| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, Ghent 9000, Belgium
2 BCCMTM/LMG Bacteria Collection, Ghent University, K.L. Ledeganckstraat 35, Ghent 9000, Belgium
Correspondence
Sabri M. Naser
Sabri.Naser{at}Ugent.be
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
-subunit (pheS) and RNA polymerase -subunit (rpoA) were sequenced and analysed for members of the Lactobacillus salivarius species group. The type strains of Lactobacillus acidipiscis and Lactobacillus cypricasei were investigated further using a third gene that encodes the -subunit of ATP synthase (atpA). The MLSA data revealed close relatedness between L. acidipiscis and L. cypricasei, with 99.8–100 % pheS, rpoA and atpA gene sequence similarities. Comparison of the 16S rRNA gene sequences of the type strains of the two species confirmed the close relatedness (99.8 % gene sequence similarity) between the two taxa. Similar phenotypes and high DNA–DNA binding values in the range of 84 to 97.5 % confirmed that L. acidipiscis and L. cypricasei are synonymous species. On the basis of the present study, it is proposed that Lactobacillus cypricasei is a later heterotypic synonym of Lactobacillus acidipiscis.
The GenBank/EMBL/DDBJ accession numbers for the partial pheS gene sequences for strains LMG 19820T, LMG 21592T, LMG 23135, CCUG 42959, CCUG 42960, CCUG 42962, LMG 9843T, LMG 14189T, LMG 22087T, LMG 21748T, LMG 9186T, LMG 10753T, LMG 10756T, LMG 21593T, LMG 9477T, LMG 6899T and LMG 6903T are AM087762, AM087687, AM168426–AM168429, AM087679, AM087760, AM087717, AM087740, AM087734, AM087737, AM087756, AM087708, AM087721, AM087746 and AM168425. Those for the rpoA partial gene sequences for strains LMG 19820T, LMG 21592T, LMG 23135, CCUG 42959, CCUG 42960, LMG 9186T and LMG 14189T are AM087849, AM087784, AM168431–AM168433, AM087831 and AM087801, respectively. Those for the atpA partial gene sequences for strains LMG 19820T and LMG 21592T are AM168424–AM168423, respectively.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
based on 11 strains isolated from fermented fish (pla-ra and pla-chom) in Thailand. L. acidipiscis utilizes D-glucose homofermentatively and produces L-lactic acid from glucose without the production of gas. L. acidipiscis strains do not grow at pH 4.0 or 8.5 and grow in 10 % NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that L. acidipiscis strains are positioned in a monophyletic cluster consisting of Lactobacillus salivarius, Lactobacillus aviarius, Lactobacillus ruminis, Lactobacillus agilis, Lactobacillus murinus, Lactobacillus animalis and Lactobacillus mali. The DNA–DNA relatedness of L. acidipiscis strains and other related Lactobacillus species was found to be in the range of 3.6 to 26.7 % (Tanasupawat et al., 2000). One year later, Lactobacillus cypricasei was described by Lawson et al. (2001) based on four strains isolated from Halloumi, a cheese produced in Cyprus. The strains were Gram-positive, non-spore-forming, facultatively anaerobic and catalase- and oxidase-negative. No growth was observed at 15 or 45 °C. Like L. acidipiscis, L. cypricasei strains formed a distinct branch within the L. salivarius species group, with L. salivarius and L. aviarius as the nearest neighbours. However, Lawson et al. (2001) did not include the related recognized species L. acidipiscis in comparisons with other species. In the present study, the relatedness between L. acidipiscis and L. cypricasei strains was investigated and revealed synonymy between the two taxa. Two reference strains of L. acidipiscis, LMG 19820T and LMG 23135, and four reference strains of L. cypricasei, LMG 21592T, CCUG 42959, CCUG 42960 and CCUG 42962, were selected for further comparative study. L. cypricasei LMG 21592T was cultivated and maintained on de Man, Rogosa and Sharpe medium (MRS; Difco). All other strains studied were cultivated and maintained on MRS (Oxoid) medium and incubated anaerobically at 37 °C, unless otherwise indicated.
The use of protein-coding gene sequence data for the determination of genomic relatedness at the bacterial species and genus levels has recently been advocated because of its ability to provide higher taxonomic resolution, since 16S rRNA gene sequence analysis may not be sufficiently specific to discriminate between closely related species (Chavagnat et al., 2002; Stackebrandt et al., 2002; Torriani et al., 2001; Ventura et al., 2003; Zeigler, 2003). Recently, the application of multilocus sequence analysis (MLSA) using the genes that code for the -subunit of bacterial phenylalanyl-tRNA synthase (pheS), the -subunit of RNA polymerase (rpoA) and the -subunit of ATP synthase (atpA) provided a robust system for the identification of Enterococcus species (Naser et al., 2005a, b). The MLSA-based identification system has proved to be valuable for the detection of synonymous species names within the genus Enterococcus (Naser et al., 2006b). In addition, relatedness between strains of Lactobacillus helveticus and Lactobacillus suntoryeus was initially evaluated using the same MLSA loci. Naser et al. (2006a) demonstrated that Lactobacillus suntoryeus is a later synonym of Lactobacillus helveticus. The pheS and rpoA genes were subsequently used for the analysis of lactobacilli of the L. salivarius species group, including the two strains of L. acidipiscis and the four reference strains of L. cypricasei. The type strains of L. acidipiscis and L. cypricasei were further investigated using the atpA gene. The primer sequences, amplification conditions and sequencing reactions were as described by Naser et al. (2005a, b). Raw sequence data were transferred to GeneBuilder (Applied Maths) where consensus sequences were determined. Consensus sequences were imported into BioNumerics 4.0 software (Applied Maths). The determined partial pheS and rpoA gene sequences (453 and 402 bp, respectively) were compared for the two L. acidipiscis strains (LMG 19820T and LMG 23135), four L. cypricasei strains (LMG 21592T, CCUG 42959, CCUG 42960 and CCUG 42962) and other members of the L. salivarius species group. Comparison of the sequences of the strains of L. cypricasei and L. acidipiscis revealed pheS and rpoA gene sequence similarities in the range of 99.8 to 100 %. The atpA partial gene sequences of L. cypricasei LMG 21592T (GenBank accession no. AM168423) and L. acidipiscis LMG 19820T (AM168424) were also compared (976 bp) and showed high relatedness. Strains of the two species had a maximum of 78 % pheS gene sequence similarity with L. mali and L. murinus, 82 % rpoA gene sequence similarity with L. agilis and 80 % atpA gene sequence similarity with L. murinus (Fig. 1). Consequently, the MLSA data indicated that L. acidipiscis and L. cypricasei might represent a single species.
|
In the original description of L. cypricasei, the authors did not determine the DNA G+C content. In the present study, we investigated this characteristic for the type strain of L. cypricasei. Cells were cultivated in MRS broth at 37 °C for 24 h. DNA was extracted from 0.5–0.75 g (wet weight) by using the protocol described by Marmur (1961) with the following modifications: (i) cells were suspended overnight in Tris/HCl buffer that contained lysozyme (8 mg ml–1) before the addition of SDS and (ii) lysed cells were treated with proteinase K (360 mg l–1; Merck) at 37 °C for 2 h. For determination of the DNA G+C content, DNA was degraded enzymically into nucleosides as described by Mesbah et al. (1989). The nucleoside mixture was then separated by HPLC using a SymmetryShield RP8 column (Waters) maintained at 37 °C. The solvent was 0.02 M NH4H2PO4 (pH 4.0) with 1.5 % acetonitrile. Non-methylated 
-phage DNA (Sigma) was used as the calibration reference. The DNA G+C content of L. cypricasei LMG 21592T was 40.1 mol%. In comparison, the DNA G+C content of L. acidipiscis LMG 19820T was 38.7 mol%.
Finally, DNA–DNA hybridizations were performed between L. cypricasei strains LMG 21592T and CCUG 42960 and L. acidipiscis strains LMG 19820T and LMG 23135. Genomic DNA was prepared according to the protocol of Pitcher et al. (1989) with the following modifications: the washed cell pellet was resuspended and lysed in a buffer (10 mM Tris/HCl, 100 mM EDTA, pH 8.0) that contained RNase (200 µg ml–1; Sigma), mutanolysin (100 U ml–1; Sigma) and lysozyme (25 mg ml–1; SERVA) for 1 h at 37 °C. The microplate method was used as described by Ezaki et al. (1989) and Goris et al. (1998) using a HTS7000 Bio Assay Reader (Perkin Elmer) for the fluorescence measurements. Biotinylated DNA was hybridized with unlabelled ssDNA, which was bound non-covalently to microplate wells. Hybridizations were performed at 36 °C in hybridization mixture (2x SSC, 5x Denhardt's solution, 2.5 % dextran sulfate, 50 % formamide, 100 µg denatured salmon sperm DNA ml–1, 1250 ng biotinylated probe DNA ml–1). Reciprocal reactions (e.g. A x B and B x A) were performed. The DNA–DNA binding values reported were the mean values of a minimum of four hybridization experiments, including the reciprocal reactions. L. acidipiscis strains LMG 19820T and LMG 23135 and L. cypricasei strains LMG 21592T and CCUG 42960 showed high DNA–DNA binding values in the range of 84 to 97.5 %, indicating clearly that the two taxa represent the same species. A DNA–DNA hybridization value of 93 % was found between the type strains of the two species.
On the basis of the evidence presented, it is concluded that Lactobacillus cypricasei is a later heterotypic synonym of Lactobacillus acidipiscis. It is proposed that the two species be united under the same name. As a rule of priority (Rules 38 and 42 of the Bacteriological Code; Lapage et al., 1992), the name Lactobacillus acidipiscis should be retained and strains of Lactobacillus cypricasei should be reclassified as such. The description of L. acidipiscis remains essentially the same except for some strain-dependent reactions, such as the production of acid from mannitol and D-ribose and the production of arginine dihydrolase.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
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.
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]
Lapage, S. P., Sneath, P. H. A., Lessel, E. F., Skerman, V. B. D., Seeliger, H. P. R. & Clark, W. A. (editors) (1992). International Code of Nomenclature of Bacteria (1990 Revision). Bacteriological Code. Washington, DC: American Society for Microbiology.
Lawson, P. A., Papademas, P., Wacher, C., Falsen, E., Robinson, R. & Collins, M. D. (2001). Lactobacillus cypricasei sp. nov., isolated from Halloumi cheese. Int J Syst Evol Microbiol 51, 45–49.[Abstract]
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
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.
Naser, S., Thompson, F. L., Hoste, B., Gevers, D., Vandemeulebroecke, K., Cleenwerck, I., Thompson, C. C., Vancanneyt, M. & Swings, J. (2005a). Phylogeny and identification of enterococci using atpA gene sequence analysis. J Clin Microbiol 43, 2224–2230.
Naser, S. M., Thompson, F. L., Hoste, B., Gevers, D., Dawyndt, P., Vancanneyt, M. & Swings, J. (2005b). Application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes. Microbiology 151, 2141–2150.
Naser, S. M., Hagen, K. E., Vancanneyt, M., Cleenwerck, I., Swings, J. & Tompkins, T. A. (2006a). 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 56, 355–360.
Naser, S. M., Vancanneyt, M., Hoste, B., Snauwaert, C., Vandemeulebroecke, K. & Swings, J. (2006b). Reclassification of Enterococcus flavescens Pompei et al. 1992 as a later synonym of Enterococcus casseliflavus (ex Vaughan et al. 1979) Collins et al. 1984, and Enterococcus saccharominimus Vancanneyt et al. 2004 as a later synonym of Enterococcus italicus Fortina et al. 2004. Int J Syst Evol Microbiol 56, 413–416.
Pitcher, D. G., Saunders, N. A. & Owen, R. G. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151–156.
Stackebrandt, E., Frederiksen, W., Garrity, G. M. & 10 other authors (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043–1047.[Abstract]
Tanasupawat, S., Shida, O., Okada, S. & Komagata, K. (2000). Lactobacillus acidipiscis sp. nov. and Weissella thailandensis sp. nov., isolated from fermented fish in Thailand. Int J Syst Evol Microbiol 50, 1479–1485.[Abstract]
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.
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.
Zeigler, D. R. (2003). Gene sequences useful for predicting relatedness of whole genomes in bacteria. Int J Syst Evol Microbiol 53, 1893–1900.
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]
|
|
|
|
 |
|
 G. Blaiotta, V. Fusco, D. Ercolini, M. Aponte, O. Pepe, and F. Villani Lactobacillus Strain Diversity Based on Partial hsp60 Gene Sequences and Design of PCR-Restriction Fragment Length Polymorphism Assays for Species Identification and Differentiation Appl. Envir. Microbiol., January 1, 2008; 74(1): 208 - 215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Naser, P. Dawyndt, B. Hoste, D. Gevers, K. Vandemeulebroecke, I. Cleenwerck, M. Vancanneyt, and J. Swings Identification of lactobacilli by pheS and rpoA gene sequence analyses Int J Syst Evol Microbiol, December 1, 2007; 57(12): 2777 - 2789. [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 | |
50 %) after 500 simulations are shown. Bar, 2 % sequence divergence.