Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis

Abstract

The ability of terminal restriction fragment (T-RFLP or TRF) profiles of 16S rRNA genes to provide useful information about the relative diversity of complex microbial communities was investigated by comparison with other methods. Four soil communities representing two pinyon rhizosphere and two between-tree (interspace) soil environments were compared by analysis of 16S rRNA gene clone libraries and culture collections (Dunbar et al., Appl. Environ. Microbiol. 65:1662-1669, 1998) and by analysis of 16S rDNA TRF profiles of community DNA. The TRF method was able to differentiate the four communities in a manner consistent with previous comparisons of the communities by analysis of 16S rDNA clone libraries. TRF profiles were not useful for calculating and comparing traditional community richness or evenness values among the four soil environments. Statistics calculated from RsaI, HhaI, HaeIII, and MspI profiles of each community were inconsistent, and the combined data were not significantly different between samples. The detection sensitivity of the method was tested. In standard PCRs, a seeded population comprising 0.1 to 1% of the total community could be detected. The combined results demonstrate that TRF analysis is an excellent method for rapidly comparing the relationships between bacterial communities in environmental samples. However, for highly complex communities, the method appears unable to provide classical measures of relative community diversity.

FIG. 1

RsaI TRF profiles of 16S rDNA amplified directly from S0, S1, C0, and C1 soil DNA samples.

FIG. 2

Dendrograms based on Jaccard similarity comparisons of the C0, C1, S0, and S1 soil communities. (A) Dendrograms based on all RsaI plus BstUI RFLP data from 16S rDNA clone libraries or only RsaI plus BstUI RFLP patterns representing members of the Acidobacterium division. (B) Dendrograms based on TRF profiles of 16S rDNAs amplified from soil DNA with conserved bacterial primers 8-27f and 1507-1492r or with specific Acidobacterium division primers YOGA31f and 1507-1492r.

FIG. 3

Sampling curves showing diversity of the C0 and S0 clone libraries assessed by RsaI plus BstUI RFLP patterns, RsaI RFLP patterns, and RsaI TRFs. Curves were constructed by rarefaction (15, 26). Curves marked with asterisks were reprinted from reference with permission from the publisher.

FIG. 4

Detection of a 16S rDNA clone (5-kb plasmid from clone S075) in a background of 1 ng of C0 soil DNA. (A) RsaI TRF profile of 16S rDNA amplified from 0.01 pg of S075 plasmid DNA. (B) RsaI TRf profile of 16S rDNA amplified from a mixture of 0.01 pg of plasmid S075 and 1 ng of C0 soil DNA. (C) RsaI TRf profile of 16S rDNA amplified from a mixture of 0.001 pg of plasmid S075 and 1 ng of C0 soil DNA. (D) RsaI TRF profile of 16S rDNA amplified from 1 ng of C0 soil DNA.