary Microbiology 59 Novel Janibacter and Bacillus species from the stratosphere guidance of Dr Vedachalam, Dr Lalji Singh of CCMB, Dr T. K. Alex, Kiran, M. D., Prakash, J. S. S., Annapoorni, S., Dube, S., Kusano, T., LEOS, Mr V. Sundararamaiah and Mr Gnana Gandhi is acknowl- Okuyama, H., Murata, N. & Shivaji, S. (2004). Psychrophilic edged. We are grateful to Dr G. Madhavan Nair, Chairman, ISRO/ Pseudomonas syringae requires trans-monounsaturated fatty acid for Secretary, DOS, for his encouragement and full support throughout growth at higher temperature. Extremophiles 8, 401–410.
the payload development and analysis. Our special thanks go to the Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in India Air Force for the helicopter recovery of the payload. We thank bacterial systematics. Methods Microbiol 19, 161–207.
Dr Jean P. Euze´by, Ecole Nationale Ve´te´rinaire, Toulouse, France, forhelp with the nomenclature of the novel species. Y. S. S. would also Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software like to thank the Department of Biotechnology, Government of India, for molecular evolutionary genetics analysis and sequence alignment.
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