Estimation of the abundance of an uncultured soil bacterial strain by a competitive quantitative PCR method.

Strain EA25 was identified in a clone library of bacterial 16S rRNA gene sequences that had been amplified from DNA extracted from soil collected in eastern Washington State. EA25 was subsequently shown to be related to members of the genera Planctomyces and Chlamydia and most closely related (93% similarity) to strain MC18, a strain identified in an Australian soil sample (W. Liesack and E. Stackebrandt, J. Bacteriol. 174:5072-5078, 1992). A competitive quantitative PCR method developed by Zachar et al. (V. Zachar, R.A. Thomas, and A.S. Goustin, Nucleic Acids Res. 21:2017-2018, 1993) was used to estimate the abundance of this uncultured strain in soil. An estimation of the abundance of EA25 was based on the number of copies of the sequence in the DNA extracted and the efficiency of the DNA extraction. In addition, amplification rates of Escherichia coli DNAs added to soil were shown to be similar to those of DNAs from laboratory cultures of E. coli. The number of EA25 16S rRNA genes was estimated to be 2.17 x 10(8) copies per g of soil, suggesting that strains similar to EA25 and the similar Australian strain could be widely distributed and present in significant numbers in soils from temperate regions. This represents the first enumeration of 16S rDNA copies from an uncultured strain in soil.