[논문]Identification and Comparison of the Nucleotide Sequence of 16S-23S rRNA Gene Intergenic Small SR(Spacer Region) of Lactobacillus rhamnosus ATCC 53103 with Those of L. casei, L. acidophilus and L. helveticus

Byun, J.R.; Yoon, Y.H.

doi:10.5713/ajas.2003.1816

[국내논문] Identification and Comparison of the Nucleotide Sequence of 16S-23S rRNA Gene Intergenic Small SR(Spacer Region) of Lactobacillus rhamnosus ATCC 53103 with Those of L. casei, L. acidophilus and L. helveticus 원문보기

Asian-Australasian journal of animal sciences, v.16 no.12, 2003년, pp.1816 - 1821

Byun, J.R. (Department of Animal Science and Technology, Chung-Ang University) , Yoon, Y.H. (Department of Animal Science and Technology, Chung-Ang University)

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Reliable PCR based identification of lactobacilli has been described utilizing the sequence of 16S-23S rRNA intergenic spacer region. Those sequence comparisons showed a high degree of difference in homology among the strains of L. rhamnosus, L. casei, L. acidophilus and L. helveticus whose 16S-23S rRNA intergenic small SR's sizes were 222 bp, 222 bp, 206 bp and 216 bp respectively. The sequence of 16S-23S rRNA intergenic spacer region of L. rhamnosus ATCC 53103 revealed the close relatedness to those of L. casei strains by the homology ranges from 95.4% to 97.2%. 16S-23S rRNA intergenic spacer region nucleotide sequence of L. acidophilus showed some distant relatedness with L. rhamnosus ATCC 53103 with the homology ranges from 40.3% to 41.8% and that with L. helveticus was shown to be 30% of homology, which exists at the most distant phylogenetic relatedness. The identification of species and strain of lactobacilli was possible on the basis of these results. The common sequences among the 17 strains were CTAAGGAA located in the initiating position of the DNA and some discrepancies were found between the same strains based on these results.

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문제 정의

Very few data are available on the SR ofLactobacillus strains. The aim of this study was to sequence the SRs of a large collection of well- characterized Lactobacillus strains and to analyze the variations among them. The second objective was to use the species -specific sequence variation of the SRs to develop a rapid molecular based method for their identification at the species level.

성능/효과

The PCR products obtained from all lactobacilli were shown in Figure 1. The sizes of 16S-23S rRNA intergenic small SR of L. rhamnosus, L. casei, L. acidophilus and L. helveticus were shown to be 222 bp, 222 bp, 206 bp and 216 bp respectively.
16S-23S rRNA intergenic spacer region nucleotide sequence of L. rhamnosus ATCC 53103 were homologous with those of two strains of L. rhamnosus by 100% homology and L. rhamnosus ATCC 53103 revealed the closest relatedness with L. casei strains with the homology ranges from 95.4% to 97.2%. The sequences ofL.
rhamnosus revealed close similarity but differentiation was possible through comparison of the sequences of 16S23S rRNA intergenic spacer region. The sequence homology between L. rhamnosus ATCC 53103 and those of 6 strains ofL. casei were compared and ranged from 95.4% to 97.2 % and the homologies between L. rhamnosus ATCC 53103 and L. acidophilus strains were shown to be 40.3% to 41.8%.
The homology of the sequences between L. rhamnosus ATCC 53103 and L. helveticus was shown to rang from 30.8% to 32.0% but the homology between three L. helveticus strains was shown to be 89.7% to 97.6%. Identification of species of lactobacilli is capable on the basis ofthese results.
acidophilus ATCC 4356 determined in this study was identical with that preserved in the gene bank with the accession number of u32971 but revealed discrepancies with the seqence of accession number z75472. The sequence of 16S-23S rRNA intergenic spacer region of L. acidophilus ATCC 4356 determined in this study and compared with that sequence the same strains of L. acidophilus from gene bank (U32971) were revealed to be almost identical with the similarity of 98.6%. The sequence of L.
The conserved region of 16S-23S rRNA intergenic spacer region of all the tested lactobacilli consisted of CTAAGGAA as an initiating sequence and had some variable nucleotide (s) in those closely related sequences of L. casei and L. rhamnosus at the position numbers 16, 17, 68, 69, 91, 126, 127, 175-180, 184 and 200.
5% or greater was considered to provide species identification and spacer region sequencing provided a rapid and accurate identification of Lactobacillus isolates from gastrointestinal, yoghurt and silage samples. The comparison of 16S-23S rRNA intergenic spacer regions of all the tested lactobacilli in this study revealed variable homology ranges depending on the species of lactobacilli; the minimum homology values were greater than 92.7%, 96.7% and 89.7% in L. casei, L. acidophilus and in L. helveticus respectively, which was lower than the value proposed by Tannock et al. (1999) as a species identity. A similarity of 90% or greater could provide an identification of species based on this study.
5% or greater was considered to provide species identification and spacer region sequencing provided a rapid and accurate identification of Lactobacillus isolates from gastrointestinal, yoghurt and silage samples. The comparison of 16S-23S rRNA intergenic spacer regions of all the tested lactobacilli in this study revealed variable homology ranges depending on the species of lactobacilli; the minimum homology values were greater than 92.7%, 96.7% and 89.7% in L. casei, L. acidophilus and in L. helveticus respectively, which was lower than the value proposed by Tannock et al. (1999) as a species identity. A similarity of 90% or greater could provide an identification of species based on this study.

후속연구

The aim of this study was to sequence the SRs of a large collection of well- characterized Lactobacillus strains and to analyze the variations among them. The second objective was to use the species -specific sequence variation of the SRs to develop a rapid molecular based method for their identification at the species level.
These relatively short sequences can be easily sequenced on both polynucleotide strands and provide reliable information for comparative work. The use of 16S-23S rRNA intergenic spacer regions of lactobacilli in identification promises to be a valuable aid in advancing our knowledge of the species composition of Lactobacillus populations. The generic character of the amplification process provides for a simple and direct genetically based diagnostic procedure for the identification oflactobacilli.

참고문헌 (11)

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Drake, M. A., C. L. Small, K. D. Spence and B. G. Swanson. 1996. Rapid detection and identification of Lactobacillus spp. in dairy products by using the polymerase chain reaction. J. Food Protec. 59:1031-1036.
Fox, G.E., J. D Wisottzky 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.
Loughney, K., E, Lund, and J. E. Dahlberg. 1982. tRNA genes are found between the 16S and 23S genes in Bacillus subtilis. Nucleic Acid Res. 10:1607-1624.
Morieira, D. and R. Amils. 1996. PCR meditated detection of the chemolithotrophic bacterium Thiobacillus cuprinus using 23S rDNA and 16S/23Sintergenic spacer region targeted oligonucleotide primers. FEMS Microbiol. Letter. 142:289-293.
Nakagawa, T., M. Shimada, H. Mukai, K. Asada, I. Kato, K. Fujino and T. Sato. 1994. Detection of alcohol-tolerant hiochi bacteria by PCR. Appl. Environ. Microbiol. 60:637-640.
Tannock, G. W., A. Tilsala-Timisjarvi, S. Rodtong, J, NG and K. Munro. 1999. Identification of Lactobacillus isolates from the gastrointestinal Tract, silage, and youghurt by 16S-23S rRNA gene intergenic spacer region sequence comparisons. Appl. Environ. Microbiol. 65(9):4264-4267.
Tilsala-Timisjarvi, A. and T. Alatossava. 1997. Development of oligonucleotide primer from the 16S-23S rRNA intergenic sequences for identifying different dairy and probiotic lactic acid bacteria by PCR. Int. J. Food Microbiol. 35:49-56.
Yeung, P. S. M., M. E. Sanders, and C. L. Kitts. 2002. Species specific identification of commercial probiotic strains. J. Dairy Sci. 85:1039-1051.
Yoon, Y, H..and B. R. Won. 2002. Antagonism against Helicobacter pylori and proteolysis of Lactobacillus helveticus CU 631 and strain identification. Asian-Aust. J. Anim. Sci. 15(7):1057-1065.

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[국내논문] Identification and Comparison of the Nucleotide Sequence of 16S-23S rRNA Gene Intergenic Small SR(Spacer Region) of Lactobacillus rhamnosus ATCC 53103 with Those of L. casei, L. acidophilus and L. helveticus 원문보기

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[국내논문] Identification and Comparison of the Nucleotide Sequence of 16S-23S rRNA Gene Intergenic Small SR(Spacer Region) of Lactobacillus rhamnosus ATCC 53103 with Those of L. casei, L. acidophilus and L. helveticus 원문보기

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