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.

[1]  U. Szewzyk,et al.  Isolation of new bacterial species from drinking water biofilms and proof of their in situ dominance with highly specific 16S rRNA probes , 1997, Applied and environmental microbiology.

[2]  N. Pace,et al.  Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[3]  J. Pinhassi,et al.  Dominant marine bacterioplankton species found among colony-forming bacteria , 1997, Applied and environmental microbiology.

[4]  Karsten Pedersen,et al.  Investigations of subterranean bacteria in deep crystalline bedrock and their importance for the disposal of nuclear waste , 1996 .

[5]  J. Sugiyama,et al.  Proposal to reclassify Zoogloea ramigera IAM 12670 (P. R. Dugan 115) as Duganella zoogloeoides gen. nov., sp. nov. , 1997, International journal of systematic bacteriology.

[6]  E. Delong,et al.  Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. , 1989, Science.

[7]  K. Pedersen,et al.  Diversity and distribution of subterranean bacteria in groundwater at Oklo in Gabon, Africa, as determined by 16S rRNA gene sequencing , 1996, Molecular ecology.

[8]  L. Shimkets,et al.  Bacterial diversity of a Carolina bay as determined by 16S rRNA gene analysis: confirmation of novel taxa , 1997, Applied and environmental microbiology.

[9]  J. Tiedje,et al.  Characterization of the Dominant and Rare Members of a Young Hawaiian Soil Bacterial Community with Small-Subunit Ribosomal DNA Amplified from DNA Fractionated on the Basis of Its Guanine and Cytosine Composition , 1998, Applied and Environmental Microbiology.

[10]  S. Ekendahl,et al.  16S rRNA gene diversity of attached and unattached bacteria in boreholes along the access tunnel to the Äspö hard rock laboratory, Sweden , 1996 .

[11]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[12]  E. Roden,et al.  Recovery of Humic-Reducing Bacteria from a Diversity of Environments , 1998, Applied and Environmental Microbiology.

[13]  J. Tiedje,et al.  Phylogenetic diversity of a bacterial community determined from Siberian tundra soil DNA. , 1997, Microbiology.

[14]  A. Kiener,et al.  Isolation of new 6-methylnicotinic-acid-degrading bacteria, one of which catalyses the regioselective hydroxylation of nicotinic acid at position C2 , 1997, Archives of Microbiology.

[15]  J. Krieger,et al.  Prokaryotic DNA sequences in patients with chronic idiopathic prostatitis , 1996, Journal of clinical microbiology.

[16]  Ross A. Overbeek,et al.  The RDP (Ribosomal Database Project) , 1997, Nucleic Acids Res..

[17]  N. Pace,et al.  Novel Division Level Bacterial Diversity in a Yellowstone Hot Spring , 1998, Journal of bacteriology.

[18]  M. Sogin,et al.  Bacterial diversity in an arctic lake: a freshwater SAR11 cluster , 1996 .

[19]  R. Zbinden,et al.  Molecular diagnosis of bacterial endocarditis by broad-range PCR amplification and direct sequencing , 1997, Journal of clinical microbiology.

[20]  W. Wade,et al.  Molecular analysis of microflora associated with dentoalveolar abscesses , 1996, Journal of clinical microbiology.

[21]  Philip Hugenholtz,et al.  Microbial Diversity in a Hydrocarbon- and Chlorinated-Solvent-Contaminated Aquifer Undergoing Intrinsic Bioremediation , 1998, Applied and Environmental Microbiology.

[22]  S. Kwok,et al.  Avoiding false positives with PCR , 1989, Nature.

[23]  S. Ekendahl,et al.  Characterization of attached bacterial populations in deep granitic groundwater from the Stripa research mine by 16S rRNA gene sequencing and scanning electron microscopy. , 1994, Microbiology.

[24]  N. Pace,et al.  Detection of DNA contamination in Taq polymerase. , 1991, BioTechniques.

[25]  G. Sarkar,et al.  Shedding light on PCR contamination , 1990, Nature.

[26]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[27]  N. Almond,et al.  Avoidance of false positives , 1990, Nature.

[28]  N. Pace.  A molecular view of microbial diversity and the biosphere. , 1997, Science.

[29]  J. Fry,et al.  Effect of sample handling on estimation of bacterial diversity in marine sediments by 16S rRNA gene sequence analysis , 1994 .