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1 Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Box 7024, SE-750 07 Uppsala, Sweden
2 Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, SE-750 07 Uppsala, Sweden
Correspondence
Stefan Roos
stefan.roos{at}mikrob.slu.se
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Published online ahead of print on 27 February 2004 as DOI 10.1099/ijs.0.3057-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Lactobacillus saerimneri GDA154T is AY255802.
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Bacterial DNA was isolated using the DNeasy Tissue kit (Qiagen). The almost complete 16S rRNA gene for the strains was amplified using PCR with domain Bacteria-specific primers (Weizenegger et al., 1992
). The resulting PCR products were purified using the Qiagen PCR Purification kit. The first part (approximately 500 bp) of the purified fragments was sequenced according to standard methods. For strain GDA154T, the whole fragment was sequenced. Primers that were used for amplification, together with additional internal primers, were also used for sequencing of the PCR products. The sequences determined from the novel isolates were used for searches in the GenBank database (http://www.ncbi.nlm.nih.gov/). Sequences representing the closest matches were retrieved and then aligned using the CLUSTAL W program (Thompson et al., 1994). For all sequences, approximately 1450 nt were used. A distance matrix was calculated using the DNADIST program of the PHYLIP package (Felsenstein, 1993) with the F84 parameter model, and a phylogenetic tree was constructed with the NEIGHBOR program. Statistical significance of the grouping was estimated by bootstrapping (100 replicates) using the programs SEQBOOT, DNADIST, NEIGHBOR and CONSENSE, all of which are from the PHYLIP package. The phylogenetic tree was displayed using the TREEVIEW program (Page, 1996).
Cell morphologies of the bacteria were observed by phase-contrast microscopy. Determination of Gram reactions was performed using the KOH method of Gregersen (1978). Sugar fermentation patterns and aesculin were determined using the API 50 CHL system (bioMérieux) in duplicate at 37 °C. Lactic acid configuration was determined using a test kit from Boehringer Mannheim. Catalase activity was determined by transferring fresh colonies from MRS agar to a glass slide and adding 5 % H2O2. Production of gas from glucose was assayed by growing the bacteria in MRS tubes containing Durham tubes. Electrophoretic analysis of whole-cell proteins was performed by the BCCM. Preparation of whole-cell protein extracts and SDS-PAGE analysis were performed as described by Pot et al. (1994). Normalized and digitized patterns were numerically analysed and clustered with the reference profiles in the SDS-PAGE protein database at the BCCM Bacteria Collection. Cell-wall analysis was performed at the DSMZ. Preparation of cell walls and determination of peptidoglycan structure were carried out using the methods described by Schleifer (1985) and Schleifer & Kandler (1972) with the modification that TLC on cellulose sheets was used instead of paper chromatography. The G+C content of the DNA was determined at the DSMZ. DNA was isolated by chromatography on hydroxyapatite by the procedure of Cashion et al. (1977) and the G+C content was determined by HPLC as described by Mesbah et al. (1989).
The partial 16S rRNA sequences (i.e. the first 500 bp) from all strains showed 100 % identity. The complete 16S rRNA sequence of GDA154T was analysed with the SIMILARITY MATRIX tool at Ribosomal Database Project II (http://rdp.cme.msu.edu/html); the highest similarity values were found to Lactobacillus salivarius, Lactobacillus aviarius and Lactobacillus mali, with 95·0, 94·8 and 94·6 % similarity, respectively. The sequence was then aligned with the sequences of all members of the L. mali subgroup and some other representatives of the Lactobacillus casei–Pediococcus group of lactobacilli, and this alignment was then used to construct a phylogenetic tree. This clearly placed the bacterium in the L. mali subgroup and confirmed the relationship to L. salivarius and L. aviarius (Fig. 1). The mean G+C content of the DNA of strain GDA154T was 42·9 mol% and the peptidoglycan type was A1
meso-diaminopimelic acid (m-DAP)-direct. The considerable difference in 16S rRNA gene sequence compared to all recognized lactobacilli shows that the isolated strains represent a novel species. This is confirmed by comparing the G+C content and peptidoglycan type with other members of the L. mali subgroup (Table 1). The strains represent a novel species, for which we propose the name Lactobacillus saerimneri sp. nov., with strain GDA154T as the type strain.
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. Furthermore, electrophoretic analysis of whole-cell proteins showed that strain GDA154T is distinct from other species in the L. mali subgroup of lactobacilli (Fig. 2).
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and Leser et al. (2002). Although many lactobacilli were detected, none has a 16S rRNA gene sequence similar to L. saerimneri. This indicates that the bacterium is a minor component in the gastrointestinal microbiota of pigs.
Description of Lactobacillus saerimneri sp. nov.
Lactobacillus saerimneri (sae.rim'ne.ri. N.L. gen. masc. n. saerimneri of Saerimner, a pig occurring in Nordic mythology, because the organism was isolated from pigs).
Gram-positive, non-motile, non-spore-forming, catalase-negative rods, 1x1·5–4 µm in size and occurring as single cells or in pairs. After anaerobic growth at 37 °C for 48 h, colonies on MRS agar are 2–3 mm in diameter; they are white with an opaque border, smooth and convex. Growth on MRS agar also occurs under aerobic conditions, but at a considerably lower rate. In MRS broth growth occurs between 15 (weak) and 45 °C. Both D- (75 %) and L-lactate (25 %) are produced. Gas is not produced from glucose. Acid is produced from D-glucose, D-fructose, D-mannose, N-acetylglucosamine (delayed reaction), sucrose, trehalose and D-turanose. Acid is not produced from glycerol, erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl 
-D-xyloside, galactose, L-sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl 
-D-mannoside, methyl -D-glucoside, amygdalin, arbutin, salicin, cellobiose, maltose, melibiose, lactose, inulin, melezitose, D-raffinose, starch, glycogen, xylitol, -gentiobiose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. Aesculin is not hydrolysed. The DNA G+C content of strain GDA154T is 42·9 mol% and the peptidoglycan type is A1 m-DAP-direct. Phylogenetic analysis of the 16S rRNA gene sequence places the species in the L. mali subgroup of lactobacilli.
The type strain is GDA154T (=LMG 22087T=DSM 16049T=CCUG 48462T).
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ACKNOWLEDGEMENTS |
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90 % strains positive; –, 