IJSEM IJSEM eTOCs
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Silva, L. R.
Right arrow Articles by Velázquez, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Silva, L. R.
Right arrow Articles by Velázquez, E.
Agricola
Right arrow Articles by Silva, L. R.
Right arrow Articles by Velázquez, E.
Int J Syst Evol Microbiol 56 (2006), 21-24; DOI  10.1099/ijs.0.46000-0
© 2006 International Union of Microbiological Societies

Acetobacter oeni sp. nov., isolated from spoiled red wine

Luis R. Silva1,2, Ilse Cleenwerck3, Raúl Rivas1, Jean Swings3,4, Martha E. Trujillo1, Anne Willems4 and Encarna Velázquez1

1 Departamento de Microbiología y Génetica, Universidad de Salamanca, Lab. 209, Edificio Departamental de Biología, Campus M. Unamuno, 37007 Salamanca, Spain
2 Departamento das Industrias Agro-Alimentares, Escola Superior Agrária de Viseu, Portugal
3 BCCM/LMG Bacteria Collection, Laboratory of Microbiology, Ghent University, Belgium
4 Faculty of Sciences, Laboratory of Microbiology, Ghent University, Belgium

Correspondence
Encarna Velázquez
evp{at}gugu.usal.es


   ABSTRACT
TOP
 ABSTRACT
MAIN TEXT
 REFERENCES
 
A bacterial strain, designated B13T, was isolated from spoiled red wine from the Dão region, Portugal. The strain was Gram-negative, strictly aerobic, rod-shaped and motile. Phylogenetic analysis on the basis of 16S rRNA gene sequences indicated that B13T belonged to the genus Acetobacter within the Alphaproteobacteria. The closest related species was Acetobacter aceti, with 98·4 % 16S rRNA gene sequence similarity. DNA–DNA hybridization showed that B13T constituted a taxon separate from the Acetobacter species with validly published names. The DNA G+C content of B13T was 58·1 mol%. Phenotypic characteristics of B13T allowed its differentiation from the recognized Acetobacter species. B13T produced 5-ketogluconic acid from glucose, but no 2-ketogluconic acid. It produced catalase, but no oxidase. It utilized glycerol, but not maltose, ethanol or methanol as carbon sources. On the basis of the results obtained, B13T represents a novel species for which the name Acetobacter oeni sp. nov. is proposed. The type strain is B13T (=LMG 21952T=CECT 5830T).

Published online ahead of print on 26 August 2005 as DOI 10.1099/ijs.0.64000-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Acetobacter oeni sp. nov. B13T is AY829472.


   MAIN TEXT
TOP
 ABSTRACT
 MAIN TEXT
REFERENCES
 
The genus Acetobacter currently comprises 23 species with validly published names, nine of which have been subsequently transferred to other genera. The Acetobacter species were delineated mainly on the basis of DNA–DNA relatedness and phylogenetic relationships (Sokollek et al., 1998Go; Lisdiyanti et al., 2000Go, 2001Go; Cleenwerck et al., 2002Go).

During a study of red wine from the Dão region, Portugal, that had been spoiled due to the presence of volatile phenols produced by Dekkera bruxellensis (Silva et al., 2004Go), a bacterial strain, B13T, was recovered. The strain was isolated by spreading 1 ml spoiled red wine samples aseptically on YEDC plates (l–1: 7 g glucose, 3 g yeast extract, 20 g calcium carbonate and 17 g agar) where it grew well and produced large haloes due to the solubilization of calcium carbonate by the large amounts of acid secreted by the strain. On this agar, colonies of the novel isolate were mucoid and opaque cream.

Strain B13T was grown on nutrient agar for 48 h at 22 °C to check for motility by phase-contrast microscopy. Gram staining of cells was carried out according to the classical Gram procedure described by Doetsch (1981)Go. Strain B13T was a Gram-negative, rod-shaped and motile organism with peritrichous flagella (0·8–0·9x3·6–5·1 µm).

DNA for 16S rRNA gene sequencing was extracted as described by Rivas et al. (2001)Go. Amplification and sequencing of the nearly complete 16S rRNA gene were performed as described previously (Rivas et al., 2003Go). The sequence determined was compared with sequences from GenBank using the BLAST program (Altschul et al., 1990Go). Phylogenetic analysis was performed using the BIONUMERICS 4.0 software package (Applied Maths). The sequence determined was aligned with similar sequences retrieved from the EMBL database. Nucleotide substitution rates were calculated using the two-parameter method of Kimura (1980)Go. The phylogenetic tree was inferred using the neighbour-joining method (Saitou & Nei, 1987Go). Bootstrap analysis based on 1000 replications was undertaken to test the robustness of the phylogenetic tree (Felsenstein, 1985Go).

The nearly complete 16S rRNA gene sequence of B13T (1484 nucleotides) was obtained. B13T was located in the Alphaproteobacteria, within the genus Acetobacter. The sequence similarities of B13T to the type strains of Acetobacter aceti, Acetobacter tropicalis, Acetobacter estunensis, Acetobacter cerevisiae, Acetobacter indonesiensis, Acetobacter malorum, Acetobacter orleanensis, Acetobacter orientalis, Acetobacter cibinongensis, Acetobacter syzygii, Acetobacter lovaniensis, Acetobacter pomorum, Acetobacter pasteurianus and Acetobacter peroxydans were 98·3, 98·1, 98·0, 97·9, 97·9, 97·8, 97·6, 97·6, 97·5, 97·4, 97·1, 97·0, 96·7 and 96·4 % respectively. Fig. 1Go shows the phylogenetic position of strain B13T within the genus Acetobacter.



View larger version (49K):
[in this window]
[in a new window]
  		Fig. 1. Neighbour-joining tree based on nearly complete 16S rRNA gene sequences of Acetobacter oeni B13T and other related species of the family Acetobacteraceae. The significance of each branch is indicated by a bootstrap value (%) calculated for 1000 subsets. Bar, 1 substitution per 100 nucleotides.

 
DNA for DNA–DNA hybridizations and DNA base composition analysis was prepared by the method of Wilson (1987)Go with minor modifications (Cleenwerck et al., 2002Go). DNA–DNA hybridizations were performed using a modification of the microplate method described by Ezaki et al. (1989)Go (Goris et al., 1998Go; Cleenwerck et al., 2002Go). The hybridization temperature was 46 °C. Reciprocal reactions (e.g. AxB and BxA) were performed and the variation between them was within the limits of this method (Goris et al., 1998Go). The DNA–DNA relatedness percentages presented are the means of a minimum of four hybridization experiments, including the reciprocal reactions. Strain B13T showed low DNA–DNA relatedness (<= 20 %) with the type strains of A. aceti (13 %), A. tropicalis (14 %), A. estunensis (20 %), A. cerevisiae (13 %), A. indonesiensis (7 %), A. malorum (5 %), A. orleanensis (6 %), A. orientalis (6 %), A. cibinongensis (7 %), A. syzygii (10 %), A. lovaniensis (15 %), A. pomorum (12 %), A. pasteurianus (16 %) and A. peroxydans (13 %). The DNA G+C content was determined by HPLC according to the method of Mesbah et al. (1989)Go. Non-methylated phage lambda DNA (Sigma) was used as the calibration reference. The DNA G+C content of B13T was 58·1 mol%.

Phenotypic characteristics were examined as described by Cleenwerck et al. (2002)Go. The phenotypic characteristics of B13T are given in the species description. Phenotypic characteristics that differentiate B13T from recognized Acetobacter species are given in Table 1Go. Strain B13T can be differentiated from the other species of the genus Acetobacter on the basis of 2- and/or 5-ketogluconic acid production from D-glucose, which are the main diagnostic characters for this genus. The ability of the strain to grow in the presence of 10 % ethanol also distinguishes strain B13T from most Acetobacter species. Moreover, strain B13T differs from the closest phylogenetically related species, A. aceti and A. estunensis, as it is unable to grow with ammonium as the sole nitrogen source with ethanol as the carbon source.


View this table:
[in this window]
[in a new window]
  		Table 1. Differential characteristics of Acetobacter oeni B13T compared with closely related species

Taxa: 1, A. cerevisiae (four strains); 2, A. malorum LMG 1746T; 3, A. pasteurianus (seven strains); 4, A. pomorum LMG 18848T; 5, A. peroxydans (two strains); 6, A. lovaniensis LMG 1617T; 7, A. orleanensis (four strains); 8, A. indonesiensis (two strains); 9, A. tropicalis (two strains); 10, A. estunensis (three strains); 11, A. aceti (four strains); 12, A. syzygii LMG 21419T; 13, A. cibinongensis LMG 21418T; 14, A. orientalis LMG 21417T; 15, A. oeni B13T. +, Positive; –, negative; W, weakly positive; V, variable; ND, not determined. Data for taxa 1–11 were taken from Cleenwerck et al. (2002)Go; data for taxa 12–15 were obtained in this study.

 
The results presented above allow the genotypic and phenotypic differentiation of B13T from the 14 Acetobacter species with validly published names. Strain B13T should therefore be classified as representing a novel species for which we propose the name Acetobacter oeni sp. nov.

Description of Acetobacter oeni sp. nov.
Acetobacter oeni (oe'ni. N.L. gen. n. oeni of wine from Gr. gen. n. oinou of wine).

Cells are motile, non-spore-forming rods, 3·6–5·1 µm in length and 0·8–0·9 µm in diameter. Gram-negative, strictly aerobic. The optimal growth temperature on YEDC is 28 °C. Colonies on YEDC are circular, convex, cream, opaque and usually 1–2 mm in diameter within 4 days growth at 28 °C. Produces 5-keto-D-gluconic acid from D-glucose but no 2-keto-D-gluconic acid. Oxidizes D-glucose in media containing ammonium nitrate as the nitrogen source, but is unable to ferment D-glucose in the same media. Produces catalase, but no oxidase. Unable to grow with ammonium as the nitrogen source with ethanol as carbon source. Growth in presence of 10 % ethanol. Utilizes glycerol as a carbon source, but not maltose or methanol. No growth in the presence of 30 % D-glucose. DNA G+C content of 58·1 mol%.

The type strain, B13T (=LMG 21952T=CECT 5830T), was isolated from spoiled red wine of the Dão region, Portugal.


   ACKNOWLEDGEMENTS
 
This work was supported by CAICYT-DGES and JCyL (Spanish Government). We wish to thank K. Engelbeen for her technical assistance with the DNA–DNA hybridizations and phenotypic tests. A. W. is grateful to the Fund for Scientific Research – Flanders for a postdoctoral fellowship.


   REFERENCES
TOP
 ABSTRACT
 MAIN TEXT
 REFERENCES
 
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef][Medline]

Cleenwerck, I., Vandemeulebroecke, K., Janssens, D. & Swings, J. (2002). Re-examination of the genus Acetobacter, with descriptions of Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. Int J Syst Evol Microbiol 52, 1551–1558.[Abstract]

Doetsch, R. N. (1981). Determinative methods of light microscopy. In Manual of Methods for General Bacteriology, pp. 21–33. Edited by P. Gerdhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg & G. B. Phillips. Washington, DC: American Society for Microbiology.

Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.[Abstract/Free Full Text]

Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]

Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998). Evaluation of a microplate DNA-DNA hybridization method compared with the initial renaturation method. Can J Microbiol 44, 1148–1153.[CrossRef]

Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]

Lisdiyanti, P., Kawasaki, H., Seki, T., Yamada, Y., Uchimura, T. & Komagata, K. (2000). Systematic study of the genus Acetobacter with descriptions of Acetobacter indonesiensis sp. nov., Acetobacter tropicalis sp. nov., Acetobacter orleanensis (Henneberg 1906) comb. nov., Acetobacter lovaniensis (Frateur 1950) comb. nov., and Acetobacter estunensis (Carr 1958) comb. nov. J Gen Appl Microbiol 46, 147–165.

Lisdiyanti, P., Kawasaki, H., Seki, T., Yamada, Y., Uchimura, T. & Komagata, K. (2001). Identification of Acetobacter strains isolated from Indonesian sources, and proposals of Acetobacter syzygii sp. nov., Acetobacter cibinongensis sp. nov., and Acetobacter orientalis sp. nov. J Gen Appl Microbiol 47, 119–131.

Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.

Rivas, R., Velázquez, E., Valverde, A., Mateos, P. F. & Martínez-Molina, E. (2001). A two primers random amplified polymorphic DNA procedure to obtain polymerase chain reaction fingerprints of bacterial species. Electrophoresis 22, 1086–1089.[CrossRef][Medline]

Rivas, R., Willems, A., Subba-Rao, N. S., Mateos, P. F., Kroppenstedt, R., Martínez-Molina, E., Gillis, M. & Velázquez, E. (2003). Description of Devosia neptuniae sp. nov. that nodulates and fixes nitrogen in symbiosis with Neptunia natans, an aquatic legume from India. Syst Appl Microbiol 26, 47–53.[CrossRef][Medline]

Saitou, N. & Nei, M. (1987). The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]

Silva, L. R., Andrade, P. B., Valentão, P., Seabra, R. M., Trujillo, M. E. & Velázquez, E. (2004). Analysis of non-coloured phenolics in red wine: effect of Dekkera bruxellensis yeast. Food Chem 89, 185–189.

Sokollek, S. J., Hertel, C. & Hammes, W. P. (1998). Description of Acetobacter oboediens sp. nov. and Acetobacter pomorum sp. nov., two new species isolated from industrial vinegar fermentations. Int J Syst Bacteriol 48, 935–940.[Abstract/Free Full Text]

Wilson, K. (1987). Preparation of genomic DNA from bacteria. In Current Protocols in Molecular Biology, pp. 2.4.1–2.4.5. Edited by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith & K. Struhl. New York: Green Publishing & Wiley-Interscience.




This article has been cited by other articles:


Home page
 Int. J. Syst. Evol. Microbiol.Home page
I. Cleenwerck, A. Gonzalez, N. Camu, K. Engelbeen, P. De Vos, and L. De Vuyst
Acetobacter fabarum sp. nov., an acetic acid bacterium from a Ghanaian cocoa bean heap fermentation
Int J Syst Evol Microbiol, September 1, 2008; 58(9): 2180 - 2185.
[Abstract] [Full Text] [PDF]

Home page
 Int. J. Syst. Evol. Microbiol.Home page
B. Ndoye, I. Cleenwerck, K. Engelbeen, R. Dubois-Dauphin, A. T. Guiro, S. Van Trappen, A. Willems, and P. Thonart
Acetobacter senegalensis sp. nov., a thermotolerant acetic acid bacterium isolated in Senegal (sub-Saharan Africa) from mango fruit (Mangifera indica L.)
Int J Syst Evol Microbiol, July 1, 2007; 57(7): 1576 - 1581.
[Abstract] [Full Text] [PDF]

Home page
 Int. J. Syst. Evol. Microbiol.Home page
I. Cleenwerck, N. Camu, K. Engelbeen, T. De Winter, K. Vandemeulebroecke, P. De Vos, and L. De Vuyst
Acetobacter ghanensis sp. nov., a novel acetic acid bacterium isolated from traditional heap fermentations of Ghanaian cocoa beans
Int J Syst Evol Microbiol, July 1, 2007; 57(7): 1647 - 1652.
[Abstract] [Full Text] [PDF]

Home page
 Int. J. Syst. Evol. Microbiol.Home page
D. Dutta and R. Gachhui
Novel nitrogen-fixing Acetobacter nitrogenifigens sp. nov., isolated from Kombucha tea.
Int J Syst Evol Microbiol, August 1, 2006; 56(Pt 8): 1899 - 1903.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Silva, L. R.
Right arrow Articles by Velázquez, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Silva, L. R.
Right arrow Articles by Velázquez, E.
Agricola
Right arrow Articles by Silva, L. R.
Right arrow Articles by Velázquez, E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS