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School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan
Correspondence
Takao Mukai
mukai{at}vmas.kitasato-u.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JCM 1039T (=KCTC 3155T) is AB107638.
A figure showing SDS-PAGE analysis of whole-cell proteins is available as supplementary material in IJSEM Online.
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described the properties of Lactobacillus acidophilus in detail. As later revealed by Johnson et al. (1980) and others (Lauer et al., 1980; Sarra et al., 1980), six genome clusters (A1–A4, B1 and B2) exist within the L. acidophilus group. Johnson et al. (1980) designated homology group A1 as L. acidophilus. Later, group B1 was designated as Lactobacillus gasseri by Lauer & Kandler (1980) and group A2 was demonstrated to be synonymous with Lactobacillus crispatus by Cato et al. (1983). Furthermore, Fujisawa et al. (1992) proposed that groups A4 and B2 were novel species (Lactobacillus gallinarum and Lactobacillus johnsonii, respectively) and that group A3 should be considered as Lactobacillus amylovorus.
Although six genome clusters of the L. acidophilus group have been designated as separate species with validly published names, they are difficult to distinguish solely on the basis of phenotypic characteristics. Consequently, many strains besides L. acidophilus (group A1) are still catalogued in culture collections as ‘L. acidophilus’, as suggested by Fujisawa et al. (1996). It has been reported that S-layers, which are composed of a single protein species with a relative molecular mass of 43 000–46 000, commonly occur in Lactobacillus isolates that belong to genome clusters A1–A4 within the L. acidophilus group, but are absent from isolates that belong to genome clusters B1 and B2 (Masuda, 1992; Mukai & Arihara, 1994; Boot et al., 1996). Our previous studies showed that cell-surface protein profiles of L. acidophilus JCM 1038 and JCM 1039 from chicken intestine were apparently different from those of reference cells that belonged to L. acidophilus group A (Mukai & Arihara, 1994). Based on our preliminary results, we suspected that these strains do not belong to L. acidophilus (group A1). Furthermore, data from a recent study by Ryu et al. (2001), who used a ribotyping technique, confirmed the results of a previous study that showed that the L. acidophilus group could be divided into two major clusters. Noticeably, the results also showed that the L. acidophilus group could be further divided by ribotyping into 14 genotypes (A1–A11 and B1–B3) with similarity levels of 50 % and that JCM 1038 and JCM 1039 belong to an independent genotype among the 14 genotypes. These ribotyping results, along with those of our previous study, suggest strongly that strains JCM 1038 and JCM 1039 should be reclassified as a different species. We have recently isolated homofermentative Lactobacillus strains from the mucosal surface of chicken small intestine that show cell-surface protein profiles similar to those observed in strains JCM 1038 and JCM 1039. In this paper, we describe the characteristics of JCM 1038, JCM 1039 and their related Lactobacillus strains isolated from the mucosa of chicken small intestine and propose that this novel species should be named Lactobacillus kitasatonis sp. nov.
Strain JCM 1039T (=KCTC 3155T) was designated as the type strain of the novel species in this study. JCM 1038 (=KCTC 3154) and JCM 1039T originated from chicken intestine; reference strains of Lactobacillus were obtained from the Japan Collection of Microorganisms (JCM, Wako, Japan). Strains KM55, KM105, KM132 and KM9212 were isolated from the small intestines of two euthanized White Leghorn hens kept in the same farm; this was performed anaerobically (AnaeroPack; Mitsubishi Gas Chemical) on Lactobacillus selective (LBS) agar (Becton Dickinson) after 48 h incubation. All further cultivation was done anaerobically on MRS agar (de Man et al., 1960) or in MRS broth at 37 °C. Cell shape, size and arrangement, Gram-stain and colonial appearance were determined by using cells grown on MRS agar plates for 2 days at 37 °C. Strains were tested for carbohydrate fermentation abilities by using the API 50 CHL system (bioMérieux). Production of gas from glucose and gluconate was examined with Durham tubes. Catalase formation, growth at 15 and 45 °C and motility were tested by previously described methods (Mitsuoka, 1969), except that MRS broth was used as the basal medium. Extraction of whole-cell proteins and their separation by SDS-PAGE were performed as described previously (Mukai & Arihara, 1994).
Chromosomal DNA from Lactobacillus strains was prepared as described previously (Luchansky et al., 1991). G+C contents of the DNA preparations were determined by the HPLC method (Kaneko et al., 1986) by using a DNA-GC kit (Yamasa Shoyu). DNA from calf thymus that had a G+C content of 42 mol% was used as a standard. DNA–DNA relatedness was determined by the method of Ezaki et al. (1989) by using photobiotin and a microplate. Almost-complete 16S rRNA gene sequences were determined for three representative strains: JCM 1038, JCM 1039T and KM9212. A fragment of the 16S rRNA gene (corresponding to positions 8–1541 in the Escherichia coli numbering system) was amplified by PCR, using conserved primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1522R [5'-AAGGAGGTGATCCA(A/G)CCGCA-3']. The reaction involved 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 2 min. PCR products were purified by using a GFX PCR DNA and Gel Band Purification kit (Amersham Biosciences). Both strands of purified fragments were sequenced directly by using a Dye Terminator Cycle Sequencing FS Ready Reaction kit (Applied Biosystems) under conditions recommended by the manufacturer and using an automated sequence analyser (model 373A; Applied Biosystems). 16S rRNA gene sequences were deposited in, and previously published 16S rRNA gene sequences were obtained from, EMBL/GenBank/DDBJ. Nucleotide substitution rates (Knuc) (Kimura, 1980) were calculated and phylogenetic trees were constructed by the neighbour-joining method (Saitou & Nei, 1987). Topology of the trees was evaluated by bootstrap analysis of the sequence data (1000 replicates) using the CLUSTAL W program (Thompson et al., 1994).
Strains JCM 1038, JCM 1039T, KM55, KM105, KM132 and KM9212 were Gram-positive, non-motile, non-spore-forming rods. These cells occurred singly, in pairs or occasionally in short chains. Colonies of these strains were circular to slightly irregular, convex, opaque, rough and white. Phenotypic characteristics that differentiate these strains from other reference strains are summarized in Table 1. All strains tested produced DL-lactic acid, did not grow at 15 °C, were catalase-negative and did not produce gas from glucose. The DNA G+C content of the six strains ranged from 37 to 40 mol% (Table 1).
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, the results of phylogenetic analysis confirmed the association between JCM 1039T and species that belong to the Lactobacillus delbrueckii group of the genus Lactobacillus (Collins et al., 1991). Strain JCM 1039T was included in a monophyletic cluster that contained L. amylovorus, L. crispatus, L. gallinarum, Lactobacillus helveticus and L. acidophilus, as shown in Fig. 1. High levels of sequence relatedness (97·5–98·6 %) were found with L. amylovorus, L. crispatus and L. gallinarum.
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, photobiotin-labelled DNA from JCM 1038, JCM 1039T and KM9212 reassociated at high levels (77–105 %) with unlabelled DNA from each strain and other strains (KM55, KM105 and KM132), indicating that they belong to a single species, whereas photobiotin-labelled DNA from JCM 1038, JCM 1039T and KM9212 reassociated at low levels with unlabelled DNA from the type strains of L. crispatus, L. gallinarum and L. amylovorus and type strains of other previously described species, indicating that they were different species.
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, express S-layer proteins on their cell surface (Masuda, 1992; Mukai & Arihara, 1994; Boot et al., 1996; Callegari et al., 1998). We analysed the cell-surface protein patterns of all six strains in comparison with those of reference strains. SDS-PAGE protein analysis by computer was not possible, due to unavailability of a computer program, and we were therefore not able to construct any relationships. However, differences in the SDS-PAGE patterns between the six strains tested and other reference strains were detected visually (see Supplementary Figure available in IJSEM Online). Considerable amounts of multiple protein bands were detected from the six strains, whereas only single protein bands, corresponding to S-layer proteins, were detected from other reference strains. On the basis of the results of this study, we propose that JCM 1038 and JCM 1039T should be reclassified in a novel species and that the four strains isolated from mucosa of chicken small intestine as well as strains JCM 1038 and JCM 1039T, which also originated in the chicken intestine, are placed in the following novel species of the genus Lactobacillus: Lactobacillus kitasatonis sp. nov.
Description of Lactobacillus kitasatonis sp. nov.
Lactobacillus kitasatonis (ki.ta.sa.to'nis. L. gen. n. kitasatonis referring to Shibasaburo Kitasato, the founder of Kitasato Institute, the father of Japanese bacteriology).
Cells are Gram-positive, non-motile, non-spore-forming rods that are generally 0·6–1·0x2·0–20·0 µm in size and occur singly, in pairs or occasionally in short chains. When the organism is grown on MRS agar at 37 °C for 2 days, colonies are 1·2–2·1 mm in diameter, circular to slightly irregular, convex, opaque, rough and white. There is no growth at 15 °C, but growth occurs at 45 °C. The organism is facultatively anaerobic and produces DL-lactic acid homofermentatively. Catalase is not produced. Milk is not curdled. Acid is produced without gas formation from glucose, mannose, maltose, galactose, sucrose, fructose or aesculin. There is no acid formation from arabinose, xylose, ribose, trehalose, melezitose, melibiose, raffinose, amygdalin or sorbitol. DNA G+C content is 37–40 mol%.
The type strain is JCM 1039T (=KCTC 3155T). The habitat of this species is the intestine of chickens.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Callegari, M. L., Riboli, B., Sanders, J. W., Cocconcelli, P. S., Kok, J., Venema, G. & Morelli, L. (1998). The S-layer gene of Lactobacillus helveticus CNRZ 892: cloning, sequence and heterologous expression. Microbiology 144, 719–726.
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