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Department of Microbiology, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja, 18071 Granada, Spain
Correspondence
Mercedes Monteoliva-Sánchez
mmonteol{at}ugr.es
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA sequence of strain E308T is DQ139839.
A 16S rRNA gene sequence-based maximum-parsimony tree is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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). The brines represent a large proportion of the total contaminating discharges from the olive industry. However, specific treatments for these wash-waters are either non-existent or ineffective (Romero Barranco et al., 2001). To obtain a better insight into the microbial community present in EWW, we analysed the microbial content. In the present study, we report the description of a novel aerobic, mesophilic, endospore-forming organism isolated from EWW that was able to grow in 0–20 % NaCl.
Initially, 180 strains were isolated from samples of EWW (provided by AgroSevilla S.A., La Roda, Sevilla, Spain) obtained at different stages of the edible olive processing. Isolation was performed by the standard dilution-plating technique on HM medium (Ventosa et al., 1982) supplemented with a balanced mixture of sea salts with 10 % (w/v) NaCl (Rodríguez-Valera et al., 1981) at 30 °C. Strains were maintained on HM medium (Ventosa et al., 1982) supplemented with 5 % (w/v) sea-salt solution (referred to here as HM 5 % sea salts medium) (Rodríguez-Valera et al., 1981). The predominant strains present in this wash-water were Gram-positive, endospore-forming bacilli. For this study, of the 180 isolated strains, four were selected on the basis of their similar physiological characteristics.
Cell morphology was examined by light microscopy and transmission electron microscopy using cells from exponentially growing cultures. Endospores were stained according to the Schaeffer–Fulton method (Smibert & Krieg, 1981) from a 10-day culture on HM 5 % sea salts medium. Growth at different temperatures (10, 20, 25, 30, 45, 55, 60 and 70 °C) and pH (2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) was determined on HM 5 % sea salts medium. Growth at different salt contents (0, 3, 5, 10, 15, 20, 25 and 30 %, w/v) was investigated on HM medium. Catalase activity was determined by bubble production in a 10 % (v/v) H2O2 solution. Oxidase activity was determined on filter paper moistened with a 1 % (w/v) aqueous solution of N,N,N',N'-tetramethyl-p-phenylenediamine. Aerobic nitrate and nitrite reduction, methyl red, indole production and Voges–Proskauer tests and hydrolysis of starch, casein, aesculin, gelatin, tyrosine and Tweens 20, 40 and 80 were all assayed according to Claus & Berkeley (1986). Utilization of various substrates as sole carbon and energy sources and acid production from carbohydrates were determined as described previously (Ventosa et al., 1982). Carbohydrates were added at 0.2 % (w/v), organic acids at 0.1 % (w/v) and alcohols at 0.1 % (v/v). Strains were incubated at 30 °C. All assays were performed in duplicate. Antimicrobial susceptibility was tested in HM 5 % sea salts medium according to the method described by Bauer et al. (1966).
Cellular fatty acid analysis was performed from strains grown on liquid HM 5 % sea salts medium for 24 h at 30 °C. Quantitative analysis of cellular fatty acid composition was performed by high-resolution GLC by the DSMZ Identification Service (Braunschweig, Germany).
DNA was purified using the method of Lind & Ursing (1986). The G+C content of genomic DNA was calculated from the midpoint value (Tm) of the thermal denaturation profile (Marmur & Doty, 1962) obtained at 260 nm with a Perkin-Elmer UV-Vis Lambda3B spectrophotometer fitted with a temperature program accessory. The Tm was determined by the graphical method described by Ferragut & Leclerc (1976) and the G+C content was estimated from this temperature using the equation of Owen & Hill (1979).
The 16S rRNA gene was amplified by PCR using standard protocols with primers 16F27 (forward; 5'-AGAGTTTGATCMTGGCTCAG-3') and 16R1525 (reverse; 5'-AAGGAGGTGWTCCARCC-3') (Pharmacia Biotech). PCR products were purified using Microcon-100 concentrators (Amicon) and sequenced directly using an Applied Biosystems 373A DNA sequencer. Taq cycle sequencing was performed with fluorescent dye-labelled dideoxynucleotides (Perkin-Elmer) and the following oligonucleotides: 16F357 (5'-CTCCTACGGGAGGCAGCAG-3'), 16R519 (5'-GWATTACCGCGGCKGCTG-3') and 16F945 (5'-GGGCCCGCACAAGCGGTGG-3').
The sequences obtained were compared with reference 16S rRNA gene sequences available in the GenBank and EMBL databases. A phylogeny analysis was made using MEGA version 3.1 (Kumar et al., 2004) after multiple alignment of the data by CLUSTAL W (Chenna et al., 2003). Distances and clustering were determined using neighbour-joining and maximum-parsimony algorithms. The stability of the clusters was ascertained by performing a bootstrap analysis (1000 replications). DNA–DNA hybridization was conducted following the method of Lind & Ursing (1986) with the modifications described by Ziemke et al. (1998).
Phenotypic characteristics common to all four strains are given in the species description. Table 1 shows differential phenotypic characteristics between the four isolates and Table 2 shows the characteristics that distinguish strain E308T from other related Virgibacillus species. The major fatty acids (greater than 1 %) found in strain E308T were iso-C14 : 0 (2.05 %), C14 : 0 (1.25 %), iso-C15 : 0 (33.71 %), anteiso-C15 : 0 (28.43 %), C15 : 0 (1.12 %), iso-C16 : 0 (3.93 %), C16 : 1
7c alcohol (1.33 %), C16 : 111c (1.69 %), C16 : 0 (3.07 %), iso-C17 : 0 (10.13 %), anteiso-C17 : 0 (9.21 %), C18 : 19c (1.15 %) and C18 : 0 (1.24 %).
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). The maximum-parsimony algorithm gave a similar result (data available as Supplementary Fig. S1 in IJSEM Online).
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On the basis of the phenotypic, genotypic, chemotaxonomic and phylogenetic data of this study, we conclude that isolates E308T, E5549, I3077 and N30129 are different strains that belong to the same species and constitute a novel species of the genus Virgibacillus, for which the name Virgibacillus olivae sp. nov. is proposed.
Description of Virgibacillus olivae sp. nov.
Virgibacillus olivae (o'li.vae. L. gen. n. olivae of an olive, referring to the isolation of the first strains from olive processing waste-water).
Cells are straight or slightly curved, rounded-ended, Gram-positive rods (1.8x0.26 µm), motile by means of peritrichous flagella, occurring singly or in pairs and occasionally in short chains. Endospore formation is slow; the endospores are nearly spherical or ellipsoidal and appear in a terminal or subterminal position. Colonies on HM 5 % sea salts medium after 24 h at 30 °C are yellow–cream, semi-translucent, oval with regular margins that may spread along the inoculation streak, slightly transparent at the edge. They have glossy surfaces and are 0.5–2 mm in diameter. Grows at 20–45 °C and pH 4–8. Halotolerant, being capable of growing in sea salts concentrations from 0 to 10 % (w/v). Optimum growth at 30 °C, pH 7 and 5 % (w/v) sea salts. Catalase and oxidase are positive. Nitrate and nitrite are reduced. Hydrolyses starch and gelatin, but not tyrosine or Tween 20, 40 or 80. Citrate is not used as a sole carbon and energy source. Indole, Voges–Proskauer and methyl red tests are negative. No growth on cetrimide agar. Able to use cellobiose, D-fructose, D-mannose, L-rhamnose and D-glucose as sole carbon and energy sources but not D-galactose or maltose. Acid is produced from D-fructose but not from D-glucose, cellobiose, D-mannose, sucrose, D-galactose, D-xylose, L-rhamnose, D-mannitol, myo-inositol or adonitol. L-Cysteine, L-histidine, L-methionine and L-valine are not used as sole sources of carbon, nitrogen and energy. Resistant to kanamycin, amikacin, clindamycin, nalidixic acid, polymyxin B, streptomycin and sulfamides. Sensitive to ampicillin, gentamicin, rifampicin, erythromycin, cephalotin and penicillin. The DNA G+C content is 30.7–33.4 mol% (Tm method).
The type strain is E308T (=LMG 23503T=DSM 18098T). The type strain and three other strains were isolated from wastewater of Spanish green olive processing. The description of the type strain is the same as that of the species. Additionally, this strain is able to grow in a medium containing 20 % (w/v) sea salts. Aesculin and casein are hydrolysed. Fumarate and propionate can be used as sole carbon and energy sources. Sensitive to tetracycline and resistant to chloramphenicol. The main fatty acids (making up 81 % of the total) are iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 0 and anteiso-C17 : 0. The DNA G+C content of the type strain is 33.4 mol% (Tm method).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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