Identifying organism involved in new and regenerated production using TAG-SIP

The coupling of stable isotope probing (SIP) with high throughput sequencing (TAG-SIP), allows examination of DNA from individual taxa for the incorporation of a specific isotopically labeled substrate, facilitating an in-depth investigation of the activity and functional diversity of in situ microbial communities. This approach was applied to the monthly San Pedro Ocean Time-series (SPOT), during April of 2014 in order to characterize the organisms involved in new and regenerated production by investigating the assimilation of ¹⁵N-NO₃^-, ¹⁵N-NH₄⁺, and ¹⁵N-urea at several light depths throughout the euphotic zone. Overall, very little variation was seen between the DNA banding patterns and density of each discrete OTU compared over multiple control treatments, i.e. unlabeled substrate was added to each control and so any disparity between the DNA banding of these OTU replicates reflects methodological variation. The lack of disparity found here further demonstrates TAG-SIP’s high precision, accuracy, and more importantly validates the TAG-SIP’s reproducibility in both gradient formation and DNA sedimentation with respect to density. The mean density of these discrete control OTU DNA bands (n=7) were then compared to those of their isotopically treated equivalent OTU after a 24h incubation in order to accurately assess and identify significant shifts in DNA density. Therefore we are confident that differences in density between control and treated sample DNA greater than the variation quantified among the controls themselves, is direct evidence of ‘heavy’ isotope incorporation, i.e. metabolic activity and growth.

Direct evidence of activity was found in a broad range of taxa, thought not every treatment yielded positive results. As expected the majority of the organisms identified as assimilators were found within the ¹⁵N-NH₄⁺ treatments. Many taxa displayed evidence of uptake in one or more but not all treatments providing evidence on which taxa are metabolizing a broad range of substrates as opposed to those which are specializing. Overall, this dataset provides unique insight into which organisms contribute to new biomass formation and ultimately to carbon export and those that help maintain the population through the recycling of nutrients.