Applied and Environmental Microbiology, January 2010, p. 609-617, Vol. 76, No. 2

Deep Sequencing of a Dimethylsulfoniopropionate-Degrading Gene (dmdA) by Using PCR Primer Pairs Designed on the Basis of Marine Metagenomic Data

Departments of Microbiology, Marine Sciences, University of Georgia, Athens, Georgia 30602

Received 1 June 2009/ Accepted 16 November 2009

In silico design and testing of environmental primer pairs with metagenomic data are beneficial for capturing a greater proportion of the natural sequence heterogeneity in microbial functionalgenes, as well as for understanding limitations of existing primer sets that were designed from more restricted sequence data. PCR primer pairs targeting 10 environmental clades and subclades of the dimethylsulfoniopropionate (DMSP) demethylase protein, DmdA, were designed using an iterative bioinformatic approach that took advantage of thousands of dmdA sequences captured in marine metagenomic data sets. Using the bioinformatically optimized primers, dmdA genes were amplified from composite free-living coastal bacterioplankton DNA (from 38 samples over 5 years and two locations) and sequenced using 454 technology. An average of 6,400 amplicons per primer pair represented more than 700 clusters of environmental dmdA sequences across allprimers, with clusters defined conservatively at >90% nucleotide sequence identity (95% amino acid identity). Degenerate and inosine-based primers did not perform better than specific primerpairs in determining dmdA richness and sometimes captured a lower degree of richness of sequences from the same DNA sample. A comparison of dmdA sequences in free-living versus particle-associated bacteria in southeastern U.S. coastal waters showed that sequence richness in some dmdA subgroups differed significantly between size fractions, though most gene clusters were shared (52 to 91%) and most sequences were affiliated with the shared clusters (90%). The availability of metagenomic sequence data has significantly enhanced the design of quantitative PCR primer pairs for this key functional gene, providing robust access to the capabilities and activities of DMSP demethylating bacteria in situ.