Measuring unbiased metatranscriptomics in suboxic waters of the central Baltic Sea using a new in situ fixation system

Abstract

An analysis of the microbial metabolism is fundamental to understanding globally important element transformations. One culture-independent approach to deduce those prokaryotic metabolic functions is to analyze metatranscriptomes. Unfortunately, since mRNA is extremely labile, it is unclear whether the abundance patterns detected in nature are vulnerable to considerable modification in situ simply due to sampling procedures. Exemplified on comparisons of metatranscriptomes retrieved from pelagic suboxic zones of the central Baltic Sea (70-120 m depth), earlier identified as areas of high aerobic ammonium oxidation activity, and quantification of specific transcripts in them, we show that different sampling techniques 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 thaumarchaeal ammonia monooxygenase transcripts that was up to 30-fold higher than that detected in samples obtained using standard oceanographic sampling 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 ecosystem based on metatranscriptomics is obvious. In consequence, our data indicate that the significance of thaumarchaeota to aerobic ammonium oxidation could yet have been considerably underestimated. Taken these results, this could in general also be the case in attempts aimed at an unbiased gene expression analysis of areas below the epipelagic zone, which cover 90% of the world's oceans.

Figure 1

Map of the central Baltic Sea including chemical profiles of the investigated pelagic redoxclines. Red dots indicate the positions of sampling stations 284 (Landsort Deep, 58°35.00′N, 18°14.06′E) and 271 (Gotland Deep, 57°19.89′N, 20°10.28′E). Embedded graphs present chemical data recorded during sampling in August 2008 and September 2009. The dashed line marks the sampling depths: 118 m (Gotland Deep 2008), 89 m (Landsort Deep 2008) and 72 m (Landsort Deep 2009). Data source for the map: Seifert T, Tauber F, Kayser B (2001). A High Resolution Spherical Grid Topography of the Baltic Sea (Revised Edition). Baltic Sea Science Conference, 25–29 November 2001; Stockholm. Poster #147 (http://www.io-warnemuende.de/iowtopo).

Figure 2

Design of the AFIS. (a) Teflon water bottle (2.7 l volume); (b) syringe (hand syringe GENIUS 450 ml) with maximal volume of 300 ml; (c) bottle lids connected with closing devices on the CTD-bottle rosette; (d) adapter between syringe and bottle, coupled with a dispenser tube; (e) escapement for the syringe plunger, directly connected with the closing devices for the bottle lids. The operating mode is based on the following principles: (1) the syringe plunger is pushed by a spring; (2) the release of the spring is coupled with the closing devices for the bottle lids; (3) the solution passes through the dispenser tube into the bottle; (4) the dispenser tube extends from the top to the bottom of the bottle; (5) the solution is equally distributed through perforations of increasing diameters made in the dispenser tube.

Figure 3

Comparison of transcript abundances in PUMP-CTD and AFIS samples in relation to their references. Colors represent the investigated Deep: black, Landsort Deep (2009); orange, Landsort Deep (2008); green, Gotland Deep (2008). Deviations in gene expression were calculated by comparing PUMP-CTD or data of fixed samples with the accordant non-fixed free-flow bottle references. A factor of 1 denotes that the abundances were equal to those of the reference (marked as a dashed line). Investigated parameters were (a) abundances based on metatranscriptomics: thaumarchaeal NH₄⁺ transporter, amoA, amoB and amoC transcripts. RT-qPCR-based abundances: (b) thaumarchaeal amoA transcripts; (c) ‘Sulfurimonas sp. GD1' recA transcripts. P, PUMP-CTD; S, stop solution; C, Carnoy fixative. ^*Based on mean±s.d. of three independent replicates, the non-parametric Mann–Whitney test showed a significant difference to the reference (P<0.01).