Propidium monoazide-denaturing gradient gel electrophoresis (PMA-DGGE) assay for the characterization of viable diatoms in marine sediments

Abstract

Molecular-based community profiles often overestimate species richness and diversity as they are incapable of distinguishing DNA signals derived from extracellular or moribund sources from that of viable organisms. Propidium monoazide (PMA), a double stranded DNA-binding dye, was used to selectively isolate the genetic signature of viable diatoms from marine cultures and environmental samples. Upon optimization of photoactivation time using a 600-W halogen lamp, PMA concentrations, and number of treatments, the compromised genetic signals from heat-killed samples, visualized by denaturing gradient gel electrophoresis, were either greatly reduced or removed entirely while those derived from intact, viable samples remained relatively unaffected. Subsequent tests illustrated that bands associated with extracellular and moribund DNA were removed from combinations of viable and nonviable cells initially derived from two sedimentary diatom isolates. In addition, viable and nonviable samples of Skeletonema marinoi that were spiked into fresh deposit-feeder (Balanoglossus aurantiacus) faecal-coil sediments demonstrated the effectiveness of the assay within an environmental matrix. Thus, we propose the use of PMA as a quick and necessary addition to any protocol relying on genetic profiles to discern changes in viable benthic diatom community structure.