Specific ribosomal DNA sequences from diverse environmental settings correlate with experimental contaminants

Abstract

Phylogenetic analysis of 16S ribosomal DNA (rDNA) clones obtained by PCR from uncultured bacteria inhabiting a wide range of environments has increased our knowledge of bacterial diversity. One possible problem in the assessment of bacterial diversity based on sequence information is that PCR is exquisitely sensitive to contaminating 16S rDNA. This raises the possibility that some putative environmental rRNA sequences in fact correspond to contaminant sequences. To document potential contaminants, we cloned and sequenced PCR-amplified 16S rDNA fragments obtained at low levels in the absence of added template DNA. 16S rDNA sequences closely related to the genera Duganella (formerly Zoogloea), Acinetobacter, Stenotrophomonas, Escherichia, Leptothrix, and Herbaspirillum were identified in contaminant libraries and in clone libraries from diverse, generally low-biomass habitats. The rRNA sequences detected possibly are common contaminants in reagents used to prepare genomic DNA. Consequently, their detection in processed environmental samples may not reflect environmentally relevant organisms.

FIG. 1

Evolutionary-distance dendrogram showing the relative positions of environmental 16S rDNA clone and strain sequences to the genera Duganella and Herbaspirillum. Bar indicates nucleotide substitution rate. The sequence length in nucleotides follows the clone or strain designation. References for clones are given in Table 1. Clones are boldfaced; the cultivated strains marine isolate BAL15 (25), arctic isolate arc21 (3), and strain Mena 23/3-3c (32) are shown in lightface. Sequences for described bacterial species (italics) were obtained from GenBank (5). The rDNA sequence from Rhodocyclus purpureus was used as an outgroup. The resolution of the tree shown here is limited by the quality of the sequence data and the short lengths (fewer than 500 nt) of many sequences. Short sequences (<500 nt) were inserted into the tree by using the parsimony insertion tool of the ARB software program (30). The contaminant clone CTHB-18 was identified in a separate negative-control extraction independently from that for the MT clone library.