1887

Abstract

Intercistronic heterogeneity of the 16S–23S rRNA internal transcribed spacer regions (ITS1) was investigated in 29 strains of fluorescent pseudomonads isolated from subterranean seeds of (hog peanut). PCR amplification of the ITS1 region generated one or two products from the strains. Sequence analysis of the amplified fragments revealed an ITS1 fragment of about 517 bp that contained genes for tRNA and tRNA in all 29 strains; an additional smaller ITS1 of 279 bp without tRNA features was detected in 15 of the strains. The length difference appeared to be due to deletions of several nucleotide blocks between the 70 bp and 359 bp positions of the alignment. The end of the deletions in the variant ITS1 type coincided with the start of antiterminator box A, which is homologous to box A of other bacteria. Phylogenetic analyses using the neighbour-joining algorithm revealed two major phylogenetic clusters, one for each of the ITS1 types. Using a single specific primer set and the DNA-intercalating dye SYBR Green I for real-time PCR and melting-curve analysis produced highly informative curves with one or two recognizable melting peaks that readily distinguished between the two ITS1 types in pure cultures. The assay was used to confirm the presence of the variant ITS1 type in the community in total DNA from root-zone soil and seed coats of hog peanut. Heterogeneity of the ITS1 region between species has potential for studying molecular systematics and population genetics of the genus , but the presence of non-identical rRNA operons within a genome may pose problems.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.028274-0
2009-08-01
2019-12-14
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/8/2630.html?itemId=/content/journal/micro/10.1099/mic.0.028274-0&mimeType=html&fmt=ahah

References

  1. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. ( 1997; ). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.[CrossRef]
    [Google Scholar]
  2. Anton, A. I., Martinez, A. J. & Rodriguez-Valera, F. ( 1998; ). Sequence diversity in the 16S–23S intergenic spacer region (ISR) of the rRNA operons in representatives of Escherichia coli ECOR collection. J Mol Evol 47, 62–72.[CrossRef]
    [Google Scholar]
  3. Boyer, S. L., Flechtner, V. R. & Johansen, J. R. ( 2001; ). Is the 16S–23S rRNA internal transcribed spacer region a good tool for use in molecular systematics and population genetics? A case study in cyanobacteria. Mol Biol Evol 18, 1057–1069.[CrossRef]
    [Google Scholar]
  4. Cho, J. C. & Tiedje, J. M. ( 2000; ). Biogeography and degree of endemicity of fluorescent Pseudomonas strains in soil. Appl Environ Microbiol 66, 5448–5456.[CrossRef]
    [Google Scholar]
  5. Chun, J., Huq, A. & Colwell, R. R. ( 1999; ). Analysis of 16S–23S rRNA intergenic spacer regions of Vibrio cholerae and Vibrio mimicus. Appl Environ Microbiol 65, 2202–2208.
    [Google Scholar]
  6. Condon, C., Philips, J., Fu, Z. Y., Squires, C. & Squires, C. L. ( 1992; ). Comparison of the expression of the seven ribosomal RNA operons in Escherichia coli. EMBO J 11, 4175–4185.
    [Google Scholar]
  7. Condon, C., Squires, C. & Squires, C. L. ( 1995a; ). Control of rRNA transcription in Escherichia coli. Microbiol Rev 59, 623–645.
    [Google Scholar]
  8. Condon, C., Liveris, D., Squires, C., Schwartz, I. & Squires, C. L. ( 1995b; ). rRNA operon multiplicity in Escherichia coli and the physiological implications of rrn inactivation. J Bacteriol 177, 4152–4156.
    [Google Scholar]
  9. Ellwood, M. & Nomura, M. ( 1980; ). Deletion of a ribosomal ribonucleic acid operon in Escherichia coli. J Bacteriol 143, 1077–1080.
    [Google Scholar]
  10. Fessehaie, A., De Boer, S. H. & Lévesque, C. A. ( 2003; ). An oligonucleotide array for the identification of and differentiation of bacteria pathogenic on potato. Phytopathology 93, 262–269.[CrossRef]
    [Google Scholar]
  11. Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A., Kirkness, E. F., Kerlavage, A. R., Bult, C. J., Tomb, J. F., Dougherty, B. A. & other authors ( 1995; ). Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 496–512.[CrossRef]
    [Google Scholar]
  12. Garrity, G. M., Bell, J. A. & Lilburn, T. ( 2005; ). Pseudomonadales Orla-Jensen 1921. In Part B: the Gammaproteobacteria, Bergey's Manual of Systematic Bacteriology, 2nd edn. Edited by G. M. Garrity, D. J. Brenner, N. R. Krieg & J. T. Staley. New York: Springer Press.
  13. Gonzalez, A. J., Landeras, E. & Mendoza, M. C. ( 2000; ). Pathovars of Pseudomonas syringae causing bacterial brown spot and halo blight in Phaseolus vulgaris L. are distinguishable by ribotyping. Appl Environ Microbiol 66, 850–854.[CrossRef]
    [Google Scholar]
  14. Gurtler, V. ( 1999; ). The role of recombination and mutation in 16S–23S rDNA spacer rearrangements. Gene 238, 241–252.[CrossRef]
    [Google Scholar]
  15. Gürtler, V. & Stanisich, V. A. ( 1996; ). New approaches to typing and identification of bacteria using the 16S–23S rDNA spacer region. Microbiology 142, 3–16.[CrossRef]
    [Google Scholar]
  16. Helps, C., Lait, P., Tasker, S. & Harbour, D. ( 2002; ). Melting curve analysis of feline calicivirus isolates detected by real-time reverse transcription PCR. J Virol Methods 106, 241–244.[CrossRef]
    [Google Scholar]
  17. Iteman, I., Rippka, R., Tandeau De Marsac, N. & Herdman, M. ( 2000; ). Comparison of conserved structural and regulatory domains within divergent 16S rRNA-23S rRNA spacer sequences of cyanobacteria. Microbiology 146, 1275–1286.
    [Google Scholar]
  18. Kimura, M. ( 1980; ). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef]
    [Google Scholar]
  19. Manceau, C. & Horvais, A. ( 1997; ). Assessment of genetic diversity among strains of Pseudomonas syringae by PCR-restriction fragment length polymorphism analysis of rRNA operons with special emphasis on P. syringae pv. tomato. Appl Environ Microbiol 63, 498–505.
    [Google Scholar]
  20. Mangold, K. A., Manson, R. U., Koay, E. S., Stephens, L., Regner, M., Thomson, R. B., Jr, , Peterson, L. R. & Kaul, K. L. ( 2005; ). Real-time PCR for detection and identification of Plasmodium spp. J Clin Microbiol 43, 2435–2440.[CrossRef]
    [Google Scholar]
  21. Milyutina, I. A., Bobrova, V. K., Matveeva, E. V., Schaad, N. W. & Troitsky, A. V. ( 2004; ). Intragenomic heterogeneity of the 16S rRNA–23S rRNA internal transcribed spacer among Pseudomonas syringae and Pseudomonas fluorescens strains. FEMS Microbiol Lett 239, 17–23.[CrossRef]
    [Google Scholar]
  22. Naimi, A., Beck, G. & Branlant, C. ( 1997; ). Primary and secondary structures of rRNA spacer regions in enterococci. Microbiology 143, 823–834.[CrossRef]
    [Google Scholar]
  23. Natalini, E. & Scortichini, M. ( 2007; ). Variability of the 16S–23S rRNA gene internal transcribed spacer in Pseudomonas avellanae strains. FEMS Microbiol Lett 271, 274–280.[CrossRef]
    [Google Scholar]
  24. Nelson, K. E., Weinel, C., Paulsen, I. T., Dodson, R. J., Hilbert, H., Martins dos Santos, V. A., Fouts, D. E., Gill, S. R., Pop, M. & other authors ( 2002; ). Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4, 799–808.[CrossRef]
    [Google Scholar]
  25. Nicolas, L., Milon, G. & Prina, E. ( 2002; ). Rapid differentiation of Old World Leishmania species by LightCycler polymerase chain reaction and melting curve analysis. J Microbiol Methods 51, 295–299.[CrossRef]
    [Google Scholar]
  26. Panangala, V. S., van Santen, V. L., Shoemaker, C. A. & Klesius, P. H. ( 2005; ). Analysis of 16S–23S intergenic spacer regions of the rRNA operons in Edwardsiella ictaluri and Edwardsiella tarda isolates from fish. J Appl Microbiol 99, 657–669.[CrossRef]
    [Google Scholar]
  27. Perneel, M., Heyrman, J., Adiobo, A., De Maeyer, K., Raaijmakers, J. M., De Vos, P. & Höfte, M. ( 2007; ). Characterization of CMR5c and CMR12a, novel fluorescent Pseudomonas strains from the cocoyam rhizosphere with biocontrol activity. J Appl Microbiol 103, 1007–1020.[CrossRef]
    [Google Scholar]
  28. Piatek, A. S., Tyagi, S., Pol, A. C., Telenti, A., Miller, L. P., Kramer, F. R. & Alland, D. ( 1998; ). Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nat Biotechnol 16, 359–363.[CrossRef]
    [Google Scholar]
  29. Posada, D. ( 2006; ). ModelTest Server: a web-based tool for the statistical selection of models of nucleotide substitution online. Nucleic Acids Res 34, W700–W703.[CrossRef]
    [Google Scholar]
  30. Rauter, C., Oehme, R., Diterich, I., Engele, M. & Hartung, T. ( 2002; ). Distribution of clinically relevant Borrelia genospecies in ticks assessed by a novel, single-run, real-time PCR. J Clin Microbiol 40, 36–43.[CrossRef]
    [Google Scholar]
  31. Ririe, K. M., Rasmussen, R. P. & Wittwer, C. T. ( 1997; ). Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245, 154–160.[CrossRef]
    [Google Scholar]
  32. Robinson, B. S., Monis, P. T. & Dobson, P. J. ( 2006; ). Rapid, sensitive, and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis. Appl Environ Microbiol 72, 5857–5863.[CrossRef]
    [Google Scholar]
  33. Saitou, N. & Nei, M. ( 1987; ). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
    [Google Scholar]
  34. Smith, L. M., Tola, E., deBoer, P. & O'Gara, F. ( 1999; ). Signalling by the fungus Pythium ultimum represses expression of two ribosomal RNA operons with key roles in the rhizosphere ecology of Pseudomonas fluorescens F113. Environ Microbiol 1, 495–502.[CrossRef]
    [Google Scholar]
  35. Tajima, F. ( 1993; ). Simple methods for testing the molecular evolutionary clock hypothesis. Genetics 135, 599–607.
    [Google Scholar]
  36. Tambong, J. T., de Cock, A. W., Tinker, N. A. & Levesque, C. A. ( 2006; ). Oligonucleotide array for identification and detection of Pythium species. Appl Environ Microbiol 72, 2691–2706.[CrossRef]
    [Google Scholar]
  37. Tambong, J. T., Mwange, K. N., Bergeron, M., Ding, T., Mandy, F., Reid, L. M. & Zhu, X. ( 2008; ). Rapid detection and identification of the bacterium Pantoea stewartii in maize by TaqMan real-time PCR assay targeting the cpsD gene. J Appl Microbiol 104, 1525–1537.[CrossRef]
    [Google Scholar]
  38. Tamura, K., Dudley, J., Nei, M. & Kumar, S. ( 2007; ). mega4: Molecular Evolutionary Genetics Analysis (mega) software version 4.0. Mol Biol Evol 24, 1596–1599.[CrossRef]
    [Google Scholar]
  39. Wolk, D. M., Schneider, S. K., Wengenack, N. L., Sloan, L. M. & Rosenblatt, J. E. ( 2002; ). Real-time PCR method for detection of Encephalitozoon intestinalis from stool specimens. J Clin Microbiol 40, 3922–3928.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.028274-0
Loading
/content/journal/micro/10.1099/mic.0.028274-0
Loading

Data & Media loading...

Supplements

vol. , part 8, pp. 2630 - 2640

[ PDF, 171 kb] Complete alignment of the 16S-23S rRNA internal transcribed spacer (ITS) region of the 29 strains isolated in this study compared to the GenBank entry (EU360313). Neighbour-joining bootstrapped (1000 replicates) consensus tree of the two copies of 16S-23S rRNA internal transcribed spacers of 29 strains obtained in this study compared with GenBank entries reported to exhibit intercistronic heterogeneity. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 1596-1599.



PDF
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