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State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, P. R. China
Correspondence
Xiuzhu Dong
dongxz{at}sun.im.ac.cn
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the partial HSP60 gene and 16S rDNA sequences of Bifidobacterium species and the complete 16S rDNA sequence of Bifidobacterium thermacidophilum subsp. porcinum reported in this study are AY166508–AY166575 and AY148470, respectively.
A list of Bifidobacterium strains used in this study and GenBank accession numbers for their HSP60 and 16S rRNA gene sequences is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Lauer, 1990; Biavati & Mattarelli, 1991; Biavati et al., 1991; Crociani et al., 1996; Meile et al., 1997; Dong et al., 2000; Hoyles et al., 2002; Jian & Dong, 2002). Conventional identification and classification of Bifidobacterium species have relied heavily on carbohydrate-fermentation patterns, which have been demonstrated to be strain-specific, rather than species-specific (Roy & Ward, 1990).
16S rDNA homology analysis has been demonstrated to be a powerful and accurate method to determine phylogenetic relationships among bacteria, including generic relationships (Olsen et al., 1994; Stackebrandt & Goebel, 1994). Sequence similarity levels among Bifidobacterium species ranged from 93 to 99 % and were even greater than 99 % within five groups (Miyake et al., 1998); this is too high to distinguish between Bifidobacterium species.
DNA–DNA hybridization has been acknowledged to be a superior method to elucidate relationships between closely related taxa, such as species. Bifidobacterial members within a species generally exhibited DNA–DNA reassociation values of >70 % (Lauer & Kandler, 1983). However, this is a laborious and inconvenient method to identify an unknown isolate.
Transaldolase gene sequence-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) patterns have also been used to differentiate bifidobacterial species isolated from humans (Requena et al., 2002); however, this method failed to discriminate Bifidobacterium catenulatum from Bifidobacterium angulatum.
The highly conserved 60 kDa heat-shock protein (HSP60) has been considered to be another useful phylogenetic marker and sequence comparisons of its gene have been used for species identification and phylogenetic analysis of the genera Staphylococcus (Kwok et al., 1999) and Bartonella (Marston et al., 1999). In our previous work (Jian et al., 2001), it was indicated that the HSP60 gene may also be a useful tool for phylogenetic study of the genus Bifidobacterium. The clustering pattern was not only similar to that of 16S rDNA, but there was also a better correlation with DNA G+C content. Furthermore, HSP60 gene sequence similarities (
84–96 %) between Bifidobacterium species were much lower than those of 16S rDNA.
To evaluate the feasibility of using HSP60 genes to identify Bifidobacterium species, 32 Bifidobacterium strains that represented eight species and subspecies were isolated from faeces of several kinds of animal (see supplementary material in IJSEM Online) by using Hungate's anaerobic roll-tube technique with TPYG (trypticase/peptone/yeast extract/glucose) medium (Scardovi, 1986). Gram-stain, anaerobic growth and catalase and fructose-6-phosphate phosphoketolase (F6PPK) activities were detected as described by Scardovi (1986). End-products of glucose fermentation were detected by GC using a model GC-14B chromatograph (Shimadzu). Biochemical characteristics were determined by using an API 50CH kit (bioMérieux). Temperature profiles were determined by using a water bath with a temperature gradient.
To confirm the identification of the 32 new isolates phylogenetically, DNA was prepared and purified as described by Marmur (1961) and 16S rDNA was amplified by PCR and sequenced by using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kits (PerkinElmer). The most similar 16S rDNA sequences in GenBank were found by searching with the BLAST program (version 2.2.1). Species identification and GenBank accession numbers of 16S rDNA are available as supplementary material in IJSEM Online.
The partial HSP60 gene (600 bp) was amplified, cloned and sequenced as described previously (Jian et al., 2001). Partial HSP60 gene sequences were also translated into protein sequences by using the PRIMER PREMIER program, version 5.00 (Biosoft International). Methods for sequence similarity calculation and phylogenetic tree construction were described previously (Jian et al., 2001).
DNA G+C content was determined by thermal denaturation (Marmur & Doty, 1962). DNA–DNA relatedness between the new isolates and reference strains was determined by using the reassociation rate method (Dong et al., 2000).
In this work, partial HSP60 genes were sequenced for 37 Bifidobacterium strains that represented 10 Bifidobacterium species, including two subspecies, with validly published names. HSP60 gene sequence homology was analysed totally for 50 bifidobacterial strains, including 12 type strains, and another Bifidobacterium infantis strain that was studied previously; a phylogenetic tree (Fig. 1) was constructed. The result showed that partial HSP60 gene sequence similarity was 96·5–100 % (mean, 98 %) among strains within a species, 95·5–97 % between Bifidobacterium pseudolongum subsp. pseudolongum JCM 1205T and nine strains of B. pseudolongum subsp. globosum and 80–96 % (mean, 88 %) among the 10 Bifidobacterium species in this study. The result was in agreement with the previous conclusion (Jian et al., 2001).
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), based on high 16S rDNA sequence similarities (
99 %) among them.
An extremely high 16S rDNA sequence similarity (98·8 %) was also determined between Bifidobacterium lactis and Bifidobacterium animalis; Cai et al. (2000) proposed that B. lactis is a junior subjective synonym of B. animalis, based on this high 16S rDNA sequence similarity and high DNA–DNA hybridization (85·5–92·3 %) between the type strains of the two species. In the current study, high partial HSP60 sequence similarity was determined among five B. animalis and two B. lactis strains: 98·9–100 % within group 1 (including B. lactis DSM 10140T, B. lactis JB-1 and B. animalis B83) and group 2 (including B. animalis JCM 1190T, B. animalis D1, B. animalis III-1 and B. animalis III-6); however, similarity levels ranged from 97·0 to 97·8 % between the two groups. Hence, B. lactis DSM 10140T and other B. lactis strains should be regarded as another subspecies of B. animalis, although they were genetically and phenotypically very similar to some B. animalis strains.
Partial HSP60 gene sequence similarity between five Bifidobacterium pseudocatenulatum and three B. catenulatum isolates (91–93 %) supported their interspecies relationship, although they had extremely high 16S rDNA gene sequence similarity (99·5 %).
Combining the results of this work and the previous study (Jian et al., 2001), a conclusion could be drawn that homology of the partial HSP60 gene of the bifidobacteria was 80–96 %, 96·5–100 % and 95·5–97 % at the interspecies, intraspecies and inter-subspecies levels, respectively. Deduced HSP60 protein sequence similarities were 87–100 %, 98·5–100 % and 98–100 % at the interspecies, intraspecies and inter-subspecies levels, respectively. Compared to 16S rDNA, the HSP60 gene has sufficient power for identification of Bifidobacterium species.
Deduced HSP60 protein sequences showed 100 % similarity among the thermophilic group that comprises B. boum, B. thermacidophilum and B. thermophilum, although interspecies DNA homologies were 92·9–95·5 %. Three signature amino acid residues at positions 156, 158 and 179 (Lys, Arg and Ser) differentiated this group from all other bifidobacteria (Asn, Lys and Thr) tested. Shared primary structures of HSP60 might relate to their thermophilic characteristics.
Based on HSP60 gene sequence similarities, two new isolates from piglet faeces, P3-14T and P3-11, were identified as a novel subspecies of B. thermacidophilum. Similarities between strain P3-14T and others were determined as follows: 97 % with B. thermacidophilum AS 1.2282T, 95·5 % with B. thermophilum JCM 1207T and 94·4 % with B. boum JCM 1211T.
Homology analysis of the complete 16S rDNA sequence (1523 bp) of P3-14T also indicated its affiliation to the thermophilic bifidobacteria: B. boum (97·7 % similarity), B. thermacidophilum (97·2 % similarity) and B. thermophilum (97·0 % similarity). Furthermore, DNA–DNA relatedness was determined to be 83·0, 58·6 and 25·5 % between isolate P3-14T and B. thermacidophilum AS 1.2282T, B. thermophilum JCM 1207T and B. boum JCM 1211T, respectively, indicating that the new isolate belonged to B. thermacidophilum.
However, compared to B. thermacidophilum, strain P3-14T grew at a relatively low temperature, as well as the fact that it fermented trehalose but not D-mannose, inulin or L-arabinose. Table 1 shows differential phenotypic characteristics of strain P3-14T, B. thermophilum JCM 1207T, B. thermacidophilum AS 1.2282T and B. boum JCM 1211T. Considering the relatively low 16S rDNA and HSP60 gene sequence similarities, different G+C contents and fermentation patterns among them, as well as the lower growth temperature of P3-14T, a novel subspecies, Bifidobacterium thermacidophilum subsp. porcinum subsp. nov., is proposed. Accordingly, B. thermacidophilum is assigned as B. thermacidophilum subsp. thermacidophilum.
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Gram-positive, non-motile, non-spore-forming, irregular rods, 0·5x3–8 µm in size after 12–24 h growth in TPYG medium at 37 °C; strictly anaerobic, no gas is formed from glucose. Optimum temperature for growth is 37–41 °C; temperature range for growth is 30–49·5 °C. Optimum initial pH is 7·0–7·2, growth is quite good at pH 4·5 but is delayed at pH 4·0. D-Fructose, galactose, D-glucose, maltose, melibiose, sucrose, D-raffinose, amidon, glycogen and D-turanose are fermented. Mannitol, rhamnose and sorbose are not fermented. Fermentation of L-arabinose, gluconate, inulin, lactose, D-mannose, methyl 
-D-glucoside, ribose, salicin, trehalose and xylose is variable. Litmus milk is not acidified or coagulated by most strains. Fermentation products from glucose are acetic and lactic acids, at a molar ratio of 2·46–4·9 : 1. DNA G+C content is 54·8–61·9 mol%.
Description of Bifidobacterium thermacidophilum subsp. thermacidophilum subsp. nov.
All strains meet the species description. Maximum temperature for growth is 49·5 °C. L-Arabinose, inulin and D-mannose are fermented. Trehalose is not fermented. Fermentation products from glucose are acetic and lactic acids at a molar ratio of 2·46–2·72 : 1. DNA G+C content is 56·85±2·05 mol%.
The type strain, 36T (=AS 1.2282T=LMG 21395T), was isolated from wastewater.
Description of Bifidobacterium thermacidophilum subsp. porcinum subsp. nov.
Bifidobacterium thermacidophilum subsp. porcinum (por.ci'num. L. neut. adj. porcinum of a hog).
All strains meet the species description. Maximum temperature for growth is 46·5 °C. Trehalose is fermented. L-Arabinose, inulin and D-mannose are not fermented. Fermentation products from glucose are acetic and lactic acids at a molar ratio of 4·9 : 1. DNA G+C content is 61·2±0·7 mol%.
The type strain, P3-14T (=AS 1.3009T=LMG 21689T), was isolated from the faeces of a piglet.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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