Comparative day/night metatranscriptomic analysis of microbial ...

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genomes (Overbeek et al., 1999) to facilitate coordinated transcription. Thus a cell’s transcript pool is anticipated to include more mRNAs from adjacent genes than what is expected from a random sampling of the genome. We tested this using the transcripts assigned to taxonomic bins for P. marinus, P. ubique and Roseobacter by counting the frequency with which transcripts from two adjacent genes on the reference strain genome (defined as � 1 gene intervening) were both present in the bin, recognizing that the wild and reference organisms will not be fully syntenic. In all cases, the transcript bins had significantly more adjacent genes than a null distribution generated from the reference genomes (Fig. 4A), suggesting that random transcript sequencing captures operon-based expression patterns in natural marine bacterioplankton communities. Predicted highly expressed genes in environmental transcript pools Genes that are frequently transcribed by a cell can be identified based on patterns in codon usage (Karlin and Mrázek, 2000). We identified predicted highly expressed (PHX) genes for the reference genomes, and then assigned PHX status to the transcripts with best hits to that reference genome based on homology. For all taxa, and in accordance with biological expectations, the environmental transcript bins had a significantly higher percentage of PHX genes than the reference genomes (Fig. 4B). This pattern was particularly evident for the Roseobacters (9% of the genes in the reference genomes are PHX versus 30% of the transcripts; 3.1-fold enrichment) and for P. marinus MIT9301 (4.6% versus 12.9%; 2.8-fold enrichment). A larger proportion of PHX transcripts were found in the day for all P. marinus bins and the Roseobacter bin (although not for P. ubique), suggesting that highly expressed genes more frequently mediate daytime-biased processes (data not shown). Metatranscriptomic comparison of day and night samples The majority of annotated transcripts (~80%) were assigned to genes related to metabolism, and in particular to three KEGG categories: amino acid transport and metabolism, energy production and conversion (particularly oxidative phosphorylation, carbon fixation and nitrogen metabolism), and carbohydrate transport (Fig. 6). Membrane transport and signal transduction pathways were also common in the community transcriptome, specifically for ABC transporters of amino acids, glycine betaine/L-proline, polyamines (spermidine and putrescine), iron and nutrients in the form of nitrate, phosphate and phosphonate. Comparative Metatranscriptomic Analysis 1365 The day/night samples allowed comparison of dominant expression patterns in the presence and absence of solar radiation in the bacterioplankton community. Among the 167 KEGG metabolic pathways represented in the annotated sequences, four pathways were better represented at night (including those for glycospingolipid biosynthesis and nucleotide sugars metabolism) and six were better represented in the day (including photosynthesis and oxidative phosphorylation) (95% confidence level; Table 2). Some KEGG pathways had significant diel differences in frequency for individual taxonomic bins. These include: histidine biosynthesis, with evidence for expression of all or nearly all genes in the pathway (both P. ubique and P. marinus at night; Fig. 7A and Fig. S1A); metabolism of glutathione, a reductant with multiple detoxifying and cytoprotective capabilities (P. marinus at night); the photosynthesis pathway (phycobilisome, photosystem I and II, cytochromes, ATP synthase) and nearly all genes involved in biosynthesis of phytoene, and subsequent conversion into carotenoids (P. marinus in the day; Fig. 7B); nucleotide sugars metabolism, glycosphingolipid biosynthesis, carotenoid biosynthesis and vitamin B6 metabolism (P. ubique in the night; Fig. S1B); and transfer of methyl groups for C1 metabolism (P. ubique and Roseobacter in the day) (Table S3). Transcript annotation based on the COG database was comparable. Among the 1577 COGs represented, statistical comparisons identified 12 that were better represented at night and 13 that were better represented in the day (Table S4). These included amino acid and nucleotide metabolism, membrane biosynthesis and polyamine dehydrogenation at night, and light-mediated energy production, protein turnover, catalase synthesis and inorganic ion transport and metabolism in the day. Statistically significant differences in the distribution of transcripts between the day and night samples were also assessed independently of KEGG and COG assignments in order to capture signals from genes not currently classified by these annotation systems. Among the additional significant functions overrepresented in the night transcriptome were those for ABC-type spermidine/putrescine transport system permeases, RNA methyltransferases and signal transduction histidine kinases. For the day transcriptome, genes encoding proteorhodopsin and an aromatic-ring hydroxylase were significantly overrepresented (Table S5). Eukaryotic sequences The majority of eukaryotic transcripts were most closely affiliated with sequences from green-lineage organisms (Viridiplantae), such as the picoeukaryotic prasinophytes Ostreococcus spp. (Derelle et al., 2006) and Micromonas spp. A large number of transcripts also appeared to be © 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1358–1375
1366 R. S. Poretsky et al. Fig. 6. The 50 most abundant KEGG pathways in the night (black) and day (gray) transcriptomes. The pathways marked with stars were significantly overexpressed in one of the pools as determined by comparisons with P < 0.05 (Rodriguez-Brito et al., 2006). most closely related to genes in Chromalveoltae (Stramenopile or Alevolate) genomes. These groups are major components of the picoeukaryotic phytoplankton (McDonald et al., 2007) and are small enough to pass the 5 mm prefilter used in this study. Gene transcripts that most closely matched reference genomes of photosynthetic eukaryotes were more abundant in the day compared with night sample. Among the most highly © 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1358–1375
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