Molecular characterization of the sequences of the 16S-23S rDNA internal spacer region (ISR) from isolates of Taylorella asinigenitalis
© Matsuda et al; licensee BioMed Central Ltd. 2008
Received: 22 July 2008
Accepted: 03 March 2009
Published: 03 March 2009
Sequence information on the 16S-23S rDNA internal spacer region (ISR) exhibits a large degree of sequence and length variation at both the genus and species levels. A primer pair for the amplification of 16S-23S rDNA ISR generated three amplicons for each of isolates of Taylorella asinigenitalis (UCD-1T, UK-1 and UK-2).
Following TA cloning and sequencing, the three isolates of T. asinigenitalis were demonstrated to possess three ISR units of different lengths. Although the three corresponding ISRs (A, B and C) were identified to be identical to each other (UK-1 and UK-2 isolates), the ISRs shared approximately 95.3–98.9% nucleotide sequence similarities between the UCD-1T and UK-1/-2 isolates. A typical order of two intercistronic tRNA genes (5'-tRNAIle-tRNAAla-3') with the different nucleotide spacers [44 through 51 base pairs (bp)] in length was identified among the isolates. The consensus sequences of the antiterminators of boxB and boxA were also identified in all ISRs. Thus, three ISRs were identified for each isolate, and therefore, at least three distinctly different ribosomal RNA operons were suggested to occur in the genome of T. asinigenitalis. This was also confirmed by Southern hybridization procedure.
The present study represents a dendrogram constructed based on the nucleotide sequence data of 16S-23S rDNA ISR for T. asinigenitalis, which may aid in the phylogenetic positioning of T. asinigenitalis within the genus Taylorella, and in the molecular discrimination of T. asinigenitalis.
In late 1997 and in early 1998, three bacterial isolates were isolated from donkey jacks (Equus asinus) in the USA and a new second species of the genus Taylorella, T. asinigenitalis, was established for these atypical organisms [5, 6]. Additional T. asinigenitalis isolates (Bd3751/05 and 115/04) were obtained more recently and were identified from the genital tract of stallions in Sweden (GenBank accession No. DQ099547) and in Spain (DQ 393780). Sequences of the nearly full-length 16S rDNA from all these five isolates of T. asinigenitalis have already been deposited in DDBJ/EMBL/GenBank (AF297174 for UK-1, AF297175 for UK-2, AF067729 for UCD-1T, DQ099547 for Bd3751/05 and DQ393780 for 115/04). It was demonstrated that the sequences were almost identical (> 99.8% similarity) among the three isolates obtained in the USA . However, no sequence information on the 16S-23S rDNA internal spacer region (ISR), which exhibits a large degree of sequence and length variation at both the genus and species levels [7, 8], of T. asinigenitalis have yet been reported.
Therefore, the aim of the present study is to clone, sequence, and analyze the 16S-23S rDNA ISRs of three isolates of T. asinigenitalis (UCD-1T, UK-1 and UK-2) and to compare the ISRs sequence data.
In the present study, three isolates of T. asinigenitalis (UCD-1T, UK-1 and UK-2; [5, 6]) were analyzed. The conditions for cell culturing have previously been described by Matsuda and colleagues [9, 10]. Genomic DNA preparation for the PCR amplification was carried out, as described already [11, 12].
The primer pair used for the 16S-23S rDNA ISR amplification of T. asinigenitalis isolates in the present study was ISR-Tef (5'-CTGGGGTGAAGTCGTAACAAG-3') for the forward primer and ISR-5r (5'-GCCAAGGCATCCACC-3'; the sequence of region 5 reported by Gurtler and Stanisich (1996) [7, 12] for the reverse primer. The ISR-Tef primer was designed in silico in the present study for PCR amplification of full-length ISR of the genus Taylorella.
PCR mastermix contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 40 mM of each dNTP, 1 μM of each primer and 0.5 unit of EX Taq™ DNA polymerase, which possesses 3'-5' exonuclease activity (Takara BioInc. Shiga, Japan). PCR was performed in a 50 μl volume, for 25 cycles at 94°C for 1 min, 65.4°C for 30 sec, 72°C for 30 sec and finally 72°C for 7 min. Purification of the amplified PCR products and TA cloning of the PCR amplicons were carried out, as described previously . Dideoxynucleotide sequencing and sequence analysis were also performed, as described previously . For accurate sequencing, multiple TA-cloned PCR products were sequenced. The sequence data of the 16S-23S rDNA ISR of T. asinigenitalis determined in the present study have been deposited in DDBJ/EMBL/GenBank (AB264283–AB264291).
Southern blot hybridization analysis of the ISR was carried out using digoxigenin (DIG) labeled fragment of the ISRA (nucleotide position 452–761:AB264283) prepared from the T. asinigenitalis UCD-1T, as a probe with Pst I digested whole genome DNAs, respectively, according to the procedure described by Sambrook et al. (2001) . Random primer extension was performed in order to prepare the fragment using a DIG-High Prime kit (Roche Applied Science, Penzberg, Germany).
Nucleotide sequences of the ISRs from the isolates of the genus Taylorella were compared to each other using CLUSTAL W software , which was incorporated in the GENETYX version 9 (GENETYX Co., Tokyo, Japan) computer software. Following this, a phylogenetic tree was constructed by an unweighted pair group mean average (UPGMA) method available on the GENETYX (version 9).
Results and discussion
The sequence corresponding to the CCUCCU sequence, a highly conserved sequence close to the 3' end of the mature 16S rRNA that is complementary to the Shine-Dalgarno sequence on mRNA [14, 15], was identified in the sequences of the amplicons of T. asinigenitalis isolates [nucleotide position 31–36; AB264283–AB264291], as well as T. equigenitalis isolates (AF408197, AB066372, AB069660, AB113653–AB113658). Thus, in the present sequencing study of the ISRs of T. asinigenitalis, CCTCCT sequences were observed in all isolates examined, where the 3' end of some bases downstream of the CCTCCT sequence may represent the probable 3' end of the 16S rRNA gene.
Consequently, the 16S-23S rDNA ISRs (A, B and C) from the three isolates of T. asinigenitalis were estimated to be approximately 837 to 955 bp. The ISR sequence data characteristically indicate that the three corresponding ISRs (A, B, and C) were identified to be identical to each other (UK-1 and UK-2 isolates). In addition, the ISR sequences shared nucleotide sequence similarities of approximately 95.3–98.9% between UCD-1T and UK-1/UK-2 isolates, respectively.
In addition, as shown in Figure 1, a dendrogram constructed, based on the nucleotide sequence information of the ISRs, demonstrated that T. asinigenitalis isolates formed a major cluster separating from T. equigenitalis isolates (Fig. 1). Thus, nucleotide sequence divergence with 16S-23S rDNA ISR for T. asinigenitalis could be useful to discriminate among the isolates and between species within the genus Taylorella.
Consequently, in the present study, it was demonstrated that three isolates of T. asinigenitalis possess three kinds of ISRs with different length, respectively. Thus, the present results may possibly indicate that T. asinigenitalis isolates carry at least three distinctly different ribosomal RNA operons in the genome.
Jang et al. (2001) previously demonstrated that pulsed-field gel electrophoresis (PFGE) profiles after digestion with Not I of the genomic DNAs from the two isolates (UK-1 and UK-2) of T. asinigenitalis were the same, but they differed from the other isolate (UCD-1T) . The present sequencing results of the 16S-23S rDNA ISR (A, B and C) clearly identified that the three ISRs were quite identical to each other [UK-1 and UK-2], but they were different from those of the UCD-1T, respectively. Thus, our present sequence informations of the ISRs are consistent with the result of PFGE analysis by Jang et al. (2001) . The two isolates (UK-1 and UK-2) of T. asinigenitalis obtained from donkey jacks in Kentucky, USA, in 1998  gave the quite identical profiles of the PFGE and the 16S-23S rDNA ISR sequences. Therefore, these two isolates may possibly be identical.
Overall, this study is the first report of 16S-23S rDNA ISR sequences and tRNA genes from T. asinigenitalis. This is also first molecular comparison of the ISRs from T. asinigenitalis with those from T. equigenitalis.
- T :
internal spacer region
polymerase chain reaction
pulsed-field gel electrophoresis.
This research was partially supported by The Promotion and Mutual Aid Corporation for Private Schools of Japan, Grant-in-Aid for Matching Fund Subsidy for Private Universities.
- Taylor CED, Rosenthal RO, Brown DFJ, Lapage SP, Hill LR, Legros RM: The causative organism of contagious equine metritis 1977: proposal for a new species to be known as Haemophilus equigenitalis. Equine Vet J. 1978, 10: 136-144.View ArticlePubMedGoogle Scholar
- Ter Laak EA, Fennema G, Jaartsveld FHJ: Contagious equine metritis in the Netherlands. Tijdschrift Diergeneeskd. 1989, 114: 189-201.Google Scholar
- Bleumink-Pluym NMC, Van Dijk L, Van Vliet AHM, Giessen Van Der JWB, Zeijst Van der BAM: Phylogenetic position of Taylorella equigenitalis determined by analysis of amplified 16S ribosomal DNA sequences. Int J Syst Bacteriol. 1993, 43: 618-621.View ArticlePubMedGoogle Scholar
- Matsuda M, Moore JE: Recent advances in molecular epidemiology and detection of Taylorella equigenitalis associated with contagious equine metritis (CEM). Vet Microbiol. 2003, 97: 111-122. 10.1016/j.vetmic.2003.08.001.View ArticlePubMedGoogle Scholar
- Katz JB, Evans LE, Hutto DL, Schroeder-Tucker LC, Carew AM, Donahue JM, Hirsh DC: Clinical, bacteriologic, serologic, and pathologic features of infections with atypical Taylorella asinigenitalis in mares. J Am Vet Assoc. 2000, 216: 1945-1948. 10.2460/javma.2000.216.1945.View ArticleGoogle Scholar
- Jang SS, Donahue JM, Arata AB, Goris J, Hansen LM, Earley DL, Vandamme PAR, Timoney PJ, Hirsh DC: Taylorella asinigenitalis sp. nov., a bacterium isolated from the genital tract of male donkeys (Equus asinus). Int J Syst Evol Microbiol. 2001, 51: 971-976.View ArticlePubMedGoogle Scholar
- Gurtler V, Stanisich VA: New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region. Microbiol. 1996, 142: 3-16.View ArticleGoogle Scholar
- Garcia-Martinez J, Acians SG, Anton AI, Rodriguez-Valera F: Use of the 16S-23S ribosomal genes spacer region in studies of prokaryotic diversity. J Microbiol Methods. 1999, 36: 55-64. 10.1016/S0167-7012(99)00011-1.View ArticlePubMedGoogle Scholar
- Matsuda M, Kaneko A, Fukuyama M, Itoh T, Shingaki M, Inoue M, Moore JE, Murphy PG, Ishida Y: First finding of urease-positive thermophilic strains of Campylobacter in river water in the Far East, namely in Japan, and their phenotypic and genotypic characterization. J Appl Bacteriol. 1996, 81: 608-612.Google Scholar
- Matsuda M, Kagawa S, Sakamoto Y, Miyajima M, Barton M, Moore JE: Detection of heterogeneous genotypes among Australian strains of Taylorella equigenitalis. Aus Vet J. 2000, 78: 56-57. 10.1111/j.1751-0813.2000.tb10362.x.View ArticleGoogle Scholar
- Sambrook J, Russell DW, Maniatis T: Molecular cloning: A Laboratory Manual. 2001, New York, Cold Spring Harbor Laboratory Press, 3Google Scholar
- Kagawa S, Nagano Y, Tazumi A, Murayama O, Millar BC, Moore JE, Matsuda M: Nucleotide sequencing and analysis of 16S rDNA and 16S-23S rDNA internal spacer region (ISR) of Taylorella equigenitalis, as an important pathogen for contagious equine metritis (CEM). Vet Res Commun. 2006, 30: 343-355. 10.1007/s11259-006-3304-6.View ArticlePubMedGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22: 4673-4680. 10.1093/nar/22.22.4673.PubMed CentralView ArticlePubMedGoogle Scholar
- Steitz JA: Genetic signals and nucleotide sequence in messenger RNA In Biological regulation and development I Gene expression. Edited by: Goldberg RF. 1979, New York, Plenum, 340-389.Google Scholar
- Benjamin L: Genes VII. 2000, New York. Oxford University PressGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.