Abstract
In order to assess the effect of genome size and number of 16S rRNA genes (rDNAs) on the quantities of PCR-generated partial 16S rDNA fragments, equimolar amounts of DNA from pairs of different species for which these parameters are known were subjected to gene amplification. The experimentally determined ratio of PCR products obtained, as determined by image analysis of SYBR-Green I-stained amplification products, corresponded well with the predicted ratio calculated from the number of rrn genes per equimolar amounts of DNA in mixtures of Escherichia coli and "Thermus thermophilus" and of Pseudomonas aeruginosa and "T. thermophilus." The values for the pair of Bacillus subtilis and "T. thermophilus" showed greater deviations from the predicted value. The dependence of the amount of 16S rDNA amplification product on these two parameters makes it impossible to quantify the number of species represented in 16S rDNA clone libraries of environmental samples as long as these two parameters are unknown for the species present.
Full Text
The Full Text of this article is available as a PDF (204.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Borges K. M., Bergquist P. L. Genomic restriction map of the extremely thermophilic bacterium Thermus thermophilus HB8. J Bacteriol. 1993 Jan;175(1):103–110. doi: 10.1128/jb.175.1.103-110.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cariello N. F., Thilly W. G., Swenberg J. A., Skopek T. R. Deletion mutagenesis during polymerase chain reaction: dependence on DNA polymerase. Gene. 1991 Mar 1;99(1):105–108. doi: 10.1016/0378-1119(91)90040-i. [DOI] [PubMed] [Google Scholar]
- Cole S. T., Saint Girons I. Bacterial genomics. FEMS Microbiol Rev. 1994 Jun;14(2):139–160. doi: 10.1111/j.1574-6976.1994.tb00084.x. [DOI] [PubMed] [Google Scholar]
- Fukunaga M., Mifuchi I. Unique organization of Leptospira interrogans rRNA genes. J Bacteriol. 1989 Nov;171(11):5763–5767. doi: 10.1128/jb.171.11.5763-5767.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Green C. J., Stewart G. C., Hollis M. A., Vold B. S., Bott K. F. Nucleotide sequence of the Bacillus subtilis ribosomal RNA operon, rrnB. Gene. 1985;37(1-3):261–266. doi: 10.1016/0378-1119(85)90281-1. [DOI] [PubMed] [Google Scholar]
- Itaya M., Tanaka T. Complete physical map of the Bacillus subtilis 168 chromosome constructed by a gene-directed mutagenesis method. J Mol Biol. 1991 Aug 5;220(3):631–648. doi: 10.1016/0022-2836(91)90106-g. [DOI] [PubMed] [Google Scholar]
- Kopczynski E. D., Bateson M. M., Ward D. M. Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms. Appl Environ Microbiol. 1994 Feb;60(2):746–748. doi: 10.1128/aem.60.2.746-748.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liesack W., Stackebrandt E. Evidence for unlinked rrn operons in the Planctomycete Pirellula marina. J Bacteriol. 1989 Sep;171(9):5025–5030. doi: 10.1128/jb.171.9.5025-5030.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Linton D., Dewhirst F. E., Clewley J. P., Owen R. J., Burnens A. P., Stanley J. Two types of 16S rRNA gene are found in Campylobacter helveticus: analysis, applications and characterization of the intervening sequence found in some strains. Microbiology. 1994 Apr;140(Pt 4):847–855. doi: 10.1099/00221287-140-4-847. [DOI] [PubMed] [Google Scholar]
- Meyerhans A., Vartanian J. P., Wain-Hobson S. DNA recombination during PCR. Nucleic Acids Res. 1990 Apr 11;18(7):1687–1691. doi: 10.1093/nar/18.7.1687. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Michaux S., Paillisson J., Carles-Nurit M. J., Bourg G., Allardet-Servent A., Ramuz M. Presence of two independent chromosomes in the Brucella melitensis 16M genome. J Bacteriol. 1993 Feb;175(3):701–705. doi: 10.1128/jb.175.3.701-705.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Muyzer G., de Waal E. C., Uitterlinden A. G. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol. 1993 Mar;59(3):695–700. doi: 10.1128/aem.59.3.695-700.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pettersson B., Johansson K. E., Uhlén M. Sequence analysis of 16S rRNA from mycoplasmas by direct solid-phase DNA sequencing. Appl Environ Microbiol. 1994 Jul;60(7):2456–2461. doi: 10.1128/aem.60.7.2456-2461.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Picard C., Ponsonnet C., Paget E., Nesme X., Simonet P. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Appl Environ Microbiol. 1992 Sep;58(9):2717–2722. doi: 10.1128/aem.58.9.2717-2722.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ratnaningsih E., Dharmsthiti S., Krishnapillai V., Morgan A., Sinclair M., Holloway B. W. A combined physical and genetic map of Pseudomonas aeruginosa PAO. J Gen Microbiol. 1990 Dec;136(12):2351–2357. doi: 10.1099/00221287-136-12-2351. [DOI] [PubMed] [Google Scholar]
- Reysenbach A. L., Giver L. J., Wickham G. S., Pace N. R. Differential amplification of rRNA genes by polymerase chain reaction. Appl Environ Microbiol. 1992 Oct;58(10):3417–3418. doi: 10.1128/aem.58.10.3417-3418.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rudner R., Chevrestt A., Buchholz S. R., Studamire B., White A. M., Jarvis E. D. Two tRNA gene clusters associated with rRNA operons rrnD and rrnE in Bacillus subtilis. J Bacteriol. 1993 Jan;175(2):503–509. doi: 10.1128/jb.175.2.503-509.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Römling U., Grothues D., Bautsch W., Tümmler B. A physical genome map of Pseudomonas aeruginosa PAO. EMBO J. 1989 Dec 20;8(13):4081–4089. doi: 10.1002/j.1460-2075.1989.tb08592.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith C. L., Econome J. G., Schutt A., Klco S., Cantor C. R. A physical map of the Escherichia coli K12 genome. Science. 1987 Jun 12;236(4807):1448–1453. doi: 10.1126/science.3296194. [DOI] [PubMed] [Google Scholar]
- Stackebrandt E., Charfreitag O. Partial 16S rRNA primary structure of five Actinomyces species: phylogenetic implications and development of an Actinomyces israelii-specific oligonucleotide probe. J Gen Microbiol. 1990 Jan;136(1):37–43. doi: 10.1099/00221287-136-1-37. [DOI] [PubMed] [Google Scholar]
- Taylor D. E., Eaton M., Yan W., Chang N. Genome maps of Campylobacter jejuni and Campylobacter coli. J Bacteriol. 1992 Apr;174(7):2332–2337. doi: 10.1128/jb.174.7.2332-2337.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tebbe C. C., Vahjen W. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl Environ Microbiol. 1993 Aug;59(8):2657–2665. doi: 10.1128/aem.59.8.2657-2665.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ulbrich N., Kumagai I., Erdmann V. A. The number of ribosomal RNA genes in Thermus thermophilus HB8. Nucleic Acids Res. 1984 Feb 24;12(4):2055–2060. doi: 10.1093/nar/12.4.2055. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weisburg W. G., Barns S. M., Pelletier D. A., Lane D. J. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991 Jan;173(2):697–703. doi: 10.1128/jb.173.2.697-703.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weller R., Weller J. W., Ward D. M. 16S rRNA sequences of uncultivated hot spring cyanobacterial mat inhabitants retrieved as randomly primed cDNA. Appl Environ Microbiol. 1991 Apr;57(4):1146–1151. doi: 10.1128/aem.57.4.1146-1151.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Widom R. L., Jarvis E. D., LaFauci G., Rudner R. Instability of rRNA operons in Bacillus subtilis. J Bacteriol. 1988 Feb;170(2):605–610. doi: 10.1128/jb.170.2.605-610.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]