Comparative day/night metatranscriptomic analysis of microbial ...

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Depth (m) 0 50 100 150 200 sequences, Proteobacteria were most abundant (41%; Fig. 3) and were dominated by a-Proteobacteria (22%), b-Proteobacteria (8%) and g-Proteobacteria (8%). Bacteroidetes (8%) and Firmicutes (12%, biased towards the day sample) were also well represented. Taxonomically binned mRNA sequences were compared with community composition data to ask whether taxa contributed to the HOT community mRNA in proportion to their representation in the microbial assemblage (i.e. whether taxa are equally transcriptionally active on a per-cell basis). Cyanobacteria dominated the transcript libraries (55% of sequences) with about twofold higher representation than in the 16S rRNA amplicons or the cell count data (Fig. 3), indicating that there is more gene expression in these autotrophic bacterioplankton than in co-occurring heterotrophs (or possibly that their transcripts are longer-lived). When relative 16S rRNA abundance was calculated among just the heterotrophic groups (i.e. with cyanobacterial sequences removed), many taxa had similar contributions to the transcript pool and amplicon pool, suggesting comparable levels of transcriptional activity on a per-gene basis within the limits of recognized biases of PCR amplification (Fig. 3). 0 200 400 600 chla (10 -3 μg l -1 ) Prochlorococcus x 10 3 cells ml -1 Synechococcus x 10 2 cells ml -1 Nanoeukaryotes x 10 2 cells ml -1 Heterotrophic bateria x 10 3 cells ml -1 Comparative Metatranscriptomic Analysis 1361 Fig. 2. Depth profiles of Prochlorococcus-like, Synechococcus-like, heterotrophic bacteria and pigmented nanoeukaryotes during the HOT-175 cruise, as determined by flow cytometry. The horizontal line indicates the mixed layer depth. The depth profile for chlorophyll a is also indicated. Data were collected through the HOT project and downloaded from the HOT Data Organization and Graphical System (http://hahana.soest.hawaii.edu/hot/hot-dogs/). Proteobacteria contributed the second largest number of transcript sequences (28%), most of which were attributed to a-Proteobacteria (19%) and g-Proteobacteria (4%). Approximately 2% of the total transcripts were of eukaryotic origin. Comparing putative taxonomic assignments of transcripts between day and night, Cyanobacteria contributed equally to the day and night transcriptome (55% versus 56%) as did a-Proteobacteria (40% versus 45% of heterotrophic transcripts) and g-Proteobacteria (11% versus 8% of heterotrophic transcripts) (Fig. 3). More detailed taxonomic assignment of transcripts was carried out for the best represented clades. The Cyanobacteria transcripts were dominated by Prochlorococcuslike sequences most similar to P. marinus AS9601, P. marinus MIT 9301 and P. marinus MIT 9312 (Table 1). The a-Proteobacteria, the most transcriptionally active among the heterotrophic groups, mostly contained sequences with similarity to the SAR11 group members P. ubique HTCC1002 and P. ubique HTCC1062 (~10% of prokaryotic transcripts). Roseobacter-like sequences were also represented and were primarily assigned to Dinoroseobacter shibae DFL 12, Jannaschia sp. CCS1, Silicibacter pomeroyi DSS-3, Roseobacter denitrificans © 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1358–1375
1362 R. S. Poretsky et al. A 16S rRNA genes mRNA B 16S rRNA genes mRNA Och 114 and Silicibacter sp. TM1040 (Table 1 and Fig. 4). These assignments do not imply that these actual species were present at the time of sample collection, but rather they represent the best current sequence matches for some of the more abundant environmental transcripts. Transcriptome coverage Cyanobacteria 18 % Cyanobacteria 55 % Cyanobacteria 21 % Cyanobacteria 56 % Other 82% Other 45% Other 79% Other 44% To estimate transcriptome coverage, 16S rRNA clone library data were used to establish a taxon-abundance model for the HOT community at an identity level of 99%. Assuming that each taxon expresses 1000 different genes at any given time (based on the Escherichia coli model; Ingraham et al., 1983) and that genome coverage Cyanobacteria Alphaproteobacteria Gammaproteobacteria Betaproteobacteria Deltaproteobacteria Epsilonproteobacteria Other Proteobacteria Actinobacteria Bacteroidetes Chlamydiae Chlorobi Chloroflexi Chrysiogenetes Acidobacteria Firmicutes Lentispaerae Planctomycetes Spirochaetes Thermotogae Verrucomicrobia Fig. 3. Contribution of taxa to the 16S rRNA amplicon pool and transcript pool for the day (A) and night (B) samples. Taxonomy is presented to the phylum level (based on NCBI taxonomy) except for Proteobacteria, which is at the subphylum level. The dashed red lines indicate cyanobacterial abundance in the night sample as determined by flow cytometric counting. follows a Lander–Waterman model (Lander and Waterman, 1988), we estimate that the most abundant taxon in the day or night sample had over 90% transcriptome coverage (i.e. 90% of the expressed genes were sequenced at least once), while the 15 most abundant taxa had more than half of their transcriptome represented (Table S2). Alternately, we determined the singletons and doubletons among the COG categories (i.e. the number of COGs containing only one or two sequences) and applied the Chao1 index of diversity to determine the theoretical abundance of COGs in the day and night. The sequencing effort captured about 80% of the COGs predicted to be present in the night transcriptome and 70% of the COGs predicted for the day transcriptome (Table S2). © 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1358–1375
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