Sampling method comparison on metranskriptom level
Measuring unbiased metatranscriptomics in the ocean
Microorganisms mediate all geochemical cycles relevant to sustaining life on Earth. An analysis of their metabolism is therefore fundamental to understanding globally important element transformations. However, most microbes are recalcitrant to cultivation, such that culture-independent methods must be used to deduce their metabolic functions. One approach that has already shown great promise in this regard is to analyze the pool of transcripts contained in natural microbial assemblages (metatranscriptomes). Unfortunately, since mRNA is extremely labile and can degrade in less than 30 sec, it is unclear whether the abundance patterns detected in nature are vulnerable to considerable modification in situ simply due to sampling procedures. Based on comparisons of metatranscriptomes retrieved from pelagic aerobic ammonium oxidation zones of 70-120 m depth and quantification of the specific transcripts in them, we show that different sampling techniques indeed significantly influence the relative abundance of transcripts presumably diagnostic of the habitat. In situ fixation using our newly developed automatic flow injection sampler resulted in an abundance of crenarchaeal ammonia monooxygenase transcripts that was up to 30-fold higher than that detected in samples obtained using standard oceanographic systems. By contrast, the abundance of transcripts indicative of cellular stress was significantly greater in non-fixed samples. Thus, the importance of in situ fixation in the reliable evaluation of distinct microbial activities in the ocean based on metatranscriptomics is obvious. This is no doubt especially the case in attempts aimed at an unbiased analysis of areas below the epipelagic zone, which cover 90% of the world's oceans.