Understanding the relationship between gene diversity and function for important environmental processes is a major ecological research goal. We applied gene-targeted metagenomics and pyrosequencing to aromatic dioxygenase genes to obtain greater sequence depth than possible by other methods. A polymerase chain reaction (PCR) primer set designed to target a 524-bp region that confers substrate specificity of biphenyl dioxygenases yielded 2000 and 604 sequences from the 5' and 3' ends of PCR products, respectively, which passed our validity criteria. Sequence alignment showed three known conserved residues, as well as another seven conserved residues not reported earlier. Of the valid sequences, 95% and 41% were assigned to 22 and 3 novel clusters in that they did not include any earlier reported sequences at 0.6 distance by complete linkage clustering for sequenced regions. The greater diversity revealed by this gene-targeted approach provides deeper insights into genes potentially important in environmental processes to better understand their ecology, functional differences and evolutionary origins. We also provide criteria for primer design for this approach, as well as guidance for data processing of diverse functional genes, as gene databases for most genes of environmental relevance are limited.

Center for Microbial Ecology Michigan State University East Lansing MI USA

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Bagnéris C, Cammack R, Mason JR. Subtle difference between benzene and toluene dioxygenases of Pseudomonas putida. Appl Environ Microbiol. 2005;71:1570–80. PubMed PMC

Dong XS, Fushinobu S, Fukuda E, Terada T, Nakamura S, Shimizu K, et al. Crystal structure of the terminal oxygenase component of cumene dioxygenase from Pseudomonas fluorescens IP01. J Bacteriol. 2005;187:2483–2490. PubMed PMC

Durbin R, Eddy S, Krogh A, Mitchison G. Biological sequence analysis: probabilistic models of proteins and nucleic acids. Cambridge University Press; 1998.

Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–1797. PubMed PMC

Finn RD, Tate J, Mistry J, Coggill PC, Sammut SJ, Hotz HR, et al. The Pfam protein families database. Nucleic Acids Res. 2008;36:D281–D288. PubMed PMC

Furusawa Y, Nagarajan V, Tanokura M, Masai E, Fukuda M, Senda T. Crystal structure of the terminal oxygenase component of biphenyl dioxygenase derived from Rhodococcus sp. strain RHA1. J Mol Biol. 2004;342:1041–1052. PubMed

Gibson DT, Parales RE. Aromatic hydrocarbon dioxygenases in environmental biotechnology. Curr Opin Biotechnol. 2000;11:236–243. PubMed

Huber JA, Mark Welch D, Morrison HG, Huse SM, Neal PR, Butterfield DA, et al. Microbial population structures in the deep marine biosphere. Science. 2007;318:97–100. PubMed

Karlsson A, Parales JV, Parales RE, Gibson DT, Eklund H, Ramaswamy S. Crystal structure of naphthalene dioxygenase: Side-on binding of dioxygen to iron. Science. 2003;299:1039–1042. PubMed

Leigh MB, Pellizari VH, Uhlik O, Sutka R, Rodrigues J, Ostrom NE, et al. Biphenyl-utilizing bacteria and their functional genes in a pine root zone contaminated with polychlorinated biphenyls (PCBs) ISME J. 2007;1:134–148. PubMed

Leigh MB, Prouzová P, Macková M, Macek T, Nagle DP, Fletcher JS. Polychlorinated biphenyl (PCB)-degrading bacteria associated with trees in a PCB-contaminated site. Appl Environ Microbiol. 2006;72:2331–2342. PubMed PMC

Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature. 2005;437:376–380. PubMed PMC

Mondello FJ, Turcich MP, Lobos JH, Erickson BD. Identification and modification of biphenyl dioxygenase sequences that determine the specificity of polychlorinated biphenyl degradation. Appl Environ Microbiol. 1997;63:3096–3103. PubMed PMC

National Research Council. The New Science of Metagenomics. The National Academies Press; Washington DC: 2007. Why Metagenomics? pp. 12–32.

Parales RE, Lee K, Resnick SM, Jiang H, Lessner DJ, Gibson DT. Substrate specificity of naphthalene dioxygenase: effect of specific amino acids at the active site of the enzyme. J Bacteriol. 2000;182:1641–1649. PubMed PMC

Rothberg JM, Leamon JH. The development and impact of 454 sequencing. Nat Biotechnol. 2008;26:1117–1124. PubMed

Schloss PD, Handelsman J. Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbiol. 2005;71:1501–1506. PubMed PMC

Sogin ML, Morrison HG, Huber JA, Welch DM, Huse SM, Neal PR, et al. Microbial diversity in the deep sea and the underexplored “rare biosphere”. Proc Natl Acad Sci U S A. 2006;103:12115–12120. PubMed PMC

Suenaga H, Watanabe T, Sato M, Ngadiman, Furukawa K. Alteration of regiospecificity in biphenyl dioxygenase by active-site engineering. J Bacteriol. 2002;184:3682–3688. PubMed PMC

Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–4680. PubMed PMC

Vardar G, Wood TK. Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for enhanced chlorinated ethene degradation and o-xylene oxidation. Appl Microbiol Biotechnol. 2005;68:510–517. PubMed

Vézina J, Barriault D, Sylvestre M. Diversity of the C-terminal portion of the biphenyl dioxygenase large subunit. J Mol Microbiol Biotechnol. 2008;15:139–151. PubMed

Zhang SW, Zhang YL, Pan Q, Cheng YM, Chou KC. Estimating residue evolutionary conservation by introducing von Neumann entropy and a novel gap-treating approach. Amino Acids. 2007;35:495–501. PubMed PMC

Predominant Biphenyl Dioxygenase From Legacy Polychlorinated Biphenyl (PCB)-Contaminated Soil Is a Part of Unusual Gene Cluster and Transforms Flavone and Flavanone

Novel PCB-degrading Rhodococcus strains able to promote plant growth for assisted rhizoremediation of historically polluted soils

Synergistic Processing of Biphenyl and Benzoate: Carbon Flow Through the Bacterial Community in Polychlorinated-Biphenyl-Contaminated Soil

Hunting Down Frame Shifts: Ecological Analysis of Diverse Functional Gene Sequences