Deep sequencing of dmdA amplicons
Deep sequencing of a DMSP-degrading gene (dmdA) in a coastal bacterioplankton community
We used 454-based pyrosequencing of PCR amplified products from Sapelo Island composited DNA samples to compare specificity and diversity of a functional gene, dmdA, using one universal, one clade, and eight subclade primer sets.  The DNA sample composites span five years, 4 seasons, and two locations at Sapelo Island (Dean Creek march and Doboy Sound inlet) and were only separated by size fraction, free-living and particle-associated.  As well, several primer types were used in the sequencing effort including, "specific", "degenerate" and "inosine" based primer pairs for comparisons.
Ten primer pairs targeting environmental clades of the dimethylsulfoniopropionate (DMSP) demethylase protein, DmdA, were designed using an iterative bioinformatic approach that took advantage of >1700 dmdA sequences captured in marine metagenomic datasets.  Using the bioinformatically-optimized primer pairs, dmdA genes were amplified from free-living coastal bacterioplankton samples and sequenced using 454 technology.  An average of 6,400 amplicons per primer pair represented almost 800 clusters of environmental dmdA sequences, with clusters defined conservatively at >90% nucleotide sequence identity (~95% protein identity).  Systematic comparisons of primer performance showed that degenerate and inosine-based primers did not perform better than specific primer sets in retrieving dmdA diversity, and sometimes captured a lower diversity of sequences from the same DNA sample.  The specific primer sets were used to compare dmdA diversity in free-living versus particle-associated bacteria in southeastern U.S. coastal waters.  Hundreds of different dmdA clusters were found in both size fractions, with Roseobacter-like and SAR11-like sequences dominating both. The free-living fraction had a higher diversity of dmdA clusters overall, though clusters retrieved by a given primer set were largely shared (52-88%) across the two size fractions, and most sequences were affiliated with these shared clusters (~90%). Despite evidence from 16S rRNA-based taxonomic surveys that free-living bacterioplankton are a considerably less diverse subset of particle-associated bacteria at this site, this seems not to be the case for a widespread bacterial gene mediating sulfur transformations.