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Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, 37134 Verona, Italy
Correspondence
Franco Dellaglio
franco.dellaglio{at}univr.it
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ABSTRACT |
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, as a heterofermentative Lactobacillus species which could be isolated from milk products, sourdough, fermenting plant material, manure, sewage and the mouth and faeces of man. Lactobacillus cellobiosus was first described by Rogosa et al. (1953) and is another heterofermentative Lactobacillus species. They share very similar phenotypic properties and both belong to the subgenus ‘Betabacterium’ Orla-Jensen of Lactobacillus.
Since the 1970s, strains belonging to L. cellobiosus and L. fermentum have been subjected to DNA–DNA hybridization studies, which proved their high relatedness (Vescovo et al., 1979). Despite the evidence that strains ATCC 11739T and ATCC 11740 of L. cellobiosus showed hybridization values higher than 70 % with L. fermentum ATCC 14932T, the two species were both included in the Approved List of Bacterial Names (Skerman et al., 1980). A later DNA–DNA hybridization study (Sriranganathan et al., 1985), in which the strains L. fermentum NCDO 215 and L. cellobiosus NCDO 927 were examined, confirmed the findings of Vescovo et al. (1979), but no reclassification was proposed. On the basis of DNA-relatedness data, Kandler & Weiss (1989) reported L. cellobiosus as a biotype of L. fermentum in Bergey's Manual of Systematic Bacteriology. As a consequence, L. cellobiosus was omitted from the phylogenetic analysis of the genus Lactobacillus (Collins et al., 1991), and the 16S rDNA sequence of L. cellobiosus has been deposited only very recently, under the designation L. fermentum (AJ575812).
Considering other literature, it is clear that the treatment of L. cellobiosus is inconsistent. Pot et al. (1994) reported that the name L. cellobiosus was invalid, while Dellaglio et al. (1994) presented it as a taxon awaiting reclassification, and Hammes & Vogel (1995) did not include it in a discussion of the Lactobacillus species. Nevertheless, no formal reclassification proposal has been presented so far, despite the availability of data supporting the close relationship between L. fermentum and L. cellobiosus. The latter name is validly published, as described in online taxonomic resources (List of Bacterial Names with Standing in Nomenclature, http://www.bacterio.cict.fr/; Bacterial Nomenclature Up-to-date, http://www.dsmz.de/bactnom/bactname.htm).
Strain depositions in major culture collections further complicate the study of the status of the species L. cellobiosus. In the ATCC (http://www.atcc.org), only two strains are available, ATCC 11739T and ATCC 11740, but they are registered as ‘Lactobacillus fermentum Beijerinck deposited as Lactobacillus cellobiosus Rogosa et al.’. In the DSMZ (http://www.dsmz.de), only strain DSM 20055T is available, with the comment ‘pro synon., Lactobacillus fermentum, never formally stated’. The Belgian Co-ordinated Collections of Microorganisms, Bacteria Collection (BCCM/LMG; http://www.belspo.be/bccm), has two strains, LMG 9846T and LMG 11441, corresponding to ATCC 11739T and ATCC 11740, respectively, with the former registered as L. cellobiosus and the latter as L. fermentum. Finally, in the Japan Collection of Microorganisms (JCM; http://www.jcm.riken.go.jp/), four strains are available: JCM 1137, JCM 2766, JCM 2767 and JCM 2768 – all registered as L. fermentum.
In the present study, the phylogenetic placement of L. cellobiosus was determined by the analysis of the 16S rDNA sequences available for the most closely related species. Moreover, for the type strains of L. fermentum and L. cellobiosus, partial sequences for the recA gene were obtained and compared to evaluate better the relatedness of the two species.
Lactobacillus fermentum LMG 6902T and L. cellobiosus DSM 20055T were grown in MRS at 37 and 30 °C, respectively. Cultures were checked for purity, and DNA was extracted by the procedure of Marmur (1961).
Small subunit (16S) rDNA sequences were aligned with the CLUSTAL X program (Thompson et al., 1997). Positions ambiguously aligned, not available or not identified (N in the sequence) were removed from all the sequences. Phylogenetic analysis was performed on the remaining 1366 positions with the TREECON program (Van de Peer & De Wachter, 1994) using Galtier and Gouy distance (Galtier & Gouy, 1995). The phylogenetic tree inferred for the considered species is shown in Fig. 1. The close relationship of L. cellobiosus and L. fermentum is supported by sequence identity: the two strains share 1361 of 1366 bp, confirming the high genetic relatedness suggested by DNA-hybridization data (Vescovo et al., 1979).
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). Protein-coding genes show greater variability, and recA has proved particularly useful in the differentiation of closely related species with almost identical 16S rDNA sequences, including lactic acid bacteria. Examples are Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus pentosus (Torriani et al., 2001), and Lactobacillus casei, Lactobacillus rhamnosus and Lactobacillus zeae (Felis et al., 2001). Accordingly, partial recA amplicons of about 730 bp were obtained by PCR with the degenerate primers recEXT-f (5'-GGC TAT GAA ACA AAT TGA AAA ACA ATW YGG NAA RGG-3') and recEXT1-r (5'-TGT TTA AAC GGT GGA GCA ACT TTR TTY TTN AC-3'). The PCR mixture (50 µl) was composed of 1x reaction buffer, 2 mM magnesium chloride, 100 µM dNTPs, 1 µM both primers, 0·08 U Taq polymerase µl–1, 5 % (v/v) DMSO and 300 ng template DNA. After an initial denaturation of 5 min at 94 °C, 30 cycles of 1 min at 94 °C, 1 min at 50 °C and 1 min at 72 °C were carried out. A final extension at 72 °C for 7 min was performed. Amplification products of the expected length of about 730 bp were obtained from both type strains. Sequencing reactions were performed at the Biomolecular Research Centre at the University of Padua. Regions of primer annealing were removed and 659 bp were used in further comparisons. A 98 % DNA sequence similarity was obtained, confirming that the two type strains belong to the same taxon. Data previously obtained for the L. casei species group (Felis et al., 2001) suggest that recA-sequence variability within a single Lactobacillus species is very low; therefore, we assumed that similarity values obtained for the type strains of L. cellobiosus and L. fermentum can be extended to all strains belonging to the two species.
Despite the high genetic relatedness, literature data (Rogosa et al., 1953; Rogosa & Hansen, 1971; Dellaglio et al., 1994) suggest that the two taxa differ in several phenotypic traits, as shown in Table 1. This heterogeneity could be an indication of an infraspecific subdivision, which was not deepened due to the unclear attribution and scarcity of strains in culture collections, as explained above.
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), the name L. fermentum is the earlier synonym and the name L. cellobiosus is the later synonym.
Emended description of Lactobacillus fermentum Beijerinck 1901
Cells are rods, 0·5–0·9 µm thick and highly variable in length, occurring singly or in pairs. Heterofermentative strains; they ferment fructose, galactose, glucose, gluconate, lactose, maltose, mannose, melibiose, raffinose, ribose and sucrose. No acid is produced from mannitol, melezitose, rhamnose, salicin or sorbitol. Some strains may produce acid from amygdalin, arabinose, cellobiose, aesculin, trehalose and xylose. Genome G+C content is 52–54 mol%. They produce L- or DL-lactic acid and NH3 from arginine, and may grow at 15 or 45 °C. Isolated from milk products, sourdough, fermenting plant material, manure, sewage and human mouth and faeces.
The type strain is ATCC 14931T (=DSM 20052T=NCDO 1750T=LMG 6902T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Collins, M. D., Rodrigues, U. M., Ash, C., Aguirre, M., Farrow, J. A. E., Martinez-Murcia, A., Phillips, B. A., Williams, A. M. & Wallbanks, S. (1991). Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16S rRNA. FEMS Microbiol Lett 77, 5–12.[CrossRef]
Dellaglio, F., de Roissart, H., Torriani, S., Curk, M. C. & Janssens, D. (1994). Caractéristiques générales des bactéries lactiques, In Bactéries Lactiques, vol. I, pp. 25–116. Edited by H. de Roissart & F. M. Luquet. Uriage, France: Lorica.
Felis, G. E., Dellaglio, F., Mizzi, L. & Torriani, S. (2001). Comparative sequence analysis of a recA gene fragment brings new evidence for a change in the taxonomy of the Lactobacillus casei group. Int J Syst Evol Microbiol 51, 2113–2117.[Abstract]
Fox, G. E., Wisotzkey, J. D. & Jurtshuk, P, Jr. (1992). How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol 42, 166–170.
Galtier, N. & Gouy, M. (1995). Inferring phylogenies from DNA sequences of unequal base compositions. Proc Natl Acad Sci U S A 92, 11317–11321.
Hammes, W. P. & Vogel, R. F. (1995). The genus Lactobacillus. In The Genera of Lactic Acid Bacteria, vol. 2, pp. 19–54. Edited by B. J. B. Wood & W. H. Holzapfel. Glasgow: Blackie Academic & Professional.
Kandler, O. & Weiss, N. (1989). Regular, nonsporeforming, Gram-positive rods. In Bergey's Manual of Systematic Bacteriology, vol. 2, pp. 1208–1234. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
Skerman, V. B. D., McGowan, V. & Sneath, P. H. A. (editors) (1980). Approved lists of bacterial names. Int J Syst Bacteriol 30, 225–420.
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Marmur, J. (1961). A procedure for the isolation of DNA from microorganisms. J Mol Biol 3, 208–218.
Pot, B., Ludwig, W., Kersters, K. & Schleifer, K.-H. (1994). Taxonomy of lactic acid bacteria. In Bacteriocins of Lactic Acid Bacteria: Microbiology, Genetics & Applications, pp. 13–90. Edited by L. De Vuyst & E. J. Vandamme. London: Blackie Academic & Professional.
Rogosa, M. & Hansen, P. A. (1971). Nomenclatural consideration of certain species of Lactobacillus Beijerinck. Request for an opinion. Int J Syst Bacteriol 21, 177–186.
Rogosa, M., Wiseman, R. F., Mitchell, J. A., Disraely, M. N. & Beaman, A. J. (1953). Species differentiation of oral lactobacilli from man including descriptions of Lactobacillus salivarius nov. spec. and Lactobacillus cellobiosus nov. spec. J Bacteriol 65, 681–699.
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Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24, 4876–4882.
Torriani, S., Felis, G. E. & Dellaglio, F. (2001). Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers. Appl Environ Microbiol 67, 3450–3454.
Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570.
Vescovo, M., Dellaglio, F., Bottazzi, V. & Sarra, P. G. (1979). Deoxyribonucleic acid homology among Lactobacillus species of the subgenus Betabacterium Orla-Jensen. Microbiologica 2, 317–330.
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