Isolation of Three Novel Bacterial Strains, Janibacter Hoylei Sp. Nov., Bacillus Isronensis Sp

1 Feb. 12-2-2009 Title Isolation of three novel bacterial strains, Janibacter hoylei sp. nov., Bacillus isronensis sp. nov. and Bacillus aryabhattai sp. nov. from cryotubes used for collecting air from upper atmosphere Authors S. Shivaji1, Preeti Chaturvedi1, Zareena Begum1, Pavan Kumar Pindi1, R. Manorama1, D. Ananth Padmanaban2, Yogesh S. Shouche3, Shrikant Pa war3, Parag Vaishampayan3, C.B.S. Dutt4, G.N. Datta4, R.K. Manchanda5, U.R. Rao4, P.M. Bhargava6, J.V. Narlikar7 Institution 1Centre for Cellular and Molecular Biology Uppal Road, Hyderabad 500 007, INDIA 2Microbial Type Culture Collection (MTCC) Institute of Microbial Technology, Sector 39A, Chandigarh 160 036 3Microbial Culture Collection, National Centre for Cell Science Pune University Campus, Ganeshkhind, Pune 411 007 4Room No 141, ISRO-HQ, Antariksha Bhawan New BEL Road, Bangalore 560 094 5Tata Institute of Fundamental Research Homi Bhabha Road, Colaba, Mumbai - 400 005, 6Anveshna, 12-13-100, Lane No. 1, Street No. 3 Tarnaka, Hyderabad 500 017 7lnter-University Centre for Astronomy and Astrophysics Ganeshkhind, Post Bag 4, Pune - 411 007, INDIA Address for correspondence Dr S Shivaji Scientist G (Director-grade Scientist) Centre for Cellular and Molecular Biology Uppal Road, Hyderabad 500 007, INDIA Email: [email protected] Telephone: 00-91 -40-27192504 Fax: 00-91-40-27160311 Running title Microbes from stratosphere Key words Bacteria, stratosphere, Bacillus, Janibacter 2 1 Three novel bacterial strains PVAS-1, B3W22 and B8W22 were isolated from cryotubes 2 used to collect air samples at altitudes between 27 and 41 km. Based on phenotypic 3 characteristics, chemotaxonomic features, DNA-DNA hybridization with the nearest 4 phylogenetic neighbours and phylogenetic analysis based on the partial 16S rRNA gene 5 sequence, three strains PVAS-1 (1196 nucleotides), B3W22 (1541 nucleotides) and 6 B8W22 (1533 nucleotides) were identified as novel species and the names suggested are 7 Janibacter hoylei sp. nov. (PVAS-17 = MTCC 8307 = DSM 21601 = CCUG 56714), 8 Bacillus isronensis sp. nov. (B3W227 = MTCC 7902 = JCM 13838) and Bacillus 9 aryabhattai sp. nov. (B8W227 = MTCC 7755 =JCM 7755). 10 11 3 12 Evidence for the occurrence of microorganisms in the upper atmosphere above 17 13 km up to 85 km altitude (Bruch, 1967; Greene et al., 1964; Lysenko, 1979) using either a 14 meteorological rocket (Imshenetsky et al., 1978; Lysenko, 1979) or a specially designed 15 direct-flow sampler (Greene et al., 1964; Rogers & Meier, 1936) or cryosamplers sent up 16 on a balloon (Harris et al., 2001; Wainwright et al., 2003; Shivaji et al., 2006) have 17 unequivocally established the presence of life forms in the upper atmosphere. 18 19 As of now, there have been, very few published studies on the quantity and nature 20 of microorganisms in the upper atmosphere (Bruch, 1967; Greene et al., 1964; Lysenko, 21 1979; Rogers & Meier, 1936; Harris et al., 2001). In these earlier reports the emphasis 22 was on detection of the microorganisms rather than identification of the species. 23 Wainwright et al. (2003) described the presence of two bacterial species {Bacillus simplex 24 and Staphylococcus pasteurii) and a fungus (Engyotontium album) where as Shivaji et 25 al. (2006) identified four new species of Bacillus namely B. aerius, B. aerophilus, B. 26 straospericus and B. altitudinus from cryogenic tubes, which were used to collect air 27 samples at altitudes of 24, 28 and 41 km. 28 29 In this paper, a polyphasic taxonomic approach was used to characterize twelve 30 bacterial strains isolated from cryotubes that were used to collect air at altitudes between 31 27 and 41 km, during a balloon flight from Hyderabad, India. Three of the twelve bacterial 32 isolates namely PVAS-1, B3W22 and B8W22 represent three novel species with two of 33 them belonging to the genus Bacillus (B3W22 and B8W22) and the other belonging to the 34 genus Janibacter (PVAS-1). 35 36 Collection of air from 24 km and above 37 The balloon with the cryosampler payload used for collecting high altitude air samples 38 (from 20-41.4 km altitude) was launched on 20th April, 2005, from the National Scientific 4 39 Balloon Facility of the Tata Institute of Fundamental Research at Hyderabad, India. The 40 cryogenic sampler comprising a 16-cryoprobe assembly is similar to the one that was 41 used earlier (Wainwright et al., 2003; Shivaji et al., 2006). All the 16 cryosampling tubes 42 were autoclaved at 120°C for 4 h and integrated into the payload in a tissue culture 43 laboratory which was UV sterilized over night. The cryotubes were also checked for 44 sterility after the assembly. For this purpose 3 cryotubes were disconnected from the 45 assembly, opened in a laminar flow hood, rinsed with sterile phosphate buffer (0.1 M, pH 46 7.2) and plated on Luria-Bertani agar and incubated at 37°C. Colonies did not appear 47 even after 1 month indicating that the cryotubes were sterile. Therefore, the isolates 48 reported in table 1 should have originated from the atmospheric samples collected at the 49 specific altitudes (Table 1). Cryotubes 1,3,5,7,9,12,13,15 were examined at the Centre for 50 Cellular and Molecular Biology, Hyderabad while cryotubes 2,4,6,8,10,14 were similarly 51 studied at the National Centre for Cell Sciences, Pune. Care was taken that both 52 laboratories followed similar protocols and there was frequent interaction and discussion 53 between the two groups to ensure homogeneity of procedure and interpretation. 54 55 Detection of viable bacteria from the air collected in the cryotubes 56 All the work related to detection of microbes in the air samples in the cryotubes 57 (Table 1) was carried out in a room with very little human traffic and inside a laminar flow 58 available exclusively for the handling of the probes. The probes were transferred to the 59 laminar flow one at a time and prior to the transfer , the surface of the cryotubes was 60 cleaned and sterilized with alcohol. All instruments used to unscrew the outlet valve of the 61 cryotube such as spanners and screw drivers were surface sterilized with alcohol and 62 flame heated prior to their use. The outlet valve of the cryotube was then connected to a 63 series of two Millipore filtration units using sterile tubing and the air was sequentially 64 filtered through a 0.45 pm filter (Millipore Catalogue #HAWP 04700) and a 0.22 pm filter 65 (Millipore Catalogue #GSWP 04700) under aseptic conditions in the laminar flow hood. 5 66 The 0.45 µm and 0.22 gm filters were then used for detection of bacteria. For this 67 purpose, each filter was cut into four quarters and one of the quarters was transferred to a 68 nutrient agar plate [0.5% (w/v) peptone, 0.3% (w/v) beef extract, 0.5% (w/v) NaCI and 69 1.5% (w/v) agar] and incubated at 15°C. After ~10 days if no growth was observed the 70 filter was transferred to a Luria-Bertani Agar (LB) plate [1% (w/v) tryptone, 0.5% (w/v) 71 yeast extract, 1.0% (w/v) NaCI and 2.0% (w/v) agar; pH adjusted to 7.2] or on to a 72 oligotrophic LB agar plate (1/10 LB) and incubated again at 15°C for 15 days. 73 Subsequently, if no colonies appeared the incubations of the filter in LB was also carried 74 out at 25°C for up to a month/However, it may be noted that when the 0.45 pm (Millipore 75 Catalogue # HVHP04700) and 0.22 pm (Catalogue #GVHP04700) filters used for filtering 76 the air were hydrophobic in nature, a thin layer of medium of the same composition was 77 applied to the surface of the media plates before the filters were placed on the surface of 78 the media. 79 80 Two of the remaining quarters of the filter were transferred to Minimal salts agar 81 [20 ml aliquot of sodium dihydrogen phosphate 3.39% (w/v), potassium phosphate 1.5% 82 (w/v), NaCI 0.25% (w/v), ammonium chloride 0.5% (w/v) and agar 2% which was 7 83 supplemented with 2 ml of sterile 20% glucose and 0.2 ml of sterile 0.1 M MgSO4 solution; 84 ,pH was adjusted to 7] for the cultivation of organisms which require low nutrients and 85 Blood agar [peptone 2.3% (w/v), corn starch 0.1% (w/v), NaCI 0.5% (w/v) and agar 1.5% 86 (w/v)] to which defibrinated blood 5% (v/v) was added (after autoclaving and cooling the 87 medium) for the cultivation of organisms which require rich media and incubated at 30°C. 88 One quarter of the filter was also incubated at 30°C on Sabourauds Agar [enzymatic 89 digest of casein 1.0% (w/v), dextrose 4% (w/v) and agar 1.5% (w/v)] for facilitating fungal 90 growth (data not shown). 91 6 92 Out of the fifteen cryotubes, from which air was sampled, a single novel bacterial 93 colony was detected only in cryotube 11, corresponding to air collected at an altitude of 94 40-41.4 km (Table 1). The filter paper from cryotube 11 when placed on Minimal salts 95 agar showed a single cream coloured colony after 24 days of incubation at 30°C and it 96 was designated as PVAS-1. 97 98 During the filtration process, plates containing fungal and bacterial media were left 99 open in the laminar flow hood from the beginning till the end of the operation to check the 100 sterility of the hood.

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