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Department of Plant Physiology and Microbiology, Vilnius University, Chiurlionio 21/27, Vilnius LT-03101, Lithuania
Correspondence
Nomeda Kuisiene
nomeda.kuisiene{at}gf.vu.lt
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ABSTRACT |
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Published online ahead of print on 21 May 2004 as DOI 10.1099/ijs.0.02976-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain N-3T is AY044055.
Graphs showing the effects of temperature, pH and salinity on the growth rate of strain N-3T are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Rainey et al., 1994). Today, there are ten species with validly published names in the genus Geobacillus (Nazina et al., 2001) embracing almost all of the 5th genetic group. Four of them have been described in the last 3 years: Geobacillus subterraneus (Nazina et al., 2001), Geobacillus uzenensis (Nazina et al., 2001), Geobacillus caldoxylosilyticus (Fortina et al., 2001) and Geobacillus toebii (Sung et al., 2002). It was shown that the novel species Bacillus vulcani also belongs to genetic group 5 (Caccamo et al., 2000). The species name Geobacillus thermodenitrificans was validated recently (Manachini et al., 2000; Nazina et al., 2001). The names of the thermophilic species ‘Bacillus caldotenax’, ‘Bacillus caldovelox’ and ‘Bacillus caldolyticus’ have not been validly published and are supposed to belong to the species Geobacillus thermoleovorans (Sunna et al., 1997; Mora et al., 1998). The latter species presumably embraces Geobacillus thermocatenulatus as well as Geobacillus kaustophilus (Sunna et al., 1997).
All species of the genus Geobacillus are very closely related phylogenetically. Intragenic similarities for the 16S rRNA gene are more than 96·5 % (Nazina et al., 2001). Quickly developing systematics for thermophilic endospore formers requires rigorous and informative methods for fast identification and grouping of strains. The significance of the application of rDNA-based fingerprinting methods (ARDRA and RSA) in this systematic group was reported previously (Blanc et al., 1997; Mora et al., 1998; Manachini et al., 2000; Caccamo et al., 2001; Fortina et al., 2001). These methods are good alternatives to more laborious techniques, such as morphological and physiological analyses, currently used for screening and identification of strains.
Strain N-3T was isolated from crude oil of the oilfield Girkaliai, which is located in Lithuania. The depth of sampling was 2000 m. The temperature of the oilfield was 60 °C and the pH was 6·5.
The isolation of thermophilic, aerobic, heterotrophic bacteria was carried out using tenfold serial dilutions of crude oil from the Lithuanian oilfield. The dilutions were inoculated on to Czapek agar. Inoculated agar plates were incubated aerobically at 60 °C for 48 h.
Cell morphology was examined under an Olympus AX70 microscope (magnification x1000) and a JEM-100S electron microscope (magnification x5000–6000) after cultivation of the strains at 60 °C on nutrient agar for 17–24 h. For bright-field microscopy, cells were stained using a Gram-staining kit (Merck). For electron microscopy, cells were prepared as described by Mignot et al. (2001). Bacterial size was determined by bright-field microscopy in living cell preparations from cultures grown on nutrient agar for 17–24 h. Colony morphology was examined under an MBS-9 microscope (magnification x4). Colour, form, transparency, type of profile, margin and surface were recorded. Results of the morphological characterization are given in the species description.
DNA extraction and amplification of the 16S rRNA gene were performed as described by Kuisiene et al. (2002). The 16S rRNA gene PCR product was extracted from agarose gel using a DNA Extraction kit (Fermentas). The purified PCR product was cloned into Escherichia coli DH5
using the InsT/Aclone PCR Product Cloning kit (Fermentas). Recombinant clones were detected by blue/white screening (Sambrook et al., 1989). Recombinant plasmid DNA was extracted as described by Birnboim & Doly (1979). The cloned 1·5 kb DNA fragments amplified by PCR were sequenced by automated DNA sequencing. The gene sequence was assembled after a minimum of 2x sequencing coverage for each base position. 16S rRNA gene sequences were edited and the G+C mol% and sequence similarity determined using DNASTAR. The sequences were aligned with the sequence of E. coli (GenBank accession no. J01695; Brosius et al., 1978) and nucleotides were determined in diagnostic positions (Ash et al., 1993).
The 16S rRNA gene sequence determined for strain N-3T was 1523 nucleotides long. The G+C content was 59·8 mol%. A number of nucleotides in potentially diagnostic positions (Ash et al., 1993) were identified. It was established that strain N-3T belongs to genetic group 5 of the endospore-forming bacteria.
The sequence of strain N-3T was most similar to that of G. thermoleovorans DSM 5366T, having 99·4 % sequence similarity. A search of the BLAST database also revealed the highest level of similarity with sequences of different strains of G. thermoleovorans. A high level of similarity was also determined for another species of genetic group 5, B. vulcani DSM 13174T (99·2 % sequence similarity). Lower sequence similarities were obtained for G. stearothermophilus DSM 22T, G. thermocatenulatus DSM 730T, G. kaustophilus DSM 7263T and G. uzenensis DSM 13551T (97·4–98·4 % sequence similarity). Geobacillus thermoglucosidasius ATCC 43742T and G. caldoxylosilyticus DSM 12041T, as well as G. toebii DSM 14590T, were the most distantly related to strain N-3T.
The 16S rRNA gene sequences of the tested strains were aligned using the CLUSTAL_X program (Thompson et al., 1997) and also manually. The size of the 16S rRNA gene used for alignment was 1415 nucleotides. A phylogenetic tree was constructed using the PHYLIP package, version 3.6a3 (Felsenstein, 2001) by the neighbour-joining method (Saitou & Nei, 1987). The evolutionary distance matrices were produced using the method of Jukes & Cantor (1969). Bootstrap analysis of the neighbour-joining data, using 1000 resamplings, was carried out to evaluate the validity and reliability of the tree topology. The tree was rooted using the X60646 sequence of Bacillus subtilis NCDO 1769T as an outgroup. All analyses were carried out using the PHYLIP package version 3.6a3 (Felsenstein, 2001). Trees were visualized using TreeView software, version 1.6.1 (Page, 1996). The phylogenetic tree (Fig. 1) shows the position of strain N-3T among the species of genetic group 5 of endospore-forming bacteria.
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Although 16S rRNA gene similarity between strain N-3T and G. thermoleovorans DSM 5366T was high, DNA–DNA relatedness was 40·0 %. DNA–DNA relatedness with B. vulcani DSM 13174T was 51·0 %. G. kaustophilus DSM 7263T was also chosen for DNA–DNA hybridization regarding the phylogenetic position of this species (Fig. 1
). DNA–DNA relatedness with this strain was 42 %. Consequently, strain N-3T could not be assigned to one of these three species (Vandamme et al., 1996; Rosselló-Mora & Amann, 2001).
DNA–DNA relatedness of strain N-3T with the reference strains of the other phylogenetically related species G. stearothermophilus DSM 22T, G. thermocatenulatus DSM 730T and G. uzenensis DSM 13551T was in the range of 32·0–52·0 %. These results showed that strain N-3T belongs to the genus Geobacillus, but represents a novel species within this genus.
The G+C content of strain N-3T was 52·5 mol%. This value is in accordance with the G+C content of the genus Geobacillus, which is 49·0–58·0 mol% (Nazina et al., 2001).
ARDRA was performed with AluI, HaeIII and TaqI as described by Kuisiene et al. (2002). ARDRA was repeated four times using different DNA extractions for amplification and different amplification products for restriction analysis. To avoid confusion with primer dimer bands, restriction fragments shorter than 80 bp were disregarded.
The strain G. thermoleovorans DSM 5366T was chosen for ARDRA as the most closely related to strain N-3T on the basis of 16S rRNA gene analysis. G. stearothermophilus DSM 22T was also included as the reference strain of the type species of the genus Geobacillus.
Restriction endonucleases AluI, HaeIII and TaqI were previously reported as suitable tools for discrimination between different species of the genus Geobacillus (Blanc et al., 1997; Mora et al., 1998; Caccamo et al., 2001; Fortina et al., 2001). In our study, these enzymes showed different restriction patterns for strain N-3T and the reference strains of species G. stearothermophilus DSM 22T and G. thermoleovorans DSM 5366T (Fig. 2).
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TaqI gel-electrophoretic profiles were identical for strains N-3T and G. stearothermophilus DSM 22T. Nevertheless, these two strains could be distinguished on the basis of AluI analysis. A fragment of 160 bp was visible in the case of G. stearothermophilus DSM 22T, while the restriction profile of strain N-3T lacked this fragment. Instead, the AluI restriction pattern of strain N-3T had a fragment of approximately 80 bp, absent from the G. stearothermophilus DSM 22T profile with this enzyme. The HaeIII patterns of these two strains also differed, although not as markedly as in the case of AluI.
In summary, our data have shown that, although strain N-3T and the species G. thermoleovorans are the closest neighbours according to 16S rRNA gene analysis, they can easily be separated on the basis of ARDRA profiles.
All physiological assays were performed in duplicate and repeated three times if the obtained results were inconsistent. Unless otherwise stated, cultures were incubated aerobically at 60 °C for 24 h. Most of the physiological tests were carried out using the methods described by Claus & Berkeley (1986). Denitrification was examined as described by Blanc et al. (1997). Hydrolysis of collagen was tested on tap-water agar plates containing 20 g collagen l–1. Resistance to streptomycin was examined on nutrient agar plates containing 10 or 50 µg streptomycin ml–1. The temperature range for growth was determined as described by Manachini et al. (2000) and retested in nutrient broth buffered with 50 mM Tris/HCl (pH 6·5) by measuring the optical density at 600 nm. To study the influence of pH on bacterial growth, nutrient broth was buffered with citrate-phosphate buffer (pH 6·0) and 50 mM Tris/HCl (pH 6·5, 7·0, 7·5, 8·0, 8·5 and 9·0). Bacterial growth in buffered medium was monitored by measuring the optical density at 600 nm using a Beckman DU-650 spectrophotometer (results for the ranges of temperature, pH and salinity are available as supplementary material in IJSEM Online). Results of the physiological characterization are given in the species description.
Description of Geobacillus lituanicus sp. nov.
Geobacillus lituanicus (li.tu.a'ni.cus. M.L. adj. lituanicus of Lithuania, referring to the Lithuanian oilfield from where the type strain was isolated).
Cells are rod-shaped, occurring in chains, motile by means of peritrichous flagella, varying in length from 4·4 to 5·8 µm and in diameter from 1·1 to 1·4 µm. Oval subterminal endospores are produced within the slightly distended sporangia. Gram staining is positive. Colonies are small, round, tawny, convex, opaque and shiny. Obligately thermophilic, the optimal growth temperature ranges between 55 and 60 °C with a minimum at 55 °C and a maximum at 70 °C. Aerobic/facultatively anaerobic chemo-organotroph; nitrate is the terminal electron acceptor under anaerobic conditions. Differentiating phenotypic characteristics are indicated in Table 1. The DNA G+C content is 52·5 mol%. Isolated from the crude oil of a high-temperature oilfield.
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ACKNOWLEDGEMENTS |
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