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1 PROIMI Planta Piloto de Procesos Industriales Microbiológicos, Av. Belgrano y Pasaje Caseros, 4000 San Miguel de Tucumán, Tucumán, Argentina
2 Cátedra de Microbiología Superior, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, San Miguel de Tucumán, Argentina
3 Cátedra de Microbiología de Alimentos, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de La Pampa, Uruguay 151, 6300 Santa Rosa, La Pampa, Argentina
Correspondence
Nelda Olivera
olivera{at}cenpat.edu.ar
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, which still lacks taxonomic standing. These results support the proposal of strain PAT 05T (=DSM 16117T=ATCC BAA-965T) as the type strain of Bacillus patagoniensis sp. nov.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain PAT 05T is AY258614.
An electron photomicrograph of a negatively stained cell of strain PAT 05T is available as supplementary material in IJSEM Online.
Present address: Centro Nacional Patagónico, Blvd Brown s/n, 9120 Puerto Madryn, Chubut, Argentina. 
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; Nielsen et al., 1994, 1995). As a result of these studies, nine novel species were described, Bacillus agaradhaerens, Bacillus clarkii, Bacillus clausii, Bacillus gibsonii, Bacillus halmapalus, Bacillus halodurans, Bacillus horikoshii, Bacillus pseudalkaliphilus and Bacillus pseudofirmus, in addition to the previously known species Bacillus cohnii and Bacillus alcalophilus. Since then, the classification of novel alkalitolerant and alkaliphilic strains has led to the proposal of species such as Bacillus vedderi (Agnew et al., 1995), Bacillus haloalkaliphilus (Fritze, 1996), Bacillus horti (Yumoto et al., 1998), Bacillus arseniciselenatis and Bacillus selenitireducens (Switzer Blum et al., 1998), Bacillus okuhidensis (Li et al., 2002) and Bacillus krulwichiae (Yumoto et al., 2003).
Naturally occurring alkaline environments harbour a wide range of alkaliphilic micro-organisms. Desert soils, such as the arid soils in north-eastern Patagonia (Argentina), are exposed to wind and water erosion, as well as salinization and alkalinization processes associated with non-irrigated lands. The physical processes that cause losses of fine material, organic matter and nutrients from the topsoil lead to the concentration of soil resources underneath remnant plant patches (Mazzarino et al., 1996). There is very limited knowledge about the microbial diversity of Patagonian arid soils, especially from vegetated soil microsites characterized by alkaline and saline conditions.
During the characterization of proteolytic micro-organisms from such soils, the strain PAT 05T was isolated from the rhizosphere of Atriplex lampa, a perennial shrub that is able to colonize alkaline and saline areas. PAT 05T is a producer of alkaline proteases that, considering their characteristics such as high optimum pH, high stability and residual activity in the presence of denaturing and chelating agents, could be a promising system enzyme for a detergent formulation (Olivera et al., 2003). This study focuses on phenotypic, phylogenetic and DNA–DNA relatedness analyses performed in order to establish the taxonomic position of strain PAT 05T.
Strain PAT 05T was originally isolated using an agar medium composed of 1 % (w/v) skimmed milk, 0·1 % (w/v) yeast extract, 5 % (w/v) NaCl and 0·1 M Na2CO3 (separately autoclaved) to provide pH 10 (Olivera et al., 2003). For routine growth, isolate PAT 05T was cultured in LB medium supplemented with 5 % (w/v) NaCl and 0·1 M Na2CO3.
Phenotypic tests were based on the methods described by Claus & Berkeley (1986) with media adjusted to approximately pH 10 according to Fritze et al. (1990). The API 50 CH gallery (bioMérieux) was used for carbohydrate utilization tests according to the procedure described by Nielsen et al. (1995). Acid production from carbohydrates was determined by the method of Hugh & Leifson (1953) using thymol blue instead of bromothymol blue at pH 10 (Yumoto et al., 2003). Doubling times at pH 6, 7, 8, 9 and 10 were evaluated in LB broth with 5 % NaCl (w/v); triplicate cultures were incubated at 200 r.p.m. and 25 °C and quantified by optical density at 600 nm. Tolerance to salt was investigated by using different NaCl concentrations in LB broth, 0·1 M Na2CO3. The effect of temperature on growth was determined in the same medium with 5 % NaCl and 0·1 M Na2CO3. Cellular morphology and size and endospores were examined by phase-contrast microscopy (Carl Zeiss Photomicroscope III). Flagellation was examined using transmission electron microscopy (JEOL CX 100) of negatively stained cells (Tesche & Schmiady, 1985).
The 16S rRNA gene sequence (corresponding to positions 27–1492 in the Escherichia coli gene) was amplified by PCR as described by DeLong (1992), using a GeneAmp model 2700 thermal cycler (Applied Biosystems). Sequencing of both strands of PCR-amplified fragments was performed using the dideoxy chain-termination method by the commercial services of GATC Biotech AG. 16S rRNA gene sequence similarity searches against the NCBI database were carried out using BLAST (Altschul et al., 1990). Sequences showing a relevant degree of similarity were imported into the CLUSTAL W program (Thompson et al., 1994), aligned and corrected manually. The percentage of similarity was calculated in the BioEdit program version 5.0.9 (Hall, 1999). Phylogenetic analyses were performed using the branch and bound parsimony algorithm with PAUP program version 4.0b10 (Swofford, 2001). Sites involving gaps were treated as missing characters. The results were evaluated with 1000-replication jackknife analysis and the length and the consistency (CI) and retention (RI) indices of the trees were calculated.
The DNA G+C content was determined by reverse-phase HPLC by the commercial services of the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). DNA–DNA hybridization analyses were also performed by the DSMZ. DNA was isolated using a French pressure cell (Thermo Spectronic) and purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). Hybridization was carried out as described by De Ley et al. (1970), with the modifications described by Huß et al. (1983) and Escara & Hutton (1980), using a model 2600 spectrophotometer equipped with a model 2527-R thermoprogrammer and plotter (Gilford Instrument Laboratories). Renaturation rates were computed with the TRANSFER.BAS program (Jahnke, 1992).
The overall biochemical and physiological characteristics (see species description) indicate that strain PAT 05T should be placed in the alkaliphilic Bacillus group. It grew as creamy white-coloured colonies and the cells were rod-shaped with peritrichous flagella (an electron photomicrograph is available as supplementary material in IJSEM Online). Subterminal oval endospores were observed in slightly swollen sporangia (Fig. 1). Strain PAT 05T did not grow under anaerobic conditions. Most of its phenotypic properties are shown in Table 1 and they are compared with those of related alkalitolerant Bacillus strains. PAT 05T grew at pH 7–10, while growth was undetectable at pH 6. Optimal growth (doubling time, td 86 min) was obtained at pH 8, although it was able to grow at pH 7 (td 99 min), pH 9 (td 88 min) and pH 10 (td 106 min). The range of temperature for growth was 5–40 °C. These results indicate that strain PAT 05T is an alkalitolerant/moderately alkaliphilic micro-organism, and its capacity to grow at low temperatures and high salinity revealed that it is also psychrotolerant and halotolerant (Table 1).
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. Analysis of 1422 bases of the 16S rRNA gene of PAT 05T confirmed that the closest match (99·6 % similarity) was to the sequence from the alkaliphilic Bacillus strain DSM 8714, which belongs to phenon 4 group a (Nielsen et al., 1994, 1995), and which still lacks taxonomic standing. The next highest similarity (98·0 %) was to Bacillus sp. WW3-SN6 (Ntougias & Russell, 2000). Similarity values in the range 95·3–89·5 % were obtained when comparing the 16S rRNA gene sequence of PAT 05T to those of type strains of alkaliphilic Bacillus species described by Nielsen et al. (1995). The highest similarity values (96·2 and 95·3 %) with strains characterized to the species level were to B. clausii SIN84 Enterogermina (Senesi et al., 2001) and the type strain of B. clausii, DSM 8716T, respectively. Levels of similarity between 16S rRNA gene sequences below 97 % suggest that the strains do not correspond to the same species (Stackebrandt & Goebel, 1994).
To characterize strain PAT 05T further, a phylogenetic tree based on its 16S rRNA gene sequence was constructed (Fig. 2). From the total of 1422 bp, 183 were parsimony informative. A single most-parsimonious tree was obtained; its length was 750 steps and the CI and RI were respectively 0·7120 and 0·6516. The phylogenetic tree revealed that PAT 05T forms a distinct clade in the alkaliphilic Bacillus tree together with Bacillus spp. DSM 8714, WW3-SN6 and DSM 8717. The taxonomic integrity of this clade was supported by the 78 % jackknife value obtained. The cladogram also showed that this clade is the sister group of the clade containing B. clausii strains, with 91 % recovery in jackknife analysis. These results confirmed that PAT 05T is closely related to taxa referred to as phenon 4 groups a and b, whose reference strains are Bacillus spp. DSM 8714 and DSM 8717, respectively (Nielsen et al., 1995). As was the case for PAT 05T, soil was the source of isolation of group 4a strains, while group 4b strains were isolated from animal manures.
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) considered a threshold value of 70 % DNA–DNA relatedness for definition of bacterial species.
All these results confirmed that strain PAT 05T should be classified in a novel species together with strains belonging to phenon 4a (Nielsen et al., 1995). We propose the name Bacillus patagoniensis sp. nov., the type strain being PAT 05T (=DSM 16117T=ATCC BAA-965T).
Description of Bacillus patagoniensis sp. nov.
Bacillus patagoniensis (pa.ta.go'ni.en.sis. N.L. masc. adj. patagoniensis pertaining to Patagonia, in Argentina, where the type strain was isolated).
Cells are aerobic rods (2·4–3·2x0·8–1·1 µm) with peritrichous flagella and they occur singly, in pairs or in chains. Endospores are observed as subterminal oval spores. Colonies are cream–white. Gram, oxidase and catalase reactions are positive. Growth occurs at pH 7–10, with an optimum at about pH 8. There is growth between 5 and 40 °C and with 15 % NaCl. Nitrate is not reduced to nitrite. Hydrolysis of casein, gelatin, starch and Tweens 20, 40 and 60 is observed, but Tween 80 and 4-methylumbelliferyl 
-D-glucuronide are not hydrolysed. Phenylalanine is not deaminated. Utilizes glycerol, D-ribose, D-glucose, D-fructose, D-mannose, L-rhamnose, D-mannitol, D-sorbitol, N-acetylglucosamine, salicin, D-cellobiose, D-maltose, sucrose, D-trehalose, D-raffinose, gentiobiose, D-turanose and potassium 2-ketogluconate but not D-arabinose, L-arabinose, D-xylose, D-galactose, L-sorbose, inositol, starch, xylitol, D-lyxose, D-arabitol or gluconate. Acid, but no gas, is produced from glycerol, D-glucose, D-mannitol, D-sorbitol, D-maltose, D-ribose, D-raffinose and D-cellobiose. The DNA G+C content of the type strain is 39·7 mol% as determined by HPLC.
The type strain is PAT 05T (=DSM 16117T=ATCC BAA-965T), isolated from the rhizosphere of the perennial shrub Atriplex lampa in north-eastern Patagonia, Argentina.
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ACKNOWLEDGEMENTS |
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