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Demonstration of the absence of intervening sequences (IVSs) within 16S rRNA genes of Taylorella equigenitalis and Taylorella asinigenitalis isolates
Research in Veterinary Science 92 (2012) 435–437
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Demonstration of the absence of intervening sequences (IVSs) within 16S rRNA genes of Taylorella equigenitalis and Taylorella asinigenitalis isolates Akihiro Tazumi a,1, Shigeyuki Nakanishi a,1, Kyohei Hayashi a, Sandrine Petry b, Erina Tasaki a, Takuya Nakajima a, Hitomi Ueno a, John E. Moore c, Beverley C. Millar c, Motoo Matsuda a,⇑ a b c
Laboratory of Molecular Biology, Graduate School of Environmental Health Sciences, Azabu University, Fuchinobe 1-17-71, Chuo-ku, Sagamihara 252-5201, Japan ANSES, Laboratoire de Pathologie Equine, Dozul 14430, France Department of Bacteriology, Northern Ireland Public Health Laboratory, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK
a r t i c l e
i n f o
Article history: Received 22 October 2010 Accepted 11 April 2011
a b s t r a c t A total of 57 Taylorella equigenitalis (n = 22) and Taylorella asinigenitalis (n = 35) isolates was shown not to carry any intervening sequences (IVSs) within 16S rRNA gene sequences. By contrast, we have already shown the genus Taylorella group to carry several kinds of IVSs within the 23S rRNA gene sequences. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: 16S rRNA gene Intervening sequence (ISR) Taylorella asinigenitalis Taylorella equigenitalis
Taylorella equigenitalis is an important pathogen responsible for contagious equine metritis (CEM) (Matsuda and Moore, 2003). CEM and its causative agent have been detected in several countries and in various breeds of horses (Crowhurst, 1977; Matsuda and Moore, 2003). After three atypical CEM isolates had been obtained from donkey jacks (Equus asinus) in the USA, a second species, Taylorella asinigenitalis, was then established (Jang et al., 2001). Most recently, other isolates of T. asinigenitalis have been reported (Baverud et al., 2006; Breuil et al., 2010). Wehavereportednucleotidesequencingandanalysesof16SrDNA and 16S–23S rDNA internal spacer region of the three representative strains (NCTC11184T, Kentucky 188 and EQ59) and homogeneity of the 16S rDNA among geographically disparate isolates of T. equigenitalis (Kagawa et al., 2006; Matsuda et al., 2006). Regarding the bacterial 23S rRNA, the excisions of intervening sequences (IVSs) from 23S rRNA (Burgin et al., 1990) and the fragmentation of 23S rRNA (Hsu et al., 1994) have been demonstrated. In T. asinigenitalis, heterogeneous IVSs were previously identified in both the first quarter and central regions within 23S rRNA gene sequences, as well as the 23S rRNA fragmentation pattern (Tazumi et al., 2007, 2008a). T. equigenitalis has also been identified ⇑ Corresponding author. Address: Laboratory of Molecular Biology, Graduate School of Environmental Health Sciences, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara 252-5201, Japan. Tel.: +81 42 769 1942; fax: +81 42 754 7661. E-mail address: [email protected] (M. Matsuda). 1 These authors contributed equally to this study and hence should be considered as joint first authors. 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.04.010
to carry IVSs within the genes and harbors fragmented 23S rRNAs (Tazumi et al., 2008b). Regarding 16S rRNA genes in the genus Taylorella organisms, no descriptions of IVS appeared. Therefore, the present study aims to clarify whether or not IVSs occur within 16S rRNA genes of T. equigenitalis and T. asinigenitalis. T. equigenitalis (n = 22) and T. asinigenitalis (n = 35) isolates shown in Table 1 were analyzed in the present study. The propagation of the isolates was carried out according to the procedures described previously (Kagawa et al., 2006). Whole genome DNA was prepared, according to the procedure described already (Tazumi et al., 2007, 2008a,b). The primer fD1 used contained the 30 end 19-nucleotide sequence of the original fD1 primer sequence (Kagawa et al., 2006). The nucleotide (G) of the 50 end of the fD1 corresponds to the nucleotide position (np) 9 bp of the 16S rRNA gene from the rrnB of Escherichia coli (Brosius et al., 1978). The rTel reverse primer was constructed based on the nucleotide sequence information of the 30 end region of the rrnB (DDBJ/EMBL/GenBank accession no. J01695) (Kagawa et al., 2006). The purified amplicons were then subjected to cycle sequencing with BigDye Terminator (Applied Biosystems, Tokyo, Japan). Sequence analysis was performed using the GENETYX-Windows computer software (version 9; GENETYX Co., Tokyo, Japan). Nucleotide sequences were compared to each other and with the accessible sequence data from some other than the T. equigenitalis and T. asinigenitalis, which were available in the databases.
436
A. Tazumi et al. / Research in Veterinary Science 92 (2012) 435–437
Table 1 Isolates of T. equigenitalis and T. asinigenitalis analyzed in the present study. Isolate no. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.
e e e e e e e e e e e e e e e e e e e e e e e a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a
T
NCTC11184 Kentucky188 EQ59 NCTC11184T N480/82 N610/88 EQ70 HH139 SS28 CEMO12 CEMO13 CEMO14 Aus 1 Aus 2 Aus 3 Aus 4 Aus 5 Aus 6 Aus 7 Fr-1 Fr-2 Fr-9 Fr-10 UK-1 UK-2 UCD-1T FSP2 FSP3 FSP6 FSP7 FSP10 FSP11 FSP12 FSP13 FSP14 FSP21 FSP22 FSP23 FSP24 FSP47 FSP73 FSP76 FSP85 FSP92 FSP93 FSP94 FSP97 FSP214 FSP235 FSP265 FSP266 FSP473 FSP474 FSP475 FSP476 FSP477 FSP479 FSP480
Breed
Sex
Country
DDBJ/EMBL/GenBank accession no.
Thoroughbred NA Thoroughbred Thoroughbred NA NA Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred NA NA French Trotter French Trotter Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Equine Donkey jack
Female Female Female Female NA NA Female Female Female Female Female Female Female Female Female Female Female Female Female NA NA Male Female Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male
England USA Japan England NA NA Japan Japan Japan Japan Japan Japan Australia Australia Australia Australia Australia Australia Australia France France France France USA USA USA France France France France France France France France France France France France France France France France France France France France France France France France France France France France France France France France
AF408197 AB066372 AB069660 X68645 X68645 X68645 AB200402 AB200397 AB200403 AB200404 AB200405 AB200406 AB200407 AB200408 AB200409 AB200410 AB200411 AB200412 AB200413 AB200398 AB200399 AB200400 AB200401 AB259167 AB297175 AB067729 AB595196 NA NA NA AB595197 NA AB595198 NA NA NA NA NA NA NA NA AB595199 NA NA NA AB595200 NA NA NA NA NA NA NA NA NA NA NA NA
T., Taylorella; e, equigenitalis; a, asinigenitalis; NCTC, National Collection of Type Cultures; NA, not available; USA, the United States of America.
The primer set for 16S rRNA gene (fD1/rTel) amplified an amplicon of about 1500 bp in length with the three reference strains of T. equigenitalis (NCTC11184T, Kentucky 188 and EQ59; AF408197, AB066372 and AB069660), as already described (Kagawa et al., 2006). Sequence differences of 16S rRNA genes among the four sequences, including the reference sequences [NCTC11184T, N480/ 82, N610/88 (X68645)] occurred at only a few nucleotide positions [Kagawa et al., 2006; see the Fig. 1A in the literature]. No IVSs were identified to occur within 16S rRNA genes among the three reference T. equigenitalis strains, based on the PCR amplification and sequencing procedures of nearly full-length 16S rRNA gene (Kagawa et al., 2006), as well as in reference sequences. Thus,
firstly, 16S rRNA genes from the five T. equigenitalis strains, did not carry any IVSs. We have already carried out cloning, sequencing and comparison of the nearly full-length 16S rRNA genes using the primer pair of fD1/rTe1 (Kagawa et al., 2006) from 17 isolates of T. equigenitalis in Japan, Australia and France (Matsuda et al., 2006). When nucleotide sequence alignment analyses of the sequences were carried out, sequence differences were demonstrated at the six loci (Matsuda et al., 2006), and no IVSs were identified within 16S rRNA gene sequences among the 17 isolates. When nucleotide sequence alignment analysis of full-length 16S rRNA gene sequences from the three American reference
A. Tazumi et al. / Research in Veterinary Science 92 (2012) 435–437
M 1 2 kbp
2.0 1.5 1.0
437
have already been completed (30 genera and 51 species), no IVSs were identified with all the isolates examined (data not shown). Thus, the 16S rRNA gene sequences may not possibly carry any IVSs within the genes from the b-subclass. The absence of IVSs within 16S rRNA genes from T. equigenitalis and T. asinigenitalis isolates suggests that these regions are highly conserved. Minor sequence variations would prevent the complete excision of the IVSs from the gene transcript. Sequence homogeneity of the 16S rDNA (99.5% or more) among geographically disparate isolates of T. equigenitalis (n = 23) (Matsuda et al., 2006), among all T. asinigenitalis examined (n = 43; 99.3–100%) and between all T. asinigenitalis and T. equigenitalis NCTC11184T sequences (Breuil et al., 2010) has already been shown. Therefore, highly conserved 16S rRNA sequences lacking the IVSs may be a useful marker for the reliable molecular identification/discrimination between these two species. Acknowledgement This research was partially supported by a research project grant awarded by the Azabu University (Research Services Division). References
Fig. 1. Agarose gel electrophoresis profiles of PCR products generated with T. asinigenitalis isolates by using a primer pair (fD1/rTel), constructed to amplify the nearly full-length 16S rRNA genes. Lane M, 1 kbp DNA ladder; lane 1, T. asinigenitalis French isolate FSP2; lanes 2 and 3.
T. asinigenitalis strains (Jang et al., 2001) with the same methodology, UK-1 (AF297774; AB259167), UK-2 (AF297175) and UCD-1T (AF067729) were carried out, no IVSs were identified (data not shown). Then, we attempted to amplify the nearly full-length 16S rRNA gene sequences from the other 32 French T. asinigenitalis isolates (Breuil et al., 2010) using the same primer pair, which was employed for the analyses of T. equigenitalis. Agarose gel electrophoresis of the 32 isolates is virtually indistinguishable from others, and only two representative profiles were shown (Fig. 1). Some nucleotide sequences from the 32 isolates were submitted to DDBJ/ EMBL/GenBank, as shown in Table 1. Thus, no IVSs were identified to occur within 16S rRNA genes with all the 32 isolates examined. Overall, in 35 T. asinigenitalis isolates, no IVSs were identified to occur within 16S rRNA gene sequences. As described above, we have already the T. asinigenitalis isolates to carry heterogeneous and multiple IVSs in the first quarter and central regions within 23S rRNA gene sequences identified (Tazumi et al., 2007, 2008a). We have also reported that all the three representative T. equigenitalis strains (NCTC11184T, Kentucky 188 and EQ59) possessed one 70 bp IVS (TeIVS2) in the central region, different from any IVSs within 23S rRNA genes found in T. asinigenitalis (Tazumi et al., 2008b). When we carried out the nucleotide sequence alignment analyses of the 16S rRNA gene sequences from at least 72 isolates belonging to the Proteobacteria b-subclass whose genome analyses
Baverud, V., Nyström, C., Johansson, K-E., 2006. Isolation and identification of Taylorella asinigenitalis from the genital tract of a stallion, first case of a natural infection. Veterinary Microbiology 11, 294–300. Breuil, M.F., Duquesne, F., Laugier, C., Petry, S., 2010. Phenotypic and 16S ribosomal RNA gene diversity of Taylorella asinigenitalis strains between 1995 to 2008. Veterinary Microbiology 10, doi: 1016/j.vetmic.2010.08.028. Brosius, J., Palmer, M.L., Kennedy, P.J., Noller, H.F., 1978. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. The Proceedings of the National Academy of Sciences, the United States of America 75, 4801–4805. Burgin, A.B., Parodos, K., Lane, D.J., Pace, N.R., 1990. The excision of intervening sequences from Salmonella 23S ribosomal RNA. Cell 60, 405–414. Crowhurst, R.C., 1977. Genital infection in mares. The Veterinary Record 100, 476. Hsu, D., Shih, L.M., Zee, Y.C., 1994. Degradation of rRNA in Salmonella strains: a novel mechanism to regulate the concentrations of rRNA and ribosomes. Journal of Bacteriology 176, 4761–4765. Jang, S.S., Donahue, J.M., Arata, A.B., Goris, J., Hansen, L.M., Earley, D.L., Vandamme, P.A.R., Timoney, P.J., Hirsh, D.C., 2001. Taylorella asinigenitalis sp. nov., a bacterium isolated from the genital tract of male donkeys (Equus asinus). International Journal of Systematic Evolution and Microbiology 51, 971–976. Kagawa, S., Nagano, Y., Tazumi, A., Murayama, O., Millar, B.C., Moore, J.E., Matsuda, M., 2006. 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). Veterinary Research Communications 30, 343–355. Matsuda, M., Moore, J.E., 2003. Recent advances in molecular epidemiology and detection of Taylorella equigenitalis associated with contagious equine metritis (CEM). Veterinary Microbiology 97, 111–122. Matsuda, M., Tazumi, A., Kagawa, S., Sekizuka, T., Murayama, O., Moore, J.E., Millar, B.C., 2006. Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis. BMC Veterinary Research 2, 1. doi:10:1186/1746-6148/2/1. Tazumi, A., Saito, S., Sekizuka, T., Murayama, O., Takamiya, S., Moore, J.E., Millar, B.C., Matsuda, M., 2007. Molecular characterization of the full-length 23S and 5S ribosomal RNA (rRNA) genes of Taylorella asinigenitalis. Antonie van Leeuwenhoek 92, 257–264. Tazumi, A., Sekizuka, T., Moore, J.E., Millar, B.C., Taneike, I., Matsuda, M., 2008a. Intervening sequences in 23S rRNA genes and 23S rRNA fragmentation in Taylorella asinigenitalis UCD-1T strain. Journal of Basic Microbiology 48, 284– 292. Tazumi, A., Sekizuka, T., Moore, J.E., Millar, B.C., Taneike, I., Matsuda, M., 2008b. Molecular characterization of intervening sequences in 23S rRNA genes and 23S rRNA fragmentation in Taylorella equigenitalis. Folia Microbiologica 53, 486– 492.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Demonstration of the absence of intervening sequences (IVSs) within 16S rRNA genes of Taylorella equigenitalis and Taylorella asinigenitalis isolates Akihiro Tazumi a,1, Shigeyuki Nakanishi a,1, Kyohei Hayashi a, Sandrine Petry b, Erina Tasaki a, Takuya Nakajima a, Hitomi Ueno a, John E. Moore c, Beverley C. Millar c, Motoo Matsuda a,⇑ a b c
Laboratory of Molecular Biology, Graduate School of Environmental Health Sciences, Azabu University, Fuchinobe 1-17-71, Chuo-ku, Sagamihara 252-5201, Japan ANSES, Laboratoire de Pathologie Equine, Dozul 14430, France Department of Bacteriology, Northern Ireland Public Health Laboratory, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK
a r t i c l e
i n f o
Article history: Received 22 October 2010 Accepted 11 April 2011
a b s t r a c t A total of 57 Taylorella equigenitalis (n = 22) and Taylorella asinigenitalis (n = 35) isolates was shown not to carry any intervening sequences (IVSs) within 16S rRNA gene sequences. By contrast, we have already shown the genus Taylorella group to carry several kinds of IVSs within the 23S rRNA gene sequences. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: 16S rRNA gene Intervening sequence (ISR) Taylorella asinigenitalis Taylorella equigenitalis
Taylorella equigenitalis is an important pathogen responsible for contagious equine metritis (CEM) (Matsuda and Moore, 2003). CEM and its causative agent have been detected in several countries and in various breeds of horses (Crowhurst, 1977; Matsuda and Moore, 2003). After three atypical CEM isolates had been obtained from donkey jacks (Equus asinus) in the USA, a second species, Taylorella asinigenitalis, was then established (Jang et al., 2001). Most recently, other isolates of T. asinigenitalis have been reported (Baverud et al., 2006; Breuil et al., 2010). Wehavereportednucleotidesequencingandanalysesof16SrDNA and 16S–23S rDNA internal spacer region of the three representative strains (NCTC11184T, Kentucky 188 and EQ59) and homogeneity of the 16S rDNA among geographically disparate isolates of T. equigenitalis (Kagawa et al., 2006; Matsuda et al., 2006). Regarding the bacterial 23S rRNA, the excisions of intervening sequences (IVSs) from 23S rRNA (Burgin et al., 1990) and the fragmentation of 23S rRNA (Hsu et al., 1994) have been demonstrated. In T. asinigenitalis, heterogeneous IVSs were previously identified in both the first quarter and central regions within 23S rRNA gene sequences, as well as the 23S rRNA fragmentation pattern (Tazumi et al., 2007, 2008a). T. equigenitalis has also been identified ⇑ Corresponding author. Address: Laboratory of Molecular Biology, Graduate School of Environmental Health Sciences, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara 252-5201, Japan. Tel.: +81 42 769 1942; fax: +81 42 754 7661. E-mail address: [email protected] (M. Matsuda). 1 These authors contributed equally to this study and hence should be considered as joint first authors. 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.04.010
to carry IVSs within the genes and harbors fragmented 23S rRNAs (Tazumi et al., 2008b). Regarding 16S rRNA genes in the genus Taylorella organisms, no descriptions of IVS appeared. Therefore, the present study aims to clarify whether or not IVSs occur within 16S rRNA genes of T. equigenitalis and T. asinigenitalis. T. equigenitalis (n = 22) and T. asinigenitalis (n = 35) isolates shown in Table 1 were analyzed in the present study. The propagation of the isolates was carried out according to the procedures described previously (Kagawa et al., 2006). Whole genome DNA was prepared, according to the procedure described already (Tazumi et al., 2007, 2008a,b). The primer fD1 used contained the 30 end 19-nucleotide sequence of the original fD1 primer sequence (Kagawa et al., 2006). The nucleotide (G) of the 50 end of the fD1 corresponds to the nucleotide position (np) 9 bp of the 16S rRNA gene from the rrnB of Escherichia coli (Brosius et al., 1978). The rTel reverse primer was constructed based on the nucleotide sequence information of the 30 end region of the rrnB (DDBJ/EMBL/GenBank accession no. J01695) (Kagawa et al., 2006). The purified amplicons were then subjected to cycle sequencing with BigDye Terminator (Applied Biosystems, Tokyo, Japan). Sequence analysis was performed using the GENETYX-Windows computer software (version 9; GENETYX Co., Tokyo, Japan). Nucleotide sequences were compared to each other and with the accessible sequence data from some other than the T. equigenitalis and T. asinigenitalis, which were available in the databases.
436
A. Tazumi et al. / Research in Veterinary Science 92 (2012) 435–437
Table 1 Isolates of T. equigenitalis and T. asinigenitalis analyzed in the present study. Isolate no. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.
e e e e e e e e e e e e e e e e e e e e e e e a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a
T
NCTC11184 Kentucky188 EQ59 NCTC11184T N480/82 N610/88 EQ70 HH139 SS28 CEMO12 CEMO13 CEMO14 Aus 1 Aus 2 Aus 3 Aus 4 Aus 5 Aus 6 Aus 7 Fr-1 Fr-2 Fr-9 Fr-10 UK-1 UK-2 UCD-1T FSP2 FSP3 FSP6 FSP7 FSP10 FSP11 FSP12 FSP13 FSP14 FSP21 FSP22 FSP23 FSP24 FSP47 FSP73 FSP76 FSP85 FSP92 FSP93 FSP94 FSP97 FSP214 FSP235 FSP265 FSP266 FSP473 FSP474 FSP475 FSP476 FSP477 FSP479 FSP480
Breed
Sex
Country
DDBJ/EMBL/GenBank accession no.
Thoroughbred NA Thoroughbred Thoroughbred NA NA Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred Thoroughbred NA NA French Trotter French Trotter Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Donkey jack Equine Donkey jack
Female Female Female Female NA NA Female Female Female Female Female Female Female Female Female Female Female Female Female NA NA Male Female Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male
England USA Japan England NA NA Japan Japan Japan Japan Japan Japan Australia Australia Australia Australia Australia Australia Australia France France France France USA USA USA France France France France France France France France France France France France France France France France France France France France France France France France France France France France France France France France
AF408197 AB066372 AB069660 X68645 X68645 X68645 AB200402 AB200397 AB200403 AB200404 AB200405 AB200406 AB200407 AB200408 AB200409 AB200410 AB200411 AB200412 AB200413 AB200398 AB200399 AB200400 AB200401 AB259167 AB297175 AB067729 AB595196 NA NA NA AB595197 NA AB595198 NA NA NA NA NA NA NA NA AB595199 NA NA NA AB595200 NA NA NA NA NA NA NA NA NA NA NA NA
T., Taylorella; e, equigenitalis; a, asinigenitalis; NCTC, National Collection of Type Cultures; NA, not available; USA, the United States of America.
The primer set for 16S rRNA gene (fD1/rTel) amplified an amplicon of about 1500 bp in length with the three reference strains of T. equigenitalis (NCTC11184T, Kentucky 188 and EQ59; AF408197, AB066372 and AB069660), as already described (Kagawa et al., 2006). Sequence differences of 16S rRNA genes among the four sequences, including the reference sequences [NCTC11184T, N480/ 82, N610/88 (X68645)] occurred at only a few nucleotide positions [Kagawa et al., 2006; see the Fig. 1A in the literature]. No IVSs were identified to occur within 16S rRNA genes among the three reference T. equigenitalis strains, based on the PCR amplification and sequencing procedures of nearly full-length 16S rRNA gene (Kagawa et al., 2006), as well as in reference sequences. Thus,
firstly, 16S rRNA genes from the five T. equigenitalis strains, did not carry any IVSs. We have already carried out cloning, sequencing and comparison of the nearly full-length 16S rRNA genes using the primer pair of fD1/rTe1 (Kagawa et al., 2006) from 17 isolates of T. equigenitalis in Japan, Australia and France (Matsuda et al., 2006). When nucleotide sequence alignment analyses of the sequences were carried out, sequence differences were demonstrated at the six loci (Matsuda et al., 2006), and no IVSs were identified within 16S rRNA gene sequences among the 17 isolates. When nucleotide sequence alignment analysis of full-length 16S rRNA gene sequences from the three American reference
A. Tazumi et al. / Research in Veterinary Science 92 (2012) 435–437
M 1 2 kbp
2.0 1.5 1.0
437
have already been completed (30 genera and 51 species), no IVSs were identified with all the isolates examined (data not shown). Thus, the 16S rRNA gene sequences may not possibly carry any IVSs within the genes from the b-subclass. The absence of IVSs within 16S rRNA genes from T. equigenitalis and T. asinigenitalis isolates suggests that these regions are highly conserved. Minor sequence variations would prevent the complete excision of the IVSs from the gene transcript. Sequence homogeneity of the 16S rDNA (99.5% or more) among geographically disparate isolates of T. equigenitalis (n = 23) (Matsuda et al., 2006), among all T. asinigenitalis examined (n = 43; 99.3–100%) and between all T. asinigenitalis and T. equigenitalis NCTC11184T sequences (Breuil et al., 2010) has already been shown. Therefore, highly conserved 16S rRNA sequences lacking the IVSs may be a useful marker for the reliable molecular identification/discrimination between these two species. Acknowledgement This research was partially supported by a research project grant awarded by the Azabu University (Research Services Division). References
Fig. 1. Agarose gel electrophoresis profiles of PCR products generated with T. asinigenitalis isolates by using a primer pair (fD1/rTel), constructed to amplify the nearly full-length 16S rRNA genes. Lane M, 1 kbp DNA ladder; lane 1, T. asinigenitalis French isolate FSP2; lanes 2 and 3.
T. asinigenitalis strains (Jang et al., 2001) with the same methodology, UK-1 (AF297774; AB259167), UK-2 (AF297175) and UCD-1T (AF067729) were carried out, no IVSs were identified (data not shown). Then, we attempted to amplify the nearly full-length 16S rRNA gene sequences from the other 32 French T. asinigenitalis isolates (Breuil et al., 2010) using the same primer pair, which was employed for the analyses of T. equigenitalis. Agarose gel electrophoresis of the 32 isolates is virtually indistinguishable from others, and only two representative profiles were shown (Fig. 1). Some nucleotide sequences from the 32 isolates were submitted to DDBJ/ EMBL/GenBank, as shown in Table 1. Thus, no IVSs were identified to occur within 16S rRNA genes with all the 32 isolates examined. Overall, in 35 T. asinigenitalis isolates, no IVSs were identified to occur within 16S rRNA gene sequences. As described above, we have already the T. asinigenitalis isolates to carry heterogeneous and multiple IVSs in the first quarter and central regions within 23S rRNA gene sequences identified (Tazumi et al., 2007, 2008a). We have also reported that all the three representative T. equigenitalis strains (NCTC11184T, Kentucky 188 and EQ59) possessed one 70 bp IVS (TeIVS2) in the central region, different from any IVSs within 23S rRNA genes found in T. asinigenitalis (Tazumi et al., 2008b). When we carried out the nucleotide sequence alignment analyses of the 16S rRNA gene sequences from at least 72 isolates belonging to the Proteobacteria b-subclass whose genome analyses
Baverud, V., Nyström, C., Johansson, K-E., 2006. Isolation and identification of Taylorella asinigenitalis from the genital tract of a stallion, first case of a natural infection. Veterinary Microbiology 11, 294–300. Breuil, M.F., Duquesne, F., Laugier, C., Petry, S., 2010. Phenotypic and 16S ribosomal RNA gene diversity of Taylorella asinigenitalis strains between 1995 to 2008. Veterinary Microbiology 10, doi: 1016/j.vetmic.2010.08.028. Brosius, J., Palmer, M.L., Kennedy, P.J., Noller, H.F., 1978. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. The Proceedings of the National Academy of Sciences, the United States of America 75, 4801–4805. Burgin, A.B., Parodos, K., Lane, D.J., Pace, N.R., 1990. The excision of intervening sequences from Salmonella 23S ribosomal RNA. Cell 60, 405–414. Crowhurst, R.C., 1977. Genital infection in mares. The Veterinary Record 100, 476. Hsu, D., Shih, L.M., Zee, Y.C., 1994. Degradation of rRNA in Salmonella strains: a novel mechanism to regulate the concentrations of rRNA and ribosomes. Journal of Bacteriology 176, 4761–4765. Jang, S.S., Donahue, J.M., Arata, A.B., Goris, J., Hansen, L.M., Earley, D.L., Vandamme, P.A.R., Timoney, P.J., Hirsh, D.C., 2001. Taylorella asinigenitalis sp. nov., a bacterium isolated from the genital tract of male donkeys (Equus asinus). International Journal of Systematic Evolution and Microbiology 51, 971–976. Kagawa, S., Nagano, Y., Tazumi, A., Murayama, O., Millar, B.C., Moore, J.E., Matsuda, M., 2006. 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). Veterinary Research Communications 30, 343–355. Matsuda, M., Moore, J.E., 2003. Recent advances in molecular epidemiology and detection of Taylorella equigenitalis associated with contagious equine metritis (CEM). Veterinary Microbiology 97, 111–122. Matsuda, M., Tazumi, A., Kagawa, S., Sekizuka, T., Murayama, O., Moore, J.E., Millar, B.C., 2006. Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis. BMC Veterinary Research 2, 1. doi:10:1186/1746-6148/2/1. Tazumi, A., Saito, S., Sekizuka, T., Murayama, O., Takamiya, S., Moore, J.E., Millar, B.C., Matsuda, M., 2007. Molecular characterization of the full-length 23S and 5S ribosomal RNA (rRNA) genes of Taylorella asinigenitalis. Antonie van Leeuwenhoek 92, 257–264. Tazumi, A., Sekizuka, T., Moore, J.E., Millar, B.C., Taneike, I., Matsuda, M., 2008a. Intervening sequences in 23S rRNA genes and 23S rRNA fragmentation in Taylorella asinigenitalis UCD-1T strain. Journal of Basic Microbiology 48, 284– 292. Tazumi, A., Sekizuka, T., Moore, J.E., Millar, B.C., Taneike, I., Matsuda, M., 2008b. Molecular characterization of intervening sequences in 23S rRNA genes and 23S rRNA fragmentation in Taylorella equigenitalis. Folia Microbiologica 53, 486– 492.