| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Dipartimento Scientifico e Tecnologico, Facoltà di Scienze MM. FF. NN., Università degli Studi di Verona, Italy
2 Nestlé Research Centre, Nestec Ltd, Lausanne, Switzerland
Correspondence
Franco Dellaglio
franco.dellaglio{at}univr.it
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
A partial phylogenetic tree, AFLPTM and RAPD profiles, and distinguishing characteristics of the subspecies of Lactobacillus delbrueckii are available as supplementary material in IJSEM Online.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). Strains belonging to the three subspecies show a high degree of DNA–DNA relatedness but can be differentiated on the basis of different phenotypic traits (Weiss et al., 1983; Kandler & Weiss, 1989).
In a previous study regarding the evolutionary structure of the species L. delbrueckii (Germond et al., 2003), a number of lactobacilli originating from five dairy fermented products from India were analysed. Among those lactobacilli, four strains isolated from traditional Indian dairy fermented (type Dahi) products were identified as L. delbrueckii on the basis of molecular and phenotypic evidence, i.e. hybridization with a probe specific for L. delbrueckii, pY85 (Delley et al., 1990), typing based on digested genomic DNA probed with ribosomal or ISL7 sequences, specific PCR amplification and the exclusive production of D-lactate from lactose. However, they could not be assigned to one of the known subspecies of L. delbrueckii because they presented atypical characteristics such as the ability of L. delbrueckii subsp. bulgaricus to degrade lactose constitutively, but no amplification of the gene encoding 
-galactosidase using specific primers for L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.
We report here the polyphasic analysis performed to define the taxonomic position of the four strains and describe a novel subspecies for L. delbrueckii.
The bacterial strains NCC725T, NCC665, NCC757 and NCC780 and the type strains of L. delbrueckii subsp. delbrueckii NCC621T (=ATCC 9649T=LMG 6412T), L. delbrueckii subsp. lactis NCC946T (=ATCC 12315T=LMG 7942T) and L. delbrueckii subsp. bulgaricus NCC641T (=ATCC 11842T=LMG 6901T) were grown in de Man–Rogosa–Sharpe (MRS) or acidified MRS (pH 5·4) medium at 37 °C.
Sugar metabolism was evaluated using the API-50CH detection kit. All strains tested fermented glucose, fructose and mannose. The ability to metabolize lactose distinguished the four strains from L. delbrueckii subsp. delbrueckii. These four strains can also be differentiated from L. delbrueckii subsp. lactis because they did not ferment both maltose and trehalose. Three of the four strains (except NCC757) fermented sucrose and not N-acetylglucosamine in contrast to type strains of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis, respectively.
DNA for specific amplification and Southern blot analysis was extracted according to the methods of Marmur (1961) and Germond et al. (2003), respectively. Ribotyping and insertion sequence (IS) typing identification results showed a closer relationship of the four Indian strains to L. delbrueckii subsp. lactis than to the other two recognized subspecies (Germond et al., 2003).
Subspecies-specific identification was performed by DNA amplification using a combination of primers designed on the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus (Torriani et al., 1999): this test allows us to differentiate between L. delbrueckii subsp. lactis and L. delbrueckii subsp. bulgaricus on the basis of the length of the amplification product, while L. delbrueckii subsp. delbrueckii should show no amplification. The four Indian strains were all negative for the assay (data not shown), even if DNA was present at the appropriate concentration and was able to be amplified, as verified by RAPD-PCR analyses.
16S rRNA gene amplifications and sequences were obtained as reported by Germond et al. (2003). Sequence alignments were obtained using the CLUSTAL_X program (Thompson et al., 1997). Phylogenetic analysis was conducted using MEGA version 2.1 (Kumar et al., 2001): distance analysis (Tamura's three-parameter distance) and neighbour-joining clustering were applied, as well as the maximum-parsimony method using default options. An analysis of the robustness of the trees was obtained by a bootstrap approach with 1000 replicates. A maximum-likelihood analysis was also performed with the program DNAML of the PHYLIP software package (Felsenstein, 1993). Accession numbers of the sequences included in the analysis are shown in Fig. A, available as supplementary material in IJSEM Online. The phylogenetic trees obtained strongly confirm that the new isolates should be classified within L. delbrueckii owing to their very low sequence divergence (identity of 99 %); the distance matrix tree (Fig. A) is available as supplementary material in IJSEM Online.
16S rRNA gene sequence similarity is not sufficient to guarantee species identity (Fox et al., 1992). Protein-encoding genes are more variable; in particular, the recA gene has proved useful in the differentiation of closely related species with almost identical 16S rRNA gene sequences, including lactic acid bacteria species such as Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus pentosus (Torriani et al., 2001) or Lactobacillus casei, Lactobacillus rhamnosus and Lactobacillus zeae (Felis et al., 2001). Therefore, part of the recA gene was amplified using the degenerate primers recEXT-f (5'-GGCTATGAAACAAATTGAAAAACAATWYGGNAARGG-3') and recEXT1-r (5'-TGTTTAAACGGTGGAGCAACTTTRTTYTTNAC-3'). The reaction mixture (50 µl) contained 300 ng template DNA, 100 µM each dNTP, 1 µM each primer, 2 mM magnesium chloride, 5 % (v/v) DMSO and 4 U Taq polymerase in a standard reaction buffer. After an initial denaturation of 5 min at 94 °C, 30 cycles of 1 min at 94 °C, 1 min at 50 °C, 1 min at 72 °C and a final extension at 72 °C for 7 min were performed. Amplification products of the expected length of about 730 bp were obtained for all strains tested.
A partial sequence of the hsp60 gene was also amplified using the degenerate primers cpn-f (5'-CTTGGGCCCAAAAGGCMGNAAYGTNGT-3') and cpn-r (5'-CCAACCGTTCTTGCAATTTTTCNCKRTCRAA-3'). The reaction mixture and amplification programme were the same as those reported above, except that an annealing temperature of 55 °C instead of 50 °C was used. Amplicons of the expected length of about 1000 bp were obtained for all the strains. Sequencing reactions were performed at the Biomolecular Research Centre (BMR), University of Padua, Italy. The partial recA and hsp60 sequences obtained for the strains were aligned as described for 16S rRNA gene sequences and showed very low divergence (identity of 99 %).
RAPD analysis was carried out using three different primers: Coc1 (5'-AGCAGCGTGG-3') (Cocconcelli et al., 1995), M13 (5'-GAGGGTGGCGGTTCT-3') (Stendid et al., 1994) and D8635 (5'-GAGCGGCCAAAGGGAGCAGAC-3') (Akopyanz et al., 1992), as described in the respective references except for the addition of 5 % (v/v) DMSO in each reaction mixture. Pattern analyses of the amplification products, separated in agarose gels and stained with ethidium bromide, were performed with the GELCOMPAR software (applied maths) with the DICE coefficient and UPGMA as calculation and clustering options. AFLPTM analysis of the four cultures was performed at the BCCMTM/LMG Bacteria Collection (Laboratorium voor Microbiologie, Universiteit Gent, Belgium). Comparison of AFLPTM (Fig. 1 and Fig. B, available as supplementary material in IJSEM Online) and RAPD profiles (Fig. C, available as supplementary material in IJSEM Online) demonstrated the genomic coherence of the four strains, their non-clonality and their close relationship with other strains of the species L. delbrueckii.
|
). These data demonstrated that the four strains belong to the same taxon, even though, owing to the standard error of the technique, the relative order of the relationships could not be evaluated.
DNA–DNA hybridization values, and 16S rRNA, recA and hsp60 gene sequence analysis, confirm that the four strains constitute a homogeneous cluster belonging to the species L. delbrueckii. However, data obtained here and in Germond et al. (2003) suggest that they cannot be assigned to any of the three recognized subspecies of L. delbrueckii: (i) ARDRA (Germond et al., 2003) and subspecies-specific PCR data support their differentiation from L. delbrueckii subsp. lactis and L. delbrueckii subsp. bulgaricus; (ii) phenotypic traits distinguish them from L. delbrueckii subsp. delbrueckii (lactose fermentation) and from L. delbrueckii subsp. lactis (maltose and trehalose metabolism) (see Table A, available as supplementary material in IJSEM Online); (iii) lactose degradation is constitutive in the four strains but is regulated in L. delbrueckii subsp. lactis (Lapierre et al., 2002); and (iv) Lactobacillus delbrueckii subsp. bulgaricus is also able constitutively to degrade lactose but the four strains are negative for the specific PCR amplification for -galactosidase (Germond et al., 2003). This evidence suggests that the four strains possess a unique genetic equipment for lactose degradation, which differs from that of the other recognized subspecies. Characterization of this equipment is beyond the scope of the present study, but it does provide proof that the four strains represent a separate subspecies. Moreover, molecular analysis, such as AFLPTM and RAPD-PCR, indicated that the four strains represent a genomic, coherent, non-clonal distinct group within the species L. delbrueckii. The data presented highlight several peculiarities to the four strains (probably a result of their geographical isolation) that fulfil the requirements for the description of a new subspecies (Rosselló-Mora & Amann, 2001) within L. delbrueckii. This description may avoid future incorrect identification of ‘atypical’ isolates of L. delbrueckii species. The name Lactobacillus delbrueckii subsp. indicus subsp. nov. is proposed for these strains. Owing to the high overall similarity with the other strains of the species, an emended description of L. delbrueckii is not required, except that the species now comprises four subspecies.
Description of Lactobacillus delbrueckii subsp. indicus subsp. nov.
Lactobacillus delbrueckii subsp. indicus (in'di.cus. L. masc. adj. indicus from India, referring to the geographical origin of the strains).
Cells are Gram-positive, catalase-negative rods with rounded ends, generally 0·5–0·8 by 2–9 µm, occurring singly, in pairs and in short chains. Non-motile. Homofermentative, produces D-lactic acid exclusively. Growth occurs at 45 °C but not at 15 °C. Glucose, fructose, mannose and lactose are fermented by all strains, while fermentation of sucrose and N-acetylglucosamine is more variable. Glycerol, erythritol, arabinose, ribose, xylose, adonitol, methyl -D-xyloside, galactose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl 
-D-mannoside, methyl -D-glucoside, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, melibiose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, fucose, arabitol, gluconate, 2-ketogluconate and 5-ketogluconate are not fermented. Arginine is not deaminated. Growth is observed in MRS plus 2·5 % (w/v) NaCl, except for strain NCC780; none of the strains grows in MRS plus 5 % (w/v) NaCl. All the strains grow in MRS at pH 3, 4 and 5, but not at pH 7·8. Metachromatic granules are often observed after methylene blue staining.
The type strain, NCC725T (=LMG 22083T=DSM 15996T), was isolated from a traditional Indian dairy fermented (type Dahi) product.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Cocconcelli, P. S., Porro, D., Galandini, S. & Senini, L. (1995). Development of RAPD protocol for typing of strains of lactic acid bacteria and enterococci. Lett Appl Microbiol 21, 376–379.[Medline]
Delley, M., Mollet, B. & Hottinger, H. (1990). DNA probe for Lactobacillus delbrueckii. Appl Environ Microbiol 56, 1967–1970.
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]
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version 3.5c. Department of Genetics, University of Washington, Seattle, USA.
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.
Germond, J.-E., Lapierre, L., Delley, M., Mollet, B., Felis, G. E. & Dellaglio, F. (2003). Evolution of the bacterial species Lactobacillus delbrueckii: a partial genomic study with reflections on prokaryotic species concept. Mol Biol Evol 20, 93–104.
Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998). Evaluation of a microplate DNA–DNA hybridisation method compared with the initial renaturation method. Can J Microbiol 44, 1148–1153.[CrossRef]
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 and J. G. Holt. Baltimore: Williams & Wilkins.
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 1244–1245.
Lapierre, L., Mollet, B. & Germond, J.-E. (2002). Regulation and adaptive evolution of lactose operon expression in Lactobacillus delbrueckii. J Bacteriol 184, 928–935.
Marmur, J. (1961). A procedure for the isolation of DNA from microorganisms. J Mol Biol 3, 208–218.
Rosselló-Mora, R. & Amann, R. (2001). The species concept for prokaryotes. FEMS Microbiol Rev 25, 39–67.[Medline]
Stendid, G., Karlsson, J. O. & Hogberg, N. (1994). Intraspecific genetic variation in Heterobasidion annosum revealed by amplification of minisatellite DNA. Mycol Res 98, 57–63.
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 25, 4876–4882.
Torriani, S., Zapparoli, G. & Dellaglio, F. (1999). Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. Appl Environ Microbiol 65, 4351–4356.
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.
Weiss, N., Schillinger, U. & Kandler, O. (1983). Lactobacillus lactis, Lactobacillus leichmannii and Lactobacillus bulgaricus, subjective synonyms of Lactobacillus delbrueckii subsp. lactis comb. nov. and Lactobacillus delbrueckii subsp. bulgaricus comb. nov. Syst Appl Microbiol 4, 552–557.
This article has been cited by other articles:
|
|
 |
|
  A. Endo and S. Okada Reclassification of the genus Leuconostoc and proposals of Fructobacillus fructosus gen. nov., comb. nov., Fructobacillus durionis comb. nov., Fructobacillus ficulneus comb. nov. and Fructobacillus pseudoficulneus comb. nov. Int J Syst Evol Microbiol, September 1, 2008; 58(9): 2195 - 2205. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Li, E. Raftis, C. Canchaya, G. F. Fitzgerald, D. van Sinderen, and P. W. O'Toole Polyphasic analysis indicates that Lactobacillus salivarius subsp. salivarius and Lactobacillus salivarius subsp. salicinius do not merit separate subspecies status. Int J Syst Evol Microbiol, October 1, 2006; 56(Pt 10): 2397 - 2403. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Dellaglio, M. Vancanneyt, A. Endo, P. Vandamme, G. E. Felis, A. Castioni, J. Fujimoto, K. Watanabe, and S. Okada Lactobacillus durianis Leisner et al. 2002 is a later heterotypic synonym of Lactobacillus vaccinostercus Kozaki and Okada 1983. Int J Syst Evol Microbiol, August 1, 2006; 56(Pt 8): 1721 - 1724. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. E. Felis, M. Vancanneyt, C. Snauwaert, J. Swings, S. Torriani, A. Castioni, and F. Dellaglio Reclassification of Lactobacillus thermotolerans Niamsup et al. 2003 as a later synonym of Lactobacillus ingluviei Baele et al. 2003. Int J Syst Evol Microbiol, April 1, 2006; 56(Pt 4): 793 - 795. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Naser, K. E. Hagen, M. Vancanneyt, I. Cleenwerck, J. Swings, and T. A. Tompkins Lactobacillus suntoryeus Cachat and Priest 2005 is a later synonym of Lactobacillus helveticus (Orla-Jensen 1919) Bergey et al. 1925 (Approved Lists 1980) Int J Syst Evol Microbiol, February 1, 2006; 56(2): 355 - 360. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
