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MIT Computer Science and Artificial Intelligence Laboratory, 32 Vassar Street, Cambridge, MA 02139, USA
Correspondence
Thomas F. Knight Jr
tk{at}mit.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Mesoplasma pleciae PS-1T is AY257485; that for the partial gyrB sequence of M. pleciae PS-1T is AY257486.
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), but its testing and interpretation are difficult and require experience and skill. Strain PS-1T, originally classified as ‘Acholeplasma pleciae’, was classified in the genus Mesoplasma based on its lack of sterol requirement and its need for fatty acid supplements. Increasingly, classification is based on genotype, especially the sequence data for the 16S rRNA gene, and, less widely, on the protein coding sequence of the DNA gyrase beta subunit gene (gyrB) (Watanabe et al., 2001).
Two differently dated lyophilized samples of Mesoplasma pleciae PS-1T (Tully, 1983; Clark & Whitcomb, 1984; Clark et al., 1986; Tully et al., 1994) obtained from the mollicute collection at the University of Florida, and a lyophilized sample of the equivalent ATCC culture, ATCC 49582T, were grown at 30 °C in M1D or SP-4 medium (Whitcomb, 1983).
Growth was quite slow (5 days) in both media, in marked contrast to my experience of much more rapid (1–2 days) growth with other Mesoplasma and Entomoplasma species.
Lysis and phenol/chloroform extraction of genomic DNA were performed using standard techniques.
PCR amplification of the 16S rRNA gene was performed (Johansson et al., 1998) using primers U1F and U8R, Qiagen Quantitect PCR mix, and cycling consisting of an initial 15 min hot start at 95 °C and 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 66 °C for 3 min. The unusually low extension temperature was found necessary to successfully amplify the very low G+C content regions found in some of the sequences (Su et al., 1996).
The approximately 1500 bp rRNA gene product was gel-purified and sequenced using primers U1F, U8R, U2F, U5R, U2R, U4R, U6F and U6R to achieve complete coverage in both directions of the entire fragment. Sequencing was done on an Applied Biosystems 310 instrument with BigDye v3.1 chemistry. Assembly was done with ABI Autoassembler software.
Novel degenerate primers for the gyrB gene were designed for the Entomoplasmatales by CLUSTAL W alignment of existing mollicute gyrB sequences, and careful selection of primers from highly conserved regions. PCR of the gyrB sequence was performed using degenerate primers gyrB-F (5'-gac ttg agg ctg tta gaa aam ghc cwg gna tgt a-3') and gyrB-R (5'-tgt ttg tca taa ttg atc wgv rty cat ytc wcc wa-3'). The resulting approximately 1700 bp fragment was gel-purified and sequenced with primers gyrB-seq-F (5'-gac ttg agg ctg tta gaa-3') and gyrB-seq-R (5'-tgt ttg tca taa ttg atc-3'). Four additional species-specific primers were chosen and synthesized to complete forward and reverse sequencing of the gyrB fragment.
16S and gyrB sequencing was performed on all three samples obtained, with identical results.
BLAST analysis of the 16S rRNA gene sequence showed closest alignment with Acholeplasma laidlawii (98 % sequence identity). All other top matches were Acholeplasma or Phytoplasma species from the order Acholeplasmatales, with no matches above 90 % identity to species outside this order. Other Mesoplasma species matched at the 82 % level.
The nucleotide alignment of the gyrB gene showed an 85 % identity to the nucleotide sequence of A. laidlawii with no similarly significant matches to any other GenBank entries.
The amino acid alignment of the partial gyrB sequence showed a 93 % identity to the sequence for A. laidlawii. Despite the presence of ten sequenced gyrB genes from other Mesoplasma and Entomoplasma species in GenBank, the next most significant match was 62 %, to the gyrB sequence of Staphylococcus epidermidis, a species quite outside the mollicute clade.
The partial amino acid sequence of the gyrB gene shows three occurrences of the amino acid tryptophan. In each case, the codon used for this amino acid is UGG, the standard bacterial codon. Coding sequences in the order Entomoplasmatales almost always use UGA as the codon for tryptophan. For example, other gyrB sequences from the Entomoplasmatales show 18 tryptophan codons, of which 17 use UGA as the codon. The single use of UGG occurs in Entomoplasma luminosum (GenBank/EMBL/DDBJ accession no. AY196995), which also has an occurrence of UGA in the same sequence.
The three occurrences of UGG and the lack of any UGA codons in the M. pleciae coding sequence strongly suggest that the organism uses UGA as a stop codon, rather than as a tryptophan codon, as would be expected from a species of the Acholeplasmatales.
I believe that the combination of 16S rRNA and gyrB nucleotide sequence similarity to Acholeplasmatales species, the lack of similarity of nucleotide and amino acid sequences to Entomoplasmatales species, and the use of UGG rather than UGA as the preferred codon for tryptophan make a compelling argument that this species is properly classified as a member of the genus Acholeplasma.
Description of Acholeplasma pleciae comb. nov.
The description is based upon that given by Tully et al. (1993) for Mesoplasma pleciae.
The type strain is PS-1T (=ATCC 49582T=NBRC 100476T); its 16S rRNA and gyrB gene sequences are available from GenBank/EMBL/DDBJ under accession numbers AY257485 and AY257486, respectively.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Clark, T. B., Tully, J. G., Rose, D. L., Henegar, R. & Whitcomb, R. F. (1986). Acholeplasmas and similar nonsterol-requiring mollicutes from insects: missing link in microbial ecology. Curr Microbiol 13, 11–16.[CrossRef]
Johansson, K.-E., Heldtander, M. U. K. & Pettersson, B. (1998). Characterization of mycoplasmas by PCR and sequence analysis with universal 16S rDNA primers. Methods Mol Biol 104, 145–165.[Medline]
Su, X., Wu, Y., Sifri, C. D. & Wellems, T. E. (1996). Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. Nucleic Acid Res 24, 1574–1575.
Tully, J. G. (1983). The Emmy Klieneberger-Nobel Award lecture. Reflections on recovery of some fastidious mollicutes with implications of the changing host patterns of these organisms. Yale J Biol Med 56, 799–813.[Medline]
Tully, J. G., Bové, J. M., Laigret, F. & Whitcomb, R. F. (1993). Revised taxonomy of the class Mollicutes: proposed elevation of a monophyletic cluster of arthropod-associated mollicutes to ordinal rank (Entomoplasmatales ord. nov.), with provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceae fam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae. Int J Syst Bacteriol 43, 378–385.
Tully, J. G., Whitcomb, R. F., Hackett, K. J., Rose, D. L., Henagar, R. B., Bové, J. M., Carle, P., Williamson, D. L. & Clark, T. B. (1994). Taxonomic descriptions of eight new non-sterol-requiring mollicutes assigned to the genus Mesoplasma. Int J Syst Bacteriol 44, 685–693.
Watanabe, K., Nelson, J., Harayama, S. & Kasai, H. (2001). ICB database: the gyrB database for identification and classification of bacteria. Nucleic Acid Res 29, 344–345.
Whitcomb, R. (1983). Culture media for spiroplasmas. In Methods in Mycoplasmology, vol. 1, Mycoplasma Characterization, pp. 147–158. Edited by S. Razin & J. G. Tully. New York: Academic Press.
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