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1 Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, California Institute of Technology, 89-2; Biotechnology and Planetary Protection Group, 4800, Oak Grove Dr., Pasadena, CA 91109, USA
2 National Research Institute of Fisheries Science, Fisheries Research Agency, Yokohama, 236-8648, Japan
Correspondence
Shariff Osman
sosman{at}jpl.nasa.gov
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ABSTRACT |
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400 strains) obtained from the JPL-SAF using species-specific PCR primer sets designed from the 16S rRNA gene sequences of strains SAFN-016T and SAFN-007T. Phylogenetic analysis of 16S rRNA gene sequences placed these novel isolates within the genus Paenibacillus. Two strains, SAFN-016T and SAFN-125, shared 98 % 16S rRNA gene sequence similarity with Paenibacillus timonensis and 97 % similarity with Paenibacillus macerans. Strain SAFN-007T showed 95.2 % 16S rRNA gene sequence similarity with Paenibacillus kobensis, its nearest phylogenetic neighbour. The results of DNA–DNA hybridization, physiological tests and biochemical analysis allowed genotypic and phenotypic differentiation of the isolates from currently recognized Paenibacillus species. Strain SAFN-007T and strains SAFN-016T and SAFN-125 are representatives of two separate novel species, for which the names Paenibacillus pasadenensis sp. nov. (type strain SAFN-007T=ATCC BAA-1211T=NBRC 101214T) and Paenibacillus barengoltzii sp. nov. (type strain SAFN-016T=ATCC BAA-1209T=NBRC 101215T) are proposed.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of the Paenibacillus strains investigated in this study are given in Table 1
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Puleo et al., 1977). During a survey of the Jet Propulsion Laboratory Spacecrat Assembly Facility (JPL-SAF) in December 2000, 30 strains of spore-forming microbes were systematically isolated and identified using both phenotypic tests and 16S rRNA gene sequence analysis (Fig. 1). Phylogenetic affiliations revealed that all of these strains were Gram-positive bacteria belonging to the genera Bacillus (23 isolates comprising nine species), Filibacter, Sporosarcina, Paenibacillus and Streptomyces. The most abundant species was Bacillus pumilus (six isolates) followed by Bacillus megaterium (three isolates). The 16S rRNA gene sequences of two isolates, SAFN-007T and SAFN-016T, exhibited >95 % similarity to members of the genus Paenibacillus. As this was the first incidence of Paenibacillus species reported in the JPL-SAF, both strains were further characterized using a polyphasic taxonomic approach.
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). Polyester swabs were used to collect samples from 200 locations at various times from both unclassified (entrance floors, ante-room and air-lock) and classified (floors, cabinet tops and air) surfaces within the JPL-SAF; specific details have been reported elsewhere (Venkateswaran et al., 2001). The collected swab samples were sonicated (25 kHz) for 2 min and aliquots were either left untreated or were subjected to heat shock (80 °C for 15 min) to kill vegetative cells. Heat-shocked samples were pour-plated in tryptic soy agar (TSA) and grown at 32 °C for 2 days. Samples not subjected to heat shock were spread-plated on R2A medium and incubated at 25 °C for 7 days.
The bacterial strains analysed in this study are shown in Table 1. A set of related type strains were received as gifts from the Agricultural Research Service Culture Collection (http://nrrl.ncaur.usda.gov) or purchased from the Swedish Culture Collection (CCUG), University of Göteborg, Sweden, and used as reference strains. All isolates were maintained in TSA stabs (Becton Dickinson) at room temperature for short-term analysis and in glycerol at –80 °C for long-term storage. Liquid cultures were grown in tryptic soy broth (TSB) (Becton Dickinson) and incubated at 32 °C with vigorous aerobic shaking for an appropriate period of time. Representative strains have been deposited in the American Type Culture Collection (ATCC) and the National Institute of Technology and Evaluation, Biological Resource Center (NBRC), Japan (Table 1).
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; Nicholson & Setlow, 1990; Schaeffer et al., 1965). Routine biochemical tests were carried out using commercially available API kits (API 20NE and API 20E), inoculated according to the manufacturer's instructions (bioMérieux). Metabolic profiling was performed using the Biolog 96-well microplate test for the oxidation of 95 different carbon sources (Biolog).
Phenotypic characteristics of the novel isolates SAFN-007T, SAFN-016T and SAFN-125 and other closely related Paenibacillus species are presented in Table 2. Strain SAFN-007T did not reduce nitrate, was positive for Voges–Proskauer tests, liquefied gelatin and assimilated arabinose, glucose, maltose, mannose, malate, mannitol and N-acetylglucosamine. However, strains SAFN-016T and SAFN-125 did not assimilate any of the carbon substrates tested apart from gluconate. Strain SAFN-007T did not assimilate gluconate as a sole carbon source. The Biolog-based carbon substrate profiles of the novel strains did not match those of any of the Bacillus or Paenibacillus species provided in the manufacturer's database. The phenotypic characteristics of Paenibacillus kobensis, the closest neighbour of strain SAFN-007T according to 16S rRNA gene sequences, differed from those of strain SAFN-007T as regards nitrate reduction, urea hydrolysis, malate assimilation and acid production from sucrose and L-arabinose (Kanzawa et al., 1995) (Table 2). These differences strongly support the conclusion of the phylogenetic analysis that strain SAFN-007T represents a novel species in the genus Paenibacillus. When strains SAFN-016T and SAFN-125 were tested, no significant colouration of tetrazolium dye was seen. This was in agreement with API test strips in which the majority of carbon substrates tested were not assimilated by either of these strains (Table 2). Significant characteristic phenotype differences between strains SAFN-016T and SAFN-125 and Paenibacillus timonensis CCUG 48216T are nitrate reduction, acid production from sugars and the assimilation of several carbohydrates, as shown in Table 2. Phenotypic characteristics of the novel Paenibacillus species are given in the species descriptions.
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), purified on Qiagen filtration columns and sequenced. The identity of a given PCR product was verified by bidirectional sequencing. The phylogenetic relationships of the organisms investigated in this study were determined by comparison of individual 16S rRNA gene sequences with other sequences in public databases using the BLAST algorithm (Altschul et al., 1990). Evolutionary trees were constructed with PAUP software, following maximum parsimony parameters (Swofford, 1990). Alignment gaps, primer regions for PCR amplification and unidentified base positions were not taken into consideration for the calculations. The robustness of the topology of the phylogenetic trees was evaluated by a bootstrap analysis with 1000 replications. GenBank accession numbers for the 16S rRNA gene sequences analysed in this study are shown in Table 1.
A phylogenetic tree based on 16S rRNA gene sequences (Fig. 2) showed that the novel isolates clustered with members of the genus Paenibacillus, the nearest neighbours being P. kobensis DSM 10249T for strain SAFN-007T and Paenibacillus timonensis CCUG 48216T for strains SAFN-016T and SAFN-0125. Strain SAFN-007T showed only 95.2 % 16S rRNA gene sequence similarity with P. kobensis DSM 10249T. Strains SAFN-016T and SAFN-125 exhibited 98 % 16S rRNA gene sequence similarities with P. timonensis CCUG 48216T and 97 % with Paenibacillus macerans NRRL B-172T and shared 99.8 % sequence similarity with each other. The phylogenetic distances between strain SAFN-007T and its nearest neighbours in the genus Paenibacillus suggest that this strain represents a novel species (Wayne, 1988). Since strains SAFN-016T and SAFN-125 are so closely related to their nearest neighbours on the basis of 16S rRNA gene sequences, further study was necessary to confirm their taxonomic position. Gene sequence similarities between the two novel Paenibacillus species and the 18 other members of the genus Paenibacillus ranged from 92 to 98 %. The two novel species showed only 86–87 % gene sequence similarity to Bacillus subtilis.
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400 strains) isolated from the JPL-SAF for similar strains. The 16S rRNA gene sequences from both novel strains, SAFN-016T and SAFN-007T, were compared with those of their closest related Paenibacillus species. Species-specific primers were designed for each group and tested against DNA extracts from both the novel strains and the type strains listed in Table 1. Isolates similar to strain SAFN-007T were screened for by using primers 172Fb (5'-CCAGATACGCGATCTTC-3') and 649R (5'-GTTTCCCGTGCGACTTGG-3'). SAFN-016T group isolates were selected for using primers 173Fa (5'-CCGGATACGCAAGTCTC-3') and 1161Rb (5'-CTAGAGTGCCCAACCTACT-3'). The PCR conditions were as follows: denaturation for 1 min at 95 °C, annealing for 2 min at 55 °C and elongation for 3 min at 72 °C for 32 cycles using a thermal cycler (MJ Research). Three additional strains produced PCR-amplified products with either the SAFN-016T- or SAFN-007T-specific primer sets. Strain SAFN-068 (isolated in March 2001) yielded a 477 bp amplicon specific for strain SAFN-007T. Strains SAFN-125 (isolated June 2001) and SAFR-173 (isolated September 2001) exhibited 988 bp amplicons specific for strain SAFN-016T. Although the PCR-amplified products from strains SAFN-068 and SAFR-173 were faint in comparison with that of strain SAFN-125 when visualized on agarose gels, the full-length 16S rRNA genes of all three isolates were amplified and sequenced. Strain SAFN-068 was identified as Paenibacillus pabuli based on 16S rRNA gene sequence similarity (99.7 %), API 20NE, 20E and Biolog analyses. Strain SAFR-173 was found to be closely related to Stenotrophomonas maltophilia (98.2 % gene sequence similarity). As the most closely related species to strain SAFN-173 was Gram-negative, phenotypic characteristics were not determined. This confirmed that strains exhibiting faint bands were not the intended sequence targets, but rather were the result of non-specific amplification. Strain SAFN-125 was the only isolate found that closely matched the 16S rRNA gene sequence of strain SAFN-016T (99.8 % similarity). Species-specific PCR screening did not reveal any additional strains for the SAFN-007T isolate.
For DNA–DNA hybridization analysis, cells were suspended in 0.1 M EDTA (pH 8.0) and cell walls were digested by lysozyme treatment (final concentration 2 mg ml–1). DNA was isolated following standard procedures (Johnson, 1981). DNA–DNA hybridization was conducted by microplate hybridization methods (Ezaki et al., 1989) with photobiotin labelling and colorimetric detection, using 1,2-phenylenediamine (Sigma) as the substrate and streptavidin–peroxidase conjugate (Boehringer Mannheim) as the colorimetric substrate (Satomi et al., 1997). Since the 16S rRNA gene sequences of strains SAFN-016T and SAFN-125 showed high similarity values with P. timonensis (98 %) and P. macerans (97 %), type strains of several Paenibacillus were included and a DNA–DNA hybridization study was performed. Due to the very low gene sequence similarity revealed between strain SAFN-007T and P. kobensis, DNA–DNA hybridization was deemed to be unnecessary. The results of this study are given in Table 3. Strain SAFN-016T exhibited 38 % DNA–DNA relatedness with P. timonensis CCUG 48216T and P. macerans NRRL B-172T. This strongly supports the claim that the isolates represent two novel species within the genus Paenibacillus.
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Description of Paenibacillus pasadenensis sp. nov.
Paenibacillus pasadenensis (pa.sa.den.en'sis. N.L. masc. adj. pasadenensis referring to Pasadena, the city in which the JPL-SAF is located).
Cells are Gram-positive rods, 0.5–0.8x3.0–5.0 µm in size and motile by means of peritrichous flagella. Ellipsoidal spores are formed in swollen sporangia. Colonies are flat, smooth, circular, entire and brownish yellow. No soluble pigment is produced on nutrient agar. Catalase and oxidase tests are positive. Acetylmethylcarbinol is produced (as determined by the Voges–Proskauer reaction). Hydrogen sulfide and indole are not produced. Nitrate is not reduced to nitrite. Gelatin is liquefied, aesculin is hydrolysed and 
-galactosidase is produced. Growth occurs in the presence of 2 % NaCl and 0.001 % lysozyme. Growth is inhibited by 3 % NaCl. Utilizes 
-cyclodextrin, D-cellobiose, D-fructose, maltose, D-melibiose, methyl -D-glucoside, D-ribose, pyruvic acid, L-alanyl glycine and L-serine. Acid is not produced from D-glucose.
The type strain, SAFN-007T (=ATCC BAA-1211T=NBRC 101214T), was isolated from the entrance floor of the JPL-SAF, Pasadena, CA, USA.
Description of Paenibacillus barengoltzii sp. nov.
Paenibacillus barengoltzii (ba.ren.gol'tzi.i. N.L. gen. n. barengoltzii referring to Jack Barengoltz, a well-known American physicist and NASA planentary protection scientist).
Cells are Gram-positive rods, 0.5–0.8x3.0–5.0 µm in size, strictly aerobic and motile by means of peritrichous flagella. Ellipsoidal spores are formed in swollen sporangia. Colonies are flat, smooth, circular, entire and brownish yellow. No soluble pigment is produced on nutrient agar. Catalase and oxidase tests are positive. Acetylmethylcarbinol is produced (as determined by the Voges–Proskauer reaction). Hydrogen sulfide and indole are not produced. Nitrate is reduced to nitrite. Gelatin is not liquefied, aesculin is hydrolysed and -galactosidase is produced. Growth occurs between 10 and 50 °C and at pH 4.5–9.0. Optimum growth occurs at 37 °C and at pH 7.0. Growth occurs in the presence of 2 % NaCl and 0.001 % lysozyme. Growth is inhibited by 5 % NaCl. Of the carbon substrates tested, only gluconate is utilized. Acid is not produced from D-glucose.
The type strain, SAFN-016T (=ATCC BAA-1209T=NBRC 101215T) was isolated from clean room floors of the JPL-SAF, Pasadena, CA, USA. Strain SAFN-125 (=ATCC BAA-1210) is a reference strain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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