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1 National Research Institute of Fisheries Science, Fisheries Research Agency, Yokohama, 236-8648, Japan
2 Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91109, USA
Correspondence
Kasthuri Venkateswaran
kjvenkat{at}jpl.nasa.gov
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and gyrB gene sequences referred to in this communication are given in Table 1
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A dendrogram based on rep-PCR fingerprinting and details of DNA–DNA hybridization among Bacillus strains isolated from the JPL spacecraft assembly facility are available as supplementary material in IJSEM Online.
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Venkateswaran et al., 2001), because of stringent maintenance. However, colonization by micro-organisms specifically adapted to such facility conditions, especially those yet to be cultured and/or characterized, is of major concern to those commissioning modern-day space-related experimentation. The search for extraterrestrial life will rely heavily on validated cleaning and bioreduction strategies to ensure that terrestrial microbial contamination does not compromise the scientific integrity of such missions. It is crucial both to minimize and eradicate such microbial contaminants and to identify and characterize the recurring, prevalent micro-organisms associated with the surfaces of spacecraft and associated environments.
Studies have repeatedly shown that extremely resilient, spore-forming members of the genus Bacillus are the most strongly represented micro-organisms in samples collected from spacecraft and facility surfaces (La Duc et al., 2003; Puleo et al., 1977). The extremely oligotrophic, low-humidity, temperature-controlled conditions of spacecraft-assembly facilities appear to select for micro-organisms able to withstand such unfavourable surroundings. During monitoring of the microbial diversity of spacecraft-associated environments over a period of 5 years (1999–2004), Bacillus pumilus was found to be the second most dominant species among the aerobic spore-forming bacteria (the predominant species being Bacillus licheniformis; La Duc et al., 2004a). Several of these B. pumilus isolates have exhibited elevated resistance to H2O2 (Venkateswaran et al., 2001; Kempf et al., 2005) and are thus considered as ‘problematic’ micro-organisms, since H2O2 is recommended for the bioreduction of spacecraft components.
In previous studies using MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) to assess similarities amongst isolates, two very distinct and consistent groups of B. pumilus were revealed (Dickinson et al., 2004). A detailed taxonomic characterization of this group of bacteria was deemed necessary to clarify the distribution of these species in spacecraft-assembly facilities. Here, we report the results of a study in which phenotypic characteristics and the results of phylogenetic analyses of 16S rRNA and gyrB gene sequences, repetitive element primer (rep)-PCR fingerprinting and DNA–DNA hybridizations for several novel isolates were compared.
The bacterial strains examined in this study are shown in Table 1. A total of 13 strains of this novel micro-organism were isolated from spacecraft and assembly-facility surfaces by standard swabbing procedures (Anonymous, 1980; La Duc et al., 2004b) at various times and locations. Comparative analyses were performed with these newly described strains, four B. pumilus strains isolated from spacecraft-assembly facilities or International Space Station hardware and two strains purchased from the American Type Culture Collection (Manassas, VA, USA), including the B. pumilus type strain ATCC 7061T. All strains were maintained in trypticase soy agar (TSA) stabs at room temperature for short-term analysis and in glycerol at –80 °C for long-term storage. Liquid cultures were grown in trypticase soy broth (TSB; Becton Dickinson) at 32 °C with vigorous aerobic shaking for an appropriate period of time.
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). A nutrient sporulation medium rich in divalent cations was used to produce spores, as described previously (Schaeffer et al., 1965; Nicholson & Setlow, 1990). The refractile nature of the spores was examined by using phase-contrast microscopy (Olympus BX-60). The optimum NaCl concentration for growth was determined in salt-free TSB supplemented with 0, 5.0, 7.5, 10.0, 12.5 and 15.0 % (w/v) NaCl, and cultures were monitored for growth for 3 weeks with incubation at 32 °C. Optimum and limiting temperatures were determined by growing cells at 4, 10, 37, 50 and 55 °C on TSA for 3 weeks. Routine biochemical tests were carried out using commercially available API kits [API 20NE, API 20E, API 50 CH (assimilation), API 50 CH (acid production) and API ZYM], which were utilized according to the instructions of the manufacturer (bioMérieux). Nutritional requirements for growth were assessed using Davis minimum medium (Becton Dickinson). Casein hydrolysis, starch hydrolysis and the production of lipase (hydrolysis of Tweens 20, 40 and 80) were tested as described by Smibert & Krieg (1994). Whole-cell fatty acid methyl ester profiles for the novel isolates and reference strains were determined using the MIDI system (Sasser, 1990). The DNA G+C content was determined by using the HPLC method of Tamaoka & Komagata (1984).
The novel isolates were Gram-positive, spore-forming rods and were aerobic, motile and oxidase- and catalase-positive and so demonstrated several morphological and physiological characteristics typical of members of the genus Bacillus (Claus & Berkeley, 1986); this indicated that all of these isolates belonged to this genus or related genera. The Biolog identification system indicated that the novel isolates were most similar to B. pumilus (Dickinson et al., 2004). Additionally, 174 phenotypic tests were performed to study the phenotype of the novel isolates. Two phenotypic characteristics, acid production from inositol and utilization of inositol, allowed the discrimination of the novel isolates from B. pumilus (Table 2). Additional phenotypic characteristics (production of acid phosphatase, Tween 80 hydrolysis, casein hydrolysis in litmus milk and utilization of raffinose) were also useful for separating these two bacterial groups, though responses to various substrates were variable among strains. Further details regarding physiological and phenotypic characteristics are discussed in the species description. The major fatty acids of the novel isolates were C15 : 0 iso (50.4–56.7 %), C15 : 0 anteiso (23.3–25.2 %), C17 : 0 iso (4.52–6.93 %) and C17 : 0 anteiso (3.71–4.69 %). All of the novel isolates and the five B. pumilus strains tested in this study had similar fatty acid methyl ester profiles. The DNA G+C contents of novel isolates FO-36bT, KL052, SAFN001 and 51-3C were 41.0, 41.0, 41.4 and 41.2 mol%, respectively. The G+C contents of B. pumilus strains ATCC 7061T and SAFR032 were 40.2 and 39.5 mol%, respectively.
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; Sambrook et al., 1989) from strains cultured in TSB supplemented with 2 % glycine. The 16S rRNA and gyrB genes were PCR-amplified with the universal primer sets described by Weisburg et al. (1991) and Yamamoto & Harayama (1995), respectively, and sequenced. The identity of a given PCR product was verified by bidirectional sequencing analysis. The phylogenetic relationships of the micro-organisms examined in this study were determined by comparing individual 16S rRNA or gyrB gene sequences with sequences in the public databases using the BLAST algorithm (Altschul et al., 1990). The multiple alignment calculation of nucleotide substitution rates (Knuc values), described by Kimura (1980), and the construction of phylogenetic trees by the neighbour-joining method (Saitou & Nei, 1987) were performed using the CLUSTAL W computer program (Thompson et al., 1994). Alignment gaps, primer regions for PCR amplification and unidentified base positions were not taken into consideration in the calculations. The topological robustness of the phylogenetic trees was evaluated by a bootstrap analysis involving 1000 replications. The GenBank nucleotide accession numbers for the 16S rRNA and gyrB gene sequences are shown in Table 1. rep-PCRs were carried out according to the manufacturers' instructions (Bacterial Barcodes); the detailed protocol of the DiversiLab System LabChip kit used can be found elsewhere (http://www.bacbarcodes.com/diversilab/35-0018-00rev6%20-LabChip.pdf). Analysis of PCR-fragment banding patterns and dendrogram construction were performed following the method of de Bruijn (1992). DNA–DNA hybridization was studied by using microplate hybridization methods (Ezaki et al., 1989) with photobiotin labelling and colorimetric detection, with 1,2-phenylenediamine (Sigma) as the substrate and a streptavidin–peroxidase conjugate (Boehringer Mannheim) as the colorimetric enzyme (Satomi et al., 1997).
Sequences of 1.4 kb of the 16S rRNA genes (covering base positions 44–1471; Escherichia coli numbering) and the 1.1 kb of the gyrB genes (covering base positions 316–1472; E. coli numbering) were used for phylogenetic analyses. The sequence similarity of 16S rRNA genes among the novel isolates was greater than 99.9 %. A phylogenetic tree based on the 16S rRNA gene (Fig. 1) showed that the novel isolates clustered with members of the genus Bacillus, the nearest neighbour being B. pumilus (99.9 % sequence similarity). Since several reports have been published showing that strains with >99 % 16S rRNA gene sequence similarity may not belong to the same species (La Duc et al., 2004c; Satomi et al., 2002; Venkateswaran et al., 1999; Stackebrandt & Goebel, 1994), comparative gyrB gene sequence analyses were carried out. As has been observed in previous studies (La Duc et al., 2004b) gyrB gene sequence-based phylogenetic topology proved more highly discriminative, grouping these strains monophyletically in a cluster separate from B. pumilus, clearly delineating them as a distinct species (Fig. 2). The sequence similarity values required to separate species on the basis of the gyrB gene vary according to the genus (Venkateswaran et al., 1999; Satomi et al., 2002, 2003, 2004). Additional reputable genetic analyses are therefore necessary to confirm the novelty of these isolates. On the basis of the results of rep-PCR fingerprinting (Supplementary Fig. S1 available in IJSEM Online), all of the novel isolates are grouped together in their own cluster, separate from the distinct cluster formed by previously described B. pumilus strains, supporting the gyrB analysis and previous MALDI-TOF MS results (Dickinson et al., 2004). The most notable difference between the protein profiles of the B. pumilus type strain (ATCC 7061T) and the proposed FO-36bT strain group is the presence of a peak at 7620 Da, found only in the FO-36bT group strains (Dickinson et al., 2004). Another difference between the two groups is that the FO-36bT group forms a tight cluster in terms of MALDI-TOF MS protein profiling. rep-PCR fingerprinting is able to resolve down to, and perhaps beyond, the species level. It has been used in recent taxonomic studies to evaluate DNA relatedness among bacterial strains (Thompson et al., 2003). The rep-PCR results generated in our work clearly distinguish the novel isolates from B. pumilus and, when coupled with supporting MALDI-TOF MS profiles (Dickinson et al., 2004), strongly suggest that these isolates should be recognized as belonging to a distinct genotype. To confirm the conclusions generated from the results of the phylogenetic analyses, DNA–DNA hybridization was performed (Supplementary Table S1 available in IJSEM Online). DNA–DNA hybridization revealed that the 13 novel isolates were all closely related (80–99 % DNA relatedness values), suggesting that they belong to the same species, but exhibited lower levels of hybridization with B. pumilus strains (54–66 %). This strongly supports the claim that these isolates represent a novel species within the genus Bacillus (Wayne et al., 1987).
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Description of Bacillus safensis sp. nov.
Bacillus safensis [sa.fen'sis. N.L. masc. adj. safensis arbitrarily derived from SAF (the spacecraft-assembly facility at the Jet Propulsion Laboratory, Pasadena, CA, USA), from where the organism was first isolated].
Cells are mesophilic, aerobic, chemoheterotrophic, Gram-positive, spore-forming rods that are motile by means of polar flagella. Cells are 0.5–0.7 µm in diameter and 1.0–1.2 µm in length. Growth occurs at 0–10 % (w/v) NaCl and at pH 5.6. Growth occurs at 10–50 °C (optimum, 30–37 °C) but not at 4 or 55 °C. Colonies are round, undulate, dull white, non-luminescent and have irregular margins on TSA plates incubated at 32 °C for 24 h. Oxidase, catalase, 
-galactosidase, -glucosidase, alkaline phosphatase, naphthol-AS-BI-phosphatase and esterase are produced, but H2S, indole, amylase, agarase, lecithinase, DNase, urease, leucine arylamidase, cystine arylamidase, valine arylamidase, trypsin, 
-galactosidase, N-acetyl--glucosamidase, -fucosidase, tryptophan deaminase, phenylalanine deaminase, arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase are not. Cells do not reduce nitrate, but do hydrolyse gelatin, aesculin and RNA. Casein hydrolysis varies among strains. Voges–Proskauer test is positive. Growth occurs on agar plates supplemented with 1 % glycine and ox gall, but does not occur in 0.0001 % lysozyme broth. Negative for gas production from D-glucose. Acid is produced from D-glucose, glycerol, L-arabinose, ribose, D-xylose, galactose, fructose, mannose, inositol, mannitol, methyl -D-mannopyranoside, methyl -D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, sucrose, trehalose, D-turanose and D-tagatose, but not from erythritol, D-arabinose, L-xylose, adonitol, methyl -D-xylopyranoside, sorbose, rhamnose, dulcitol, sorbitol, inulin, melezitose, raffinose, starch, glycogen, xylitol, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. Reactions for lactose, melibiose and gentiobiose vary among strains. Citrate, malate, D-glucose, glycerol, L-arabinose, ribose, D-xylose, galactose, fructose, mannose, inositol, mannitol, methyl -D-mannopyranoside, methyl -D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, sucrose, trehalose, gentiobiose, D-turanose, D-tagatose, gluconate, lactate, L-aspartate and L-glutamate are readily utilized as energy sources. Erythritol, D-arabinose, L-xylose, adonitol, methyl -D-xylopyranoside, sorbose, dulcitol, sorbitol, inulin, lactose, melezitose, starch, glycogen, xylitol, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-ketogluconate, 5-ketogluconate, capric acid, adipic acid, phenylacetic acid, propionate and glycine are not utilized as energy sources. Rhamnose utilization varies among strains. The DNA G+C content is 41.0–41.4 mol%. The chain composition of the whole-cell fatty acids is primarily C15 : 0 iso, C15 : 0 anteiso, C17 : 0 iso and C17 : 0 anteiso.
The type strain, FO-36bT (=ATCC BAA-1126T=NBRC 100820T), was isolated from the spacecraft-assembly facility of the Jet Propulsion Laboratory, Pasadena, CA, USA. Strains SAFN-001 (=ATCC BAA-1128=NBRC 100821), KL-052 (=ATCC BAA-1129=NBRC 100822) and 51-3C (=ATCC BAA-1127=NBRC 100823) are reference strains.
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ACKNOWLEDGEMENTS |
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