1887

Abstract

As well as intraspecific heterogeneity, intragenomic heterogeneity between 16S rRNA gene copies has been described for a range of bacteria. Due to the wide use of 16S rRNA gene sequence analysis for taxonomy, identification and metagenomics, evaluating the extent of these heterogeneities in natural populations is an essential prerequisite. We investigated inter- and intragenomic 16S rRNA gene heterogeneity of the variable region V3 in a population of 149 clinical isolates of spp. of human origin and in 13 type or reference strains using PCR-temporal temperature gel electrophoresis (TTGE). 16S rRNA gene diversity was high in the studied population, as 45 different banding patterns were observed. Intragenomic heterogeneity was demonstrated for 110 (74 %) isolates and 8 (61.5 %) type or reference strains displaying two or three different gene copies. Polymorphic nucleotide positions accounted for 0.5–2.5 % of the sequence and were scattered in helices H16 and H17 of the rRNA molecule. Some of them changed the secondary structure of H17. Phylotaxonomic structure of the population based on the single-copy housekeeping gene was compared with TTGE patterns. The intragenomic V3 heterogeneity, as well as recombination events between strains or isolates of different clades, impaired the 16S rRNA-based identification for some species. Such approaches should be conducted in other bacterial populations to optimize the interpretation of 16S rRNA gene sequences in taxonomy and/or diversity studies.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.038224-0
2010-07-01
2019-10-17
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/7/2080.html?itemId=/content/journal/micro/10.1099/mic.0.038224-0&mimeType=html&fmt=ahah

References

  1. Aas, J. A., Paster, B. J., Stokes, L. N., Olsen, I. & Dewhirst, F. E. ( 2005; ). Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 43, 5721–5732.[CrossRef]
    [Google Scholar]
  2. Acinas, S. G., Marcelino, L. A., Klepac-Ceraj, V. & Polz, M. F. ( 2004; ). Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons. J Bacteriol 186, 2629–2635.[CrossRef]
    [Google Scholar]
  3. Adilakshmi, T., Ramaswamy, P. & Woodson, S. A. ( 2005; ). Protein-independent folding pathway of the 16S rRNA 5′ domain. J Mol Biol 351, 508–519.[CrossRef]
    [Google Scholar]
  4. Arif, N., Do, T., Byun, R., Sheehy, E., Clark, D., Gilbert, S. C. & Beighton, D. ( 2008; ). Veillonella rogosae sp. nov., an anaerobic, Gram negative coccus isolated from dental plaque. Int J Syst Evol Microbiol 58, 581–584.[CrossRef]
    [Google Scholar]
  5. Bhatti, M. A. & Frank, M. O. ( 2000; ). Veillonella parvula meningitis: case report and review of Veillonella infections. Clin Infect Dis 31, 839–840.[CrossRef]
    [Google Scholar]
  6. Boo, T. W., Cryan, B., O'Donnell, A. & Fahy, G. ( 2005; ). Prosthetic valve endocarditis caused by Veillonella parvula. J Infect 50, 81–83.[CrossRef]
    [Google Scholar]
  7. Byun, R., Carlier, J.-P., Jacques, N. A., Marchandin, H. & Hunter, N. ( 2007; ). Veillonella denticariosi sp. nov., isolated from human carious dentine. Int J Syst Evol Microbiol 57, 2844–2848.[CrossRef]
    [Google Scholar]
  8. Case, R. J., Boucher, Y., Dahllöf, I., Holmström, C., Doolittle, W. F. & Kjelleberg, S. ( 2007; ). Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies. Appl Environ Microbiol 73, 278–288.[CrossRef]
    [Google Scholar]
  9. Cilia, V., Lafay, B. & Christen, R. ( 1996; ). Sequence heterogeneities among 16S ribosomal RNA sequences, and their effect on phylogenetic analyses at the species level. Mol Biol Evol 13, 451–461.[CrossRef]
    [Google Scholar]
  10. Coenye, T. & Vandamme, P. ( 2003; ). Intragenomic heterogeneity between multiple 16S ribosomal RNA operons in sequenced bacterial genomes. FEMS Microbiol Lett 228, 45–49.[CrossRef]
    [Google Scholar]
  11. Conville, P. S. & Witebsky, F. G. ( 2007; ). Analysis of multiple differing copies of the 16S rRNA gene in five clinical isolates and three type strains of Nocardia species and implications for species assignment. J Clin Microbiol 45, 1146–1151.[CrossRef]
    [Google Scholar]
  12. Dahllöf, I., Baillie, H. & Kjelleberg, S. ( 2000; ). rpoB-based microbial community analysis avoids limitations inherent in 16S rRNA gene intraspecies heterogeneity. Appl Environ Microbiol 66, 3376–3380.[CrossRef]
    [Google Scholar]
  13. Favier, C. F., Vaughan, E. E., De Vos, W. M. & Akkermans, A. D. ( 2002; ). Molecular monitoring of succession of bacterial communities in human neonates. Appl Environ Microbiol 68, 219–226.[CrossRef]
    [Google Scholar]
  14. Felsenstein, J. ( 1997; ). An alternating least squares approach to inferring phylogenies from pairwise distances. Syst Biol 46, 101–111.[CrossRef]
    [Google Scholar]
  15. Graf, J. ( 1999; ). Diverse restriction fragment length polymorphism patterns of the PCR-amplified 16S rRNA genes in Aeromonas veronii strains and possible misidentification of Aeromonas species. J Clin Microbiol 37, 3194–3197.
    [Google Scholar]
  16. Guindon, S. & Gascuel, O. ( 2003; ). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52, 696–704.[CrossRef]
    [Google Scholar]
  17. Hall, T. A. ( 1999; ). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98.
    [Google Scholar]
  18. Huson, D. H. & Bryant, D. ( 2006; ). Application of phylogenetic networks in evolutionary studies. Mol Biol Evol 23, 254–267.
    [Google Scholar]
  19. Janse, I., Bok, J. & Zwart, G. ( 2004; ). A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis. J Microbiol Methods 57, 279–281.[CrossRef]
    [Google Scholar]
  20. Jensen, S., Frost, P. & Torsvik, V. L. ( 2009; ). The nonrandom microheterogeneity of 16S rRNA genes in Vibrio splendidus may reflect adaptation to versatile lifestyles. FEMS Microbiol Lett 294, 207–215.[CrossRef]
    [Google Scholar]
  21. Könönen, E., Kanervo, A., Takala, A., Asikainen, S. & Jousimies-Somer, H. ( 1999; ). Establishment of oral anaerobes during the first year of life. J Dent Res 78, 1634–1639.[CrossRef]
    [Google Scholar]
  22. López-López, A., Benlloch, S., Bonfá, M., Rodríguez-Valera, F. & Mira, A. ( 2007; ). Intragenomic 16S rDNA divergence in Haloarcula marismortui is an adaptation to different temperatures. J Mol Evol 65, 687–696.[CrossRef]
    [Google Scholar]
  23. Marchandin, H., Jean-Pierre, H., Carrière, C., Canovas, F., Darbas, H. & Jumas-Bilak, E. ( 2001; ). Prosthetic joint infection due to Veillonella dispar. Eur J Clin Microbiol Infect Dis 20, 340–342.
    [Google Scholar]
  24. Marchandin, H., Teyssier, C., Siméon de Buochberg, M., Jean-Pierre, H., Carrière, C. & Jumas-Bilak, E. ( 2003; ). Intra-chromosomal heterogeneity between the four 16S rRNA gene copies in the genus Veillonella: implications for phylogeny and taxonomy. Microbiology 149, 1493–1501.[CrossRef]
    [Google Scholar]
  25. Marchandin, H., Teyssier, C., Jumas-Bilak, E., Robert, M., Artigues, A.-C. & Jean-Pierre, H. ( 2005; ). Molecular identification of the first human isolate belonging to the Veillonella rattiVeillonella criceti group based on 16S rDNA and dnaK gene sequencing. Res Microbiol 156, 603–607.[CrossRef]
    [Google Scholar]
  26. Marriott, D., Stark, D. & Harkness, J. ( 2007; ). Veillonella parvula discitis and secondary bacteremia: a rare infection complicating endoscopy and colonoscopy? J Clin Microbiol 45, 672–674.[CrossRef]
    [Google Scholar]
  27. Martínez-Murcia, A. J., Antón, A. I. & Rodríguez-Valera, F. ( 1999; ). Patterns of sequence variation in two regions of the 16S rRNA multigene family of Escherichia coli. Int J Syst Bacteriol 49, 601–610.[CrossRef]
    [Google Scholar]
  28. Morandi, A., Zhaxybayeva, O., Gogarten, J. P. & Graf, J. ( 2005; ). Evolutionary and diagnostic implications of intragenomic heterogeneity in the 16S rRNA gene in Aeromonas trains. J Bacteriol 187, 6561–6564.[CrossRef]
    [Google Scholar]
  29. Moreno, C., Romero, J. & Espejo, R. T. ( 2002; ). Polymorphism in repeated 16S rRNA genes is a common property of type strains and environmental isolates of the genus Vibrio. Microbiology 148, 1233–1239.
    [Google Scholar]
  30. Muyzer, G. ( 1999; ). DGGE/TGGE a method for identifying genes from natural ecosystems. Curr Opin Microbiol 2, 317–322.[CrossRef]
    [Google Scholar]
  31. Neefs, J. M., Van de Peer, Y., De Rijk, P., Chapelle, S. & De Wachter, R. ( 1993; ). Compilation of small ribosomal subunit RNA structures. Nucleic Acids Res 21, 3025–3049.[CrossRef]
    [Google Scholar]
  32. Ninet, B., Monod, M., Emler, S., Pawlowski, J., Metral, C., Rohner, P., Auckenthaler, R. & Hirschel, B. ( 1996; ). Two different 16S rRNA genes in a mycobacterial strain. J Clin Microbiol 34, 2531–2536.
    [Google Scholar]
  33. Nübel, U., Engelen, B., Felske, A., Snaidr, J., Wieshuber, A., Amann, R. I., Ludwig, W. & Backhaush, H. ( 1996; ). Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacteriol 178, 5636–5643.
    [Google Scholar]
  34. Ogier, J. C., Son, O., Gruss, A., Tailliez, P. & Delacroix-Buchet, A. ( 2002; ). Identification of the bacterial microflora in dairy products by temporal temperature gradient gel electrophoresis. Appl Environ Microbiol 68, 3691–3701.[CrossRef]
    [Google Scholar]
  35. Paster, B. J., Boches, S. K., Galvin, J. L., Ericson, R. E., Lau, C. N., Levanos, V. A., Sahasrabudhe, A. & Dewhirst, F. E. ( 2001; ). Bacterial diversity in human subgingival plaque. J Bacteriol 183, 3770–3783.[CrossRef]
    [Google Scholar]
  36. Preza, D., Olsen, I., Aas, J. A., Willumsen, T., Grinde, B. & Paster, B. J. ( 2008; ). Bacterial profiles of root caries in elderly patients. J Clin Microbiol 46, 2015–2021.[CrossRef]
    [Google Scholar]
  37. Roudière, L., Lorto, S., Tallagranda, E., Marchandin, H., Jeannot, J.-L. & Jumas-Bilak, E. ( 2007; ). Empreinte moléculaire des communautés bactériennes et hétérogénéité intraspécifique de l'ADNr 16S: est-il raisonnable d'occulter le problème? Pathol Biol (Paris) 55, 434–440 (in French).[CrossRef]
    [Google Scholar]
  38. Roudière, L., Jacquot, A., Marchandin, H., Aujoulat, F., Devine, R., Zorgniotti, I., Jean-Pierre, H., Picaud, J.-C. & Jumas-Bilak, E. ( 2009; ). Optimized PCR-temporal temperature gel electrophoresis compared to cultivation to assess diversity of gut microbiota in neonates. J Microbiol Methods 79, 156–165.[CrossRef]
    [Google Scholar]
  39. Santoyo, G. & Romero, D. ( 2005; ). Gene conversion and concerted evolution in bacterial genomes. FEMS Microbiol Rev 29, 169–183.
    [Google Scholar]
  40. Satokari, R. M., Vaughan, E. E., Akkermans, A. D. L., Saarela, M. & De Vos, W. M. ( 2001; ). Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis. Appl Environ Microbiol 67, 504–513.[CrossRef]
    [Google Scholar]
  41. Stackebrandt, E. & Ebers, J. ( 2006; ). Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 33, 152–155.
    [Google Scholar]
  42. Stackebrandt, E. & Goebel, B. M. ( 1994; ). Taxonomic note: a place for DNA–DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.[CrossRef]
    [Google Scholar]
  43. Sundquist, A., Bigdeli, S., Jalili, R., Druzin, M. L., Waller, S., Pullen, K. M., El-Sayed, Y. Y., Taslimi, M. M., Batzoglou, S. & Ronaghi, M. ( 2007; ). Bacterial flora-typing with targeted, chip-based Pyrosequencing. BMC Microbiol 7, 108 [CrossRef]
    [Google Scholar]
  44. Teyssier, C., Marchandin, H., Siméon de Buochberg, M., Ramuz, M. & Jumas-Bilak, E. ( 2003; ). Atypical 16S rRNA gene copies in Ochrobactrum intermedium strains reveal a large genomic rearrangement by recombination between rrn copies. J Bacteriol 185, 2901–2909.[CrossRef]
    [Google Scholar]
  45. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. ( 1997; ). The clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.[CrossRef]
    [Google Scholar]
  46. Ueda, K., Seki, T., Kudo, T., Yoshida, T. & Kataoka, M. ( 1999; ). Two distinct mechanisms cause heterogeneity of 16S rRNA. J Bacteriol 181, 78–82.
    [Google Scholar]
  47. van Berkum, P., Terefework, Z., Paulin, L., Suomalainen, S., Lindström, K. & Eardly, B. D. ( 2003; ). Discordant phylogenies within the rrn loci of Rhizobia. J Bacteriol 185, 2988–2998.[CrossRef]
    [Google Scholar]
  48. Vásquez, A., Ahrné, S., Pettersson, B. & Molin, G. ( 2001; ). Temporal temperature gradient gel electrophoresis (TTGE) as a tool for identication of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus. Lett Appl Microbiol 32, 215–219.[CrossRef]
    [Google Scholar]
  49. Wang, M., Ahrné, S., Antonsson, M. & Molin, G. ( 2004; ). T-RFLP combined with principal component analysis and 16S rRNA gene sequencing: an effective strategy for comparison of fecal microbiota in infants of different ages. J Microbiol Methods 59, 53–69.[CrossRef]
    [Google Scholar]
  50. Yap, W. H., Zhang, Z. & Wang, Y. ( 1999; ). Distinct types of rRNA operons exist in the genome of the actinomycete Thermomonospora chromogena and evidence for horizontal transfer of an entire rRNA operon. J Bacteriol 181, 5201–5209.
    [Google Scholar]
  51. Zilberstein, B., Quintanilha, A. G., Santos, M. A., Pajecki, D., Moura, E. G., Alves, P. R., Maluf Filho, F., de Souza, J. A. & Gama-Rodrigues, J. ( 2007; ). Digestive tract microbiota in healthy volunteers. Clinics 62, 47–54.
    [Google Scholar]
  52. Zuker, M. ( 2003; ). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31, 3406–3415.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.038224-0
Loading
/content/journal/micro/10.1099/mic.0.038224-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error