Previous Article | Next Article 
Applied and Environmental Microbiology, August 2001, p. 3450-3454, Vol. 67, No. 8
Dipartimento Scientifico e Tecnologico,
Facoltà di Scienze MM.FF.NN., Università degli Studi di
Verona, 37134 Verona,1 and DOFATA,
Università degli Studi di Catania, 95124 Catania,2 Italy
Received 12 February 2001/Accepted 27 April 2001
In this study, we succeeded in differentiating Lactobacillus
plantarum, Lactobacillus pentosus, and
Lactobacillus paraplantarum by means of
recA gene sequence comparison. Short homologous regions of about 360 bp were amplified by PCR with degenerate consensus primers, sequenced, and analyzed, and 322 bp were considered for the
inference of phylogenetic trees. Phylograms, obtained by parsimony, maximum likelihood, and analysis of data matrices with the
neighbor-joining model, were coherent and clearly separated the three
species. The validity of the recA gene and RecA protein
as phylogenetic markers is discussed. Based on the same sequences,
species-specific primers were designed, and a multiplex PCR protocol
for the simultaneous distinction of these bacteria was
optimized. The sizes of the amplicons were 318 bp for L.
plantarum, 218 bp for L. pentosus, and 107 bp
for L. paraplantarum. This strategy permitted the
unambiguous identification of strains belonging to L.
plantarum, L. pentosus, and L.
paraplantarum in a single reaction, indicating its
applicability to the speciation of isolates of the L.
plantarum group.
The species Lactobacillus
plantarum, Lactobacillus pentosus, and
Lactobacillus paraplantarum are genotypically closely
related and show highly similar phenotypes. The genetic heterogeneity of the L. plantarum group has been demonstrated by Dellaglio
et al. (8) on the basis of DNA-DNA hybridization data:
three groups were identified which were later classified as L. plantarum sensu stricto (2), L. pentosus
(28), and L. paraplantarum
(7).
Despite the importance of these species for fermented foods of plant,
animal, and fish origin, their correct identification is complicated by
the ambiguous response of traditional physiological tests and molecular
methods: Fourier transform infrared spectroscopy of lactobacilli from
breweries was not able to differentiate spectra from L. plantarum and L. pentosus (6). Randomly
amplified polymorphic DNA-PCR and related numerical analysis gave
satisfying results, but the methods were applied only to L. plantarum and L. pentosus (27). Finally,
two papers reported the selection of species-specific PCR primers for
the L. plantarum group; however, in one case specificity towards L. paraplantarum was not checked (21),
and in the other the primers did not guarantee sufficient specificity
(3). Satisfying results have been obtained by
Southern-type hybridization with a pyrDFE probe
(4), randomly amplified polymorphic DNA-PCR, and
AFLP (Keygene NV, Wageningen, The Netherlands) (S. Torriani, F. Clementi, F. Dellaglio, B. Hoste, M. Vancanneyt, and J. J. Swings,
Proc. Sixth Symp. Lactic Acid Bacteria, poster B2, 1999), but such
methods are not suitable for routine identification requirements. The
difficulty of correct identification of these species and the
increasing interest in some of their properties, e.g., probiotic activities (13) and tannin degradation (19),
indicates the need for a simple and reliable molecular method for the
definite differentiation of L. plantarum, L. paraplantarum, and L. pentosus.
PCR using species-specific oligonucleotides designed based on
phylogenetic molecular markers could be a useful approach, since these
molecules are ubiquitous and relatively highly conserved. For this
purpose, 16S ribosomal DNA sequences are not suitable because of the
high identity value (>99%) shared by L. plantarum and
L. pentosus (5, 21). Consequently, the
definition of phylogenetic distances is also not feasible by such a
classical approach for L. plantarum group species. It has
been proposed that the recA gene could be used as a
phylogenetic marker (11, 17), and it has already given
satisfying results for many bacterial genera, including bifidobacteria
(15).
RecA is a small protein (352 amino acids in Escherichia
coli) implicated in homologous DNA recombination, SOS induction,
and DNA damage-induced mutagenesis (11). This panoply of
functions implies multiple biochemical activities, including DNA
binding (double and single stranded), pairing and exchange of
homologous DNA, ATP hydrolysis, and coproteolitic cleavage of the LexA,
In this study, short recA gene sequences were obtained and
analyzed in order to infer a phylogenetic classification scheme, still
lacking for the three species of the L. plantarum group. Moreover, species-specific primers were designed based on the obtained
sequences and were used in a multiplex PCR assay.
Bacterial strains and cultivation.
The bacterial strains
tested for inferring phylogeny were L. paraplantarum LMG
16673T, L. paraplantarum LMG 18402, L. paraplantarum NIRD P2, L. pentosus LMG
10755T, L. pentosus LMG 18401, L. plantarum ATCC 14917T, L. plantarum B41, and L. plantarum NCFB 340.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3450-3454.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Differentiation of Lactobacillus
plantarum, L. pentosus, and L.
paraplantarum by recA Gene Sequence Analysis and
Multiplex PCR Assay with recA Gene-Derived
Primers
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
cI, and UmuD proteins (11). Due to its fundamental
role, the recA gene is ubiquitous, and its gene product has
been proposed as a phylogenetic marker for distantly related species
(11, 17).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-D-thiogalactopyranoside, and 80 µg of
5-bromo-4-chloro-3-indolyl--D-galactopyranoside/ml.
Lactobacilli and E. faecium were grown in MRS broth (Oxoid)
for 16 h at 37°C, B. breve was grown in MRS broth
with 0.05% cysteine added for 48 h at 37°C in anaerobiosis
obtained with Anaerocult A (Merck, Darmstadt, Germany), A. aceti was grown in YPM broth (0.5% yeast extract, 0.3% peptone,
2.5% mannitol; pH not adjusted) for 3 days at 30°C, and L. lactis and S. thermophilus were grown in M17 broth
(Oxoid) for 24 h at 37°C.
DNA preparation. Genomic DNA of lactic acid bacteria was extracted as described by Marmur (18). In genomic DNA extraction from A. aceti the method was modified by the addition of 2× hexadecyltrimethylammonium bromide solution (2% hexadecyltrimethylammonium bromide, 100 mM Tris [pH 8], 20 mM EDTA [pH 8], 1.4 mM NaCl, and 1% polyvinylpyrrolidone), with incubation at 65°C for 10 min.
recA gene fragment amplifications. For the amplification of recA gene regions, 300 ng of chromosomal DNA from each Lactobacillus strain was added to a 50-µl PCR mixture (2.5 U of Sigma-Aldrich REDTaq DNA polymerase in 1× REDTaq reaction buffer, 50 µM each primer, 100 µM deoxynucleotide triphosphates) and submitted to initial denaturation of 1 min at 94°C followed by 30 cycles of 1 min at 94°C, 1 min and 30 s at 55°C, and 1 min at 72°C in a GeneAmp PCR System 2400 (Perkin-Elmer). The primers used for the amplification were 5'-TT(C,T) AT(A,T,C) GA(C,T) GC(A,T,C,G) GA(A,G) CA(C,T) GC-3' (forward primer, corresponding to amino acid region 92 to 98 of the E. coli RecA protein [10]) and 5'-CC(A,T) CC(A,T) G(T,G)(A,T) GT(A,T,C) GT(C,T) TC(A,T,C,G) GG-3'(reverse primer, corresponding to amino acid region 206 to 211 of the E. coli RecA protein [9]). The expected size of the amplicons was about 360 bp.
Amplification products were purified from 2% agarose gel by the QIAEX II Gel Extraction System (Qiagen GmbH, Hilden, Germany).Cloning of PCR products. Three microliters of a suspension containing 30 ng of purified amplicons of recA gene fragments (360 bp) was added to a ligation mixture containing 50 ng of pGEM-T vector (Promega Corp., Madison, Wis.) and 3 U of T4 DNA ligase (Promega) in 1× ligation buffer and incubated overnight at 4°C. Two microliters of the ligation mixture was used to transform E. coli XL1-Blue by electroporation in an E. coli Pulser apparatus (Bio-Rad Laboratories, Richmond, Calif.). Recombinant clones carrying the insert were selected by blue-white screening. Plasmids containing DNA fragments were extracted with the Quiaprep Spin Miniprep kit (Qiagen). The presence of the insert was verified by restriction analysis with enzyme BglII (Takara Shuzo Co., Ltd., Otsu, Shiga, Japan). Restriction was carried out for 1 h at 37°C in 1× reaction buffer with 10 U of enzyme in a final volume of 20 µl.
Sequencing. Sequencing reactions were performed at Centro Genoma Vegetale-ENEA CR Casaccia, Rome, Italy, on purified plasmids with primers T7 and SP6 (both strands for each sequence).
Analysis of sequence data and construction of phylogenetic trees. The identities of sequences obtained were verified by a BLASTX (1) search against major molecular databases. Putative amino acid sequences were deduced by the GeneDoc program version 2.5.000 (K. B. Nicholas and H. B. Nicholas, unpublished data).
The nucleotide and putative amino acid sequences of the recA gene fragments were aligned with the CLUSTAL W program (26). Phylogenetic trees were constructed using programs in the PHYLIP software package version 3.5c. Calculation of distance matrices was carried out by DNADIST and PROTDIST programs on nucleotide and putative amino acid sequences, respectively, using the default models. Trees were inferred using the NEIGHBOR program, which implements the neighbor-joining method of Saitou and Nei (22). Phylogenetic trees from gene sequences were also constructed with the DNAML (maximum likelihood) and DNAPARS (parsimony) programs. The "jumble" option in program menus was always selected to test different sequence orders in inferring phylogenies to minimize the influence of sequence order on the tree topology (16). A bootstrap confidence analysis was performed by generating 1,000 replicated data sets with the SEQBOOT program, constructing trees with DNADIST and NEIGHBOR, and generating the consensus tree with the CONSENSE program (all in the PHYLIP software package version 3.5c).Reference sequences used in phylogenetic analysis. The following bacterial recA gene sequences were tested as outgroups in phylogenetic analysis: accession number M81465 (Acholeplasma laidlawii), AJ006705 (Acidothermus cellulolyticus), M29680 (Anabaena variabilis), AF214780 (Arthrobacter globiformis), X52132 (Bacillus subtilis), AF094756 (B.breve), U61497 (Clostridium perfringens), AJ006707 (Frankia alni), AJ006706 (Geodermatophilus obscurus), AJ286124 (L. paracasei subsp. paracasei NCFB 151T), AJ286116 (Lactobacillus zeae LMG 17315T), M88106 (L. lactis), L22073 (Mycoplasma mycoides), U30293 (Ruminococcus albus), U33924 (Spirulina platensis), L25893 (Staphylococcus aureus), U21934 (Streptococcus pyogenes), and M29495 (Synechococcus sp.).
Multiplex PCR assay. A multiplex PCR assay (20 µl) was performed with the recA gene-based primers paraF (5'-GTC ACA GGC ATT ACG AAA AC-3'), pentF (5'-CAG TGG CGC GGT TGA TAT C-3'), planF (5'-CCG TTT ATG CGG AAC ACC TA-3'), and pREV (5'-TCG GGA TTA CCA AAC ATC AC-3'). The PCR mixture was composed of 1.5 mM MgCl2, the primers paraF, pentF, and pREV (0.25 µM each), 0.12 µM primer planF, 12 µM deoxynucleotide triphosphates (3 µM each), 0.025 U of Taq (Polymed, Florence, Italy)/µl, 1× PCR buffer (Polymed), and 5 ng of DNA/µl. PCRs were performed on a Perkin-Elmer GeneAmp PCR System 2400 with initial denaturation at 94°C for 3 min, 30 cycles of denaturation at 94°C (30 s), annealing at 56°C (10 s), and elongation at 72°C (30 s), and final extension at 72°C for 5 min. The PCR products were visualized on a 2% agarose gel in 1× TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.2) buffer. Purified amplicons were sequenced using the designed multiplex species-specific primers to confirm their identities.
Nucleotide sequence accession numbers. The EMBL-GenBank-DDBJ accession numbers assigned to the recA internal region for the lactic acid bacterial strains investigated in this study are AJ286120 (L. paraplantarum LMG 16673T), AJ271963 (L. paraplantarum LMG 18402), AJ271964 (L. paraplantarum NIRD P2), AJ286118 (L. pentosus LMG 10755T), AJ292254 (L. pentosus LMG 18401), AJ286119 (L. plantarum ATCC 14917T), AJ271962 (L. plantarum B41), and AJ292255 (L. plantarum NCFB 340).
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Identification and alignment of recA sequences. Sequence identities were verified by a BLASTX (1) search against available molecular databases. Moreover, the presence of Asp-100, Tyr-103, Asp-144, and Ser-145 (E. coli numbering) in the putative translation fragments further confirms their identity, since those residues are considered functionally important (25).
In the phylogenetic analysis, sequence fragments of 322 bp were considered, excluding regions of primer annealing. In these regions, point mutations, likely due to primer degeneration, were indeed observed. Sequence alignment did not introduce any gaps, and there were no ambiguous positions that could bias the inference of phylogeny. This was not surprising, since the sequences were derived from closely related species.Phylogenetic analysis.
Different factors known to influence
the phylogenetic analysis, i.e., the choice of outgroup sequences
(24) and bioinformatic methods (14), were
considered. recA gene sequences of several reference
organisms, which are available in major databases, were selected as
outgroups to evaluate the resulting classification scheme. Congruent
results were obtained with all reference sequences tested (data not
shown). An example of a phylogenetic tree obtained with B. subtilis as the outgroup and L. paracasei subsp.
paracasei NCFB 151T and L. zeae LMG 17315T as reference sequences is
shown in Fig. 1.
|
|
Primer design and multiplex PCR assay.
The 322-bp
recA fragment sequences were aligned, and four regions were
selected for the design of species-specific primers. A single reverse
primer (pREV) and three forward primers (paraF, pentF, and planF) were
used in the PCR tests. The designed pREV primer partially overlapped (5 bases; replacing an N with a G) the degenerate primer used by Duwat et
al. (9). Numbering the fragment sequences from 1 to 322, the forward specific primers were located as follows: planF, the
specific primer for L. plantarum, nucleotide positions 9 to
28; pentF, the specific primer for L. pentosus, nucleotide
positions 109 to 127; and paraF, the specific primer for L. paraplantarum, nucleotide positions 220 to 239. Consequently, the
expected sizes of the amplicons were 318 bp for L. plantarum, 218 bp for L. pentosus, and 107 bp for
L. paraplantarum. To avoid the presence of aspecific bands,
the multiplex PCR protocol was optimized in relation to the temperature
and time of annealing and the relative concentrations of the primers.
The amplification of DNA from all of the L. plantarum, L. pentosus, and L. paraplantarum strains produced only
the expected product, while none of the negative controls produced any
amplicon. The identities of the three fragments were confirmed by
sequencing. An example of the results is shown in Fig.
2.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Phylogenetic analysis of partial recA gene sequences permitted a clear distinction among L. plantarum, L. pentosus, and L. paraplantarum. This assignment relies only on sequence properties, since results produced by several outgroups and different methods prove the robustness of recA phylogenetic analysis. The high bootstrap values obtained are much more significant considering that the recA sequences of two facultatively heterofermentative lactobacilli belonging to the same 16S rRNA phylogenetic group were included in the analysis.
From the comparative analysis of gene sequences and those of the corresponding amino acids, some particularities emerged concerning nucleotide substitutions and identity values. Nearly all nucleotide mutations are synonymous substitutions; consequently, identity values for gene sequences are variable and intraspecific groups are clearly distinguishable, while for amino acid sequences there is little variability. A maximum of three nonsynonymous nucleotide substitutions occurred, which produced the following amino acid replacements: isoleucine (L. paraplantarum strains) instead of valine at amino acid position 4, isoleucine (L. paraplantarum LMG 18402) instead of threonine at position 23, and alanine (L. plantarum B41) instead of valine at position 66. The sole nonconservative substitution was the introduction of isoleucine with a nonpolar side chain instead of threonine with a polar one. The remaining substitutions involved exchanges between nonpolar amino acids.
From a taxonomic point of view, our results confirm that short recA gene sequences have high discriminating power for species difficult to differentiate, as already demonstrated for Bifidobacterium infantis and Bifidobacterium longum (15). At present, the most reliable method for the definition of a species is DNA-DNA hybridization, and it has also been stated that there is a correspondence between these data and the degree of identity of 16S rRNA sequences (23). It is generally accepted that species having 70% or greater DNA similarity usually have more than 97% 16S rRNA sequence identity (23). On the other hand, the identity of 16S rRNA sequences may not be sufficient to guarantee that two taxa belong to the same species (12). This suggests that 16S ribosomal DNA sequence analysis is not the appropriate method to replace DNA reassociation to delineate species and measure intraspecies relationships (23). In the present study, the species rank was indicated by a range of recA identity values of 83.5 to 86.7%, which corresponds to DNA hybridization homologies of 15 to 56% (7). In light of our results and those of previous studies (e.g., reference 15), the recA gene can be proposed as a new method for inferring relationships among very closely related species. recA gene analysis can have a double potential as a phylogenetic marker: its amino acid sequence can be used to infer phylogenies between distantly related taxa, thus avoiding the disadvantages of rRNAs, e.g., overestimation of the relatedness of species with similar nucleotide frequencies, nonindependence of substitution patterns at different sites, and bias derived from different GC contents of the organisms (11). Furthermore, the nucleotide sequence can be used to evaluate phylogenetic relationships and relative taxonomic positions of recently diverged species with similar GC contents and nucleotide frequencies. In fact, at the nucleotide level, recA can tolerate mutations which do not or only slightly alter its product. These mutations give us information about recent evolutionary history, which is too recent to be fixed in slowly diverging sequences like 16S rRNAs (20).
By exploiting the variable nucleotide regions of recA gene fragments, we have also succeeded in designing a set of species-specific PCR primers. These can be used separately as primer pairs in different species-specific PCRs but also in a multiplex assay. The optimization of this multiplex PCR makes it possible to assign one isolate to a definite species of the L. plantarum group in a single step rather than with three distinct reactions. This assay is rapid (less than 2 h), reliable for its high-specificity reaction conditions, and complete, since all three species of the group are investigated.
The clear distinction obtained with short gene sequences validates the possibility of using the recA gene as a phylogenetic-taxonomic marker for closely related species and opens new possibilities for a rapid and reliable identification of lactic acid bacteria of importance for food.
| |
ACKNOWLEDGMENTS |
|---|
We are grateful to Luca Mizzi, Dipartimento di Genetica e di Biologia dei Microrganismi, Università di Milano, Milan, Italy, for his skillful assistance in phylogenetic analysis and to Franca Rossi for valuable revision of the manuscript.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Dipartimento Scientifico e Tecnologico, Facoltà di Scienze MM.FF.NN., Università degli Studi di Verona, Strada Le Grazie, 15, Ca' Vignal 2-37134 Verona, Italy. Phone: 39 045 8027917. Fax: 39 045 8027928. E-mail: dellaglio{at}sci.univr.it.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. |
Altschul, S. F.,
T. L. Madden,
A. A. Schäffer,
J. Zhang,
Z. Zhang,
W. Miller, and D. J. Lipman.
1997.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res.
25:3389-3402 |
| 2. | Bergey, D. H., F. C. Harrison, R. S. Breed, B. W. Hammer, and F. M. Huntoon. 1923. Lactobacillus plantarum,, p. 250. In R. E. Buchanan, and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 1st ed. The Williams & Wilkins Co., Baltimore, Md. |
| 3. | Berthier, F., and S. D. Ehrlich. 1998. Rapid species identification within two groups of closely related lactobacilli using PCR primers that target the 16S/23S rRNA spacer region. FEMS Microbiol. Lett. 161:97-106[CrossRef][Medline]. |
| 4. |
Bringel, F.,
M.-C. Curk, and J.-C. Hubert.
1996.
Characterization of lactobacilli by Southern-type hybridization with a Lactobacillus plantarum pyrDFE probe.
Int. J. Syst. Bacteriol.
46:588-594 |
| 5. | Collins, M. D., U. M. Rodrigues, C. Ash, M. Aguirre, J. A. E. Farrow, A. Martinez-Murcia, B. A. Phillips, A. M. Williams, and S. Wallbanks. 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]. |
| 6. | Curk, M.-C., F. Peladan, and J.-C. Hubert. 1994. Fourier transform infrared (FTIR) spectroscopy for identifying Lactobacillus species. FEMS Microbiol. Lett. 123:241-248[CrossRef]. |
| 7. |
Curk, M.-C.,
J.-C. Hubert, and F. Bringel.
1996.
Lactobacillus paraplantarum sp. nov., a new species related to Lactobacillus plantarum.
Int. J. Syst. Bacteriol.
46:595-598 |
| 8. | Dellaglio, F., V. Bottazzi, and M. Vescovo. 1975. Deoxyribonucleic acid homology among Lactobacillus species of the subgenus Streptobacterium Orla-Jensen. Int. J. Syst. Bacteriol. 25:160-172. |
| 9. |
Duwat, P.,
S. D. Ehrlich, and A. Gruss.
1992.
Use of degenerate primers for polymerase chain reaction cloning and sequencing of the Lactococcus lactis subsp. lactis recA gene.
Appl. Environ. Microbiol.
58:2674-2678 |
| 10. |
Dybvig, K.,
S. K. Hollingshead,
D. G. Heath,
D. B. Clewell,
F. Sun, and A. Woodard.
1992.
Degenerate oligonucleotide primers for enzymatic amplification of recA sequences from gram-positive bacteria and mycoplasmas.
J. Bacteriol.
174:2729-2732 |
| 11. | Eisen, J. A. 1995. The RecA protein as a model molecule for the molecular systematic studies of bacteria: comparison of trees of RecAs and 16S RNA from the same species. J. Mol. Evol. 41:1105-1123[Medline]. |
| 12. |
Fox, G. E.,
J. D. Wisotzkey, and P. Jurtshuk.
1992.
How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity.
Int. J. Syst. Bacteriol.
42:166-170 |
| 13. | Herias, M. V., C. Hessle, E. Telemo, T. Midtvedt, L. A. Hanson, and A. E. Wold. 1999. Immunomodulatory effects of Lactobacillus plantarum colonizing the intestine of gnotobiotic rats. Clin. Exp. Immunol. 116:283-290[CrossRef][Medline]. |
| 14. | Kim, J. 1993. Improving the accuracy of phylogenetic estimation by combining different methods. Syst. Biol. 42:331-340[CrossRef]. |
| 15. | Kullen, M. J., L. J. Brady, and D. J. O'Sullivan. 1997. Evaluation of using a short region of the recA gene for the rapid and sensitive speciation of dominant bifidobacteria in the human large intestine. FEMS Microbiol. Lett. 154:377-383[Medline]. |
| 16. | Lake, J. A. 1991. The order of sequence alignment can bias the selection of tree topology. Mol. Biol. Evol. 8:378-385[Medline]. |
| 17. | Lloyd, A. T., and P. M. Sharp. 1993. Evolution of the recA gene and the molecular phylogeny of bacteria. J. Mol. Evol. 37:399-407[Medline]. |
| 18. | Marmur, J. 1961. A procedure for the isolation of DNA from microorganisms. J. Mol. Biol. 3:208-218. |
| 19. |
Osawa, R.,
K. Kuroiso,
S. Goto, and A. Shimizu.
2000.
Isolation of tannin-degrading lactobacilli from humans and fermented foods.
Appl. Environ. Microbiol.
66:3093-3097 |
| 20. |
Palys, T.,
L. K. Nakamura, and F. M. Cohan.
1997.
Discovery and classification of ecological diversity in the bacterial world: the role of DNA sequence data.
Int. J. Syst. Bacteriol.
47:1145-1156 |
| 21. | Quere, F., A. Deschamps, and M. C. Urdaci. 1997. DNA probe and PCR-specific reaction for Lactobacillus plantarum. J. Appl. Microbiol. 82:783-790[CrossRef][Medline]. |
| 22. | Saitou, N., and M. Nei. 1987. Reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425[Abstract]. |
| 23. |
Stackebrandt, E., and B. M. Goebel.
1994.
Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology.
Int. J. Syst. Bacteriol.
44:846-849 |
| 24. | Stackebrandt, E., and W. Ludwig. 1994. The importance of using outgroup reference organisms in phylogenetic studies: the Atopobium case. Syst. Appl. Microbiol. 17:39-43. |
| 25. | Story, R. M., and T. A. Steitz. 1992. Structure of the recA protein-ADP complex. Nature 355:374-376[CrossRef][Medline]. |
| 26. |
Thompson, J. D.,
D. G. Higgins, and T. J. Gibson.
1994.
CLUSTAL W: improving the sensitivity of multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice.
Nucleic Acids Res.
22:4673-4680 |
| 27. | Van Reenen, C. A., and L. M. T. Dicks. 1996. Evaluation of numerical analysis of Random Amplified Polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus plantarum and Lactobacillus pentosus. Curr. Microbiol. 32:183-187[CrossRef][Medline]. |
| 28. |
Zanoni, P.,
J. A. E. Farrow,
B. A. Phillips, and M. D. Collins.
1987.
Lactobacillus pentosus (Fred, Peterson and Anderson) sp. nov., nom. rev.
Int. J. Syst. Bacteriol.
37:339-341 |
Applied and Environmental Microbiology, August 2001, p. 3450-3454, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3450-3454.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Watanabe, K., Fujimoto, J., Tomii, Y., Sasamoto, M., Makino, H., Kudo, Y., Okada, S.
(2009). Lactobacillus kisonensis sp. nov., Lactobacillus otakiensis sp. nov., Lactobacillus rapi sp. nov. and Lactobacillus sunkii sp. nov., heterofermentative species isolated from sunki, a traditional Japanese pickle. Int. J. Syst. Evol. Microbiol.
59: 754-760
[Abstract] [Full Text] -
Siragusa, S., Di Cagno, R., Ercolini, D., Minervini, F., Gobbetti, M., De Angelis, M.
(2009). Taxonomic Structure and Monitoring of the Dominant Population of Lactic Acid Bacteria during Wheat Flour Sourdough Type I Propagation Using Lactobacillus sanfranciscensis Starters. Appl. Environ. Microbiol.
75: 1099-1109
[Abstract] [Full Text] -
Martin-Platero, A. M., Valdivia, E., Maqueda, M., Martin-Sanchez, I., Martinez-Bueno, M.
(2008). Polyphasic Approach to Bacterial Dynamics during the Ripening of Spanish Farmhouse Cheese, Using Culture-Dependent and -Independent Methods. Appl. Environ. Microbiol.
74: 5662-5673
[Abstract] [Full Text] -
Endo, A., Okada, S.
(2008). 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.
58: 2195-2205
[Abstract] [Full Text] -
Franciosi, E., Settanni, L., Carlin, S., Cavazza, A., Poznanski, E.
(2008). A Factory-Scale Application of Secondary Adjunct Cultures Selected from Lactic Acid Bacteria During Puzzone di Moena Cheese Ripening. J DAIRY SCI
91: 2981-2991
[Abstract] [Full Text] -
Blaiotta, G., Fusco, V., Ercolini, D., Aponte, M., Pepe, O., Villani, F.
(2008). Lactobacillus Strain Diversity Based on Partial hsp60 Gene Sequences and Design of PCR-Restriction Fragment Length Polymorphism Assays for Species Identification and Differentiation. Appl. Environ. Microbiol.
74: 208-215
[Abstract] [Full Text] -
Naser, S. M., Dawyndt, P., Hoste, B., Gevers, D., Vandemeulebroecke, K., Cleenwerck, I., Vancanneyt, M., Swings, J.
(2007). Identification of lactobacilli by pheS and rpoA gene sequence analyses. Int. J. Syst. Evol. Microbiol.
57: 2777-2789
[Abstract] [Full Text] -
O'Keefe, S. J. D., Chung, D., Mahmoud, N., Sepulveda, A. R., Manafe, M., Arch, J., Adada, H., van der Merwe, T.
(2007). Why Do African Americans Get More Colon Cancer than Native Africans?. J. Nutr.
137: 175S-182S
[Abstract] [Full Text] -
Groenewald, W. H., Van Reenen, C. A., Todorov, S. D., Toit, M. D., Witthuhn, R. C., Holzapfel, W. H., Dicks, L. M.T.
(2006). Identification of Lactic Acid Bacteria from Vinegar Flies Based on Phenotypic and Genotypic Characteristics. Am. J. Enol. Vitic.
57: 519-525
[Abstract] [Full Text] -
Naser, S. M., Vancanneyt, M., Hoste, B., Snauwaert, C., Swings, J.
(2006). Lactobacillus cypricasei Lawson et al. 2001 is a later heterotypic synonym of Lactobacillus acidipiscis Tanasupawat et al. 2000.. Int. J. Syst. Evol. Microbiol.
56: 1681-1683
[Abstract] [Full Text] -
Felis, G. E., Vancanneyt, M., Snauwaert, C., Swings, J., Torriani, S., Castioni, A., Dellaglio, F.
(2006). Reclassification of Lactobacillus thermotolerans Niamsup et al. 2003 as a later synonym of Lactobacillus ingluviei Baele et al. 2003.. Int. J. Syst. Evol. Microbiol.
56: 793-795
[Abstract] [Full Text] -
Perez Pulido, R., Ben Omar, N., Abriouel, H., Lucas Lopez, R., Martinez Canamero, M., Galvez, A.
(2005). Microbiological Study of Lactic Acid Fermentation of Caper Berries by Molecular and Culture-Dependent Methods. Appl. Environ. Microbiol.
71: 7872-7879
[Abstract] [Full Text] -
Bringel, F., Castioni, A., Olukoya, D. K., Felis, G. E., Torriani, S., Dellaglio, F.
(2005). Lactobacillus plantarum subsp. argentoratensis subsp. nov., isolated from vegetable matrices. Int. J. Syst. Evol. Microbiol.
55: 1629-1634
[Abstract] [Full Text] -
Settanni, L., van Sinderen, D., Rossi, J., Corsetti, A.
(2005). Rapid Differentiation and In Situ Detection of 16 Sourdough Lactobacillus Species by Multiplex PCR. Appl. Environ. Microbiol.
71: 3049-3059
[Abstract] [Full Text] -
Dellaglio, F., Felis, G. E., Castioni, A., Torriani, S., Germond, J.-E.
(2005). Lactobacillus delbrueckii subsp. indicus subsp. nov., isolated from Indian dairy products. Int. J. Syst. Evol. Microbiol.
55: 401-404
[Abstract] [Full Text] -
Dellaglio, F., Torriani, S., Felis, G. E.
(2004). Reclassification of Lactobacillus cellobiosus Rogosa et al. 1953 as a later synonym of Lactobacillus fermentum Beijerinck 1901. Int. J. Syst. Evol. Microbiol.
54: 809-812
[Abstract] [Full Text] -
Ventura, M., Zink, R.
(2003). Comparative Sequence Analysis of the tuf and recA Genes and Restriction Fragment Length Polymorphism of the Internal Transcribed Spacer Region Sequences Supply Additional Tools for Discriminating Bifidobacterium lactis from Bifidobacterium animalis. Appl. Environ. Microbiol.
69: 7517-7522
[Abstract] [Full Text] -
Ventura, M., Canchaya, C., Meylan, V., Klaenhammer, T. R., Zink, R.
(2003). Analysis, Characterization, and Loci of the tuf Genes in Lactobacillus and Bifidobacterium Species and Their Direct Application for Species Identification. Appl. Environ. Microbiol.
69: 6908-6922
[Abstract] [Full Text] -
Ennahar, S., Cai, Y., Fujita, Y.
(2003). Phylogenetic Diversity of Lactic Acid Bacteria Associated with Paddy Rice Silage as Determined by 16S Ribosomal DNA Analysis. Appl. Environ. Microbiol.
69: 444-451
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2010 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»