Gene-targeted-metagenomics reveals extensive diversity of aromatic dioxygenase genes in the environment

Abstract

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.

Figure 1

Shannon entropy (H′) at each alignment position and conserved residues among obtained sequences and/or reference sequences for (a) BPHD-f3 sequences and (b) BPHD-r1 sequences. Open circles (○) indicate entropy of reference sequences and filled circles (●) indicate entropy of obtained sequences. The corresponding position numbers and the residues of bphA1 from B. xenovorans LB400 are indicated. The ratio of residues conserved in either set with >95% are shown. The residues highly conserved only among obtained sequences are indicated with [*].

Figure 2

Clustering of valid sequences at different distance levels by Complete Linkage Clustering based on amino acid sequences. The numbers of OTUs of valid sequences (solid line, BPHD-f3 sequences; dashed line, BPHD-r1 sequences) at each distance are shown. The arrow indicates the distance level used for the distribution analysis.

Figure 3

Pairwise distance to the closest reference sequence(s) for each valid sequence and the number of sequences in each cluster. Symbols are the median of the distances in the cluster. Error bar indicates the range of the distance.