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% PHX Genes Frequency Frequency Based on these coverage estimates, increased sequencing depth would have been required to fully capture some specialized processes carried out by rarer members of the HOT community, but frequently transcribed genes from abundant taxa were well represented. In support of this, transcript mapping to the three P. mari- Number of Adjacent Genes Fig. 4. Evidence for prokaryotic gene expression patterns in the community transcriptome based on P. marinus, P. ubique and Roseobacter genome bins. A. Operon-based expression was evaluated by comparing the number of adjacent transcripts (closed circles) to the number of adjacent genes found in 1000 random samples of the same size from the reference genome (black lines). B. Preferential representation of transcripts from genes predicted to be highly expressed was evaluated by comparing the per cent of PHX genes in the reference genome (grey bar) to the per cent in the transcript pool (black bar). Differences between transcript pools and reference genomes were significant for both operon and PHX analyses (Wilcoxon signed-rank test; P < 0.05). Table 1. Number of sequences from the community transcriptome with highest homology to the listed reference genomes, as determined by top BLASTX hit to RefSeq. Night Day Prochlorococcus marinus str. MIT 9301 6309 6292 Prochlorococcus marinus str. AS9601 3214 2849 Pelagibacter ubique HTCC1002 2541 1851 Prochlorococcus marinus str. MIT 9312 1430 1264 Pelagibacter ubique HTCC1062 1308 944 Dinoroseobacter shibae DFL 12 48 34 Jannaschia sp. CCS1 41 27 Silicibacter pomeroyi DSS-3 39 30 Roseobacter denitrificans Och 114 30 28 Silicibacter sp. TM1040 19 26 Comparative Metatranscriptomic Analysis 1363 nus and two P. ubique reference genomes showed sequences with homology to approximately half the genes, at coverage depths ranging from 1 to nearly 500 hits per gene (Fig. 5). Moreover, many of the reference genes with the greatest coverage are those mediating metabolic processes expected to be dominant in the HOT bacterioplankton community (e.g. the photosynthesis genes psaA and psaB, the light-harvesting complex and RuBisCo, ammonium transporters and transcriptionrelated genes; Fig. 5). Other genes on the reference genomes for which there is similarly deep transcript coverage (e.g. proteorhodopsin, Na+/solute symporters, colicin V production and several hypothetical proteins) can be hypothesized to also represent dominant metabolic activities (Fig. 5). Operon signature in environmental transcript pools Genes that encode steps in the same metabolic pathway are frequently clustered into operons in prokaryotic © 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1358–1375
1364 R. S. Poretsky et al. Occurences 30 25 20 15 10 5 0 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 500 450 400 150 100 50 425 400 75 50 25 80 60 40 20 Hypothetical protein Photosytem II PsbJ protein Ammonium transporter family Photosystem II PsbB (CP47) Ribulose bisphosphate carboxylase MIT9312 Photosystem II D2 Cytochrome b559, beta subunit MIT9301 Protoporphyrin IX magnesium chelatase, subunit chlH 0 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 AS9601 0 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 HTCC 1002 0 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 35 30 25 20 15 10 5 A E B D C Photosystem II PsbA (D1) lipoprotein precursor Bacteriorhodopsin light-harvesting complex protein Na+/solute symporter AcrB/AcrD/AcrF family protein (Acriflavin resistance) Chromosome segregation SMC family protein 30S ribosomal protein S1 excinuclease ABC subunit C heat shock protein a HTCC 1062 Integral membrane protein, interacts with FtsH Ribosomal protein L14 Ribosomal protein L20 Photosystem I PsaA Hypothetical protein Elongation factor Tu Photosystem I PsaB 30S ribosomal protein S3 Photosystem II reaction center Z DNA-directed RNA polymerase beta prime chain octaprenyl-diphosphate synthase translation elongation factor EF-G adenylylsulfate reductase 0 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 Fig. 5. Mapping of transcripts to five reference genomes. A–C are P. marinus strains; D–E are P. ubique strains. The x-axis shows gene number in the reference genome. Shaded areas represent possible hypervariable regions with few mapped transcripts. © 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1358–1375
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