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Comparative genome-wide identification of transcriptional start sites in two environmentally relevant cyanobacteria reveals unexpected diversity in the transcriptome architecture

ABSTRACT: Prochlorococcus is a genus of abundant and ecologically important marine cyanobacteria. Here, we present the comprehensive comparison of the structure and composition of the transcriptomes of two closely related Prochlorococcus strains, which, despite their similarities, have adapted their gene pool to specific environmental constraints. We used large-scale strand-specific cDNA sequencing, microarray RNA profiling and computational analyses to characterize the transcriptomes of Prochlorococcus sp. MED4 and Prochlorococcus sp. MIT9313, representatives of the two major ecotypes adapted to high and low light conditions, respectively. We present genome-wide maps of transcriptional start sites (TSS) for both organisms. Our data suggest antisense transcription for three quarters of all genes. A direct comparison revealed very little conservation in the use of TSS and the nature of non-coding transcripts between both strains. We conclude that the major transcriptional output from these highly streamlined genomes consists of antisense RNA and that a hitherto unrecognized high degree of variability exists in the transcriptional architecture of these rapidly evolving prokaryotes. Furthermore, we detected extremely short 5’ untranslated regions with a median length of only 27 nt and 29 nt for MED4 and MIT9313, respectively, and noticed the absence of the Shine-Dalgarno motif, which is suggestive of alternative mechanisms for the initiation of translation. For 8 % of all protein-coding genes, the median length to the initiator codon is 10 nt or shorter, suggesting that leaderless translation and ribosomal protein S1-dependent translation constitute the primary avenues for protein synthesis in Prochlorococcus.

ORGANISM(S): Prochlorococcus phage P-SSM4 Prochlorococcus marinus str. MIT 9313 Tiamatvirus PSSP7 Prochlorococcus marinus subsp. pastoris str. CCMP1986

PROVIDER: GSE53065 | GEO | 2013/12/07

SECONDARY ACCESSION(S): PRJNA230785

REPOSITORIES: GEO

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Project description:Prochlorococcus is a genus of abundant and ecologically important marine cyanobacteria. Here, we present the comprehensive comparison of the structure and composition of the transcriptomes of two closely related Prochlorococcus strains, which, despite their similarities, have adapted their gene pool to specific environmental constraints. We used large-scale strand-specific cDNA sequencing, microarray RNA profiling and computational analyses to characterize the transcriptomes of Prochlorococcus sp. MED4 and Prochlorococcus sp. MIT9313, representatives of the two major ecotypes adapted to high and low light conditions, respectively. We present genome-wide maps of transcriptional start sites (TSS) for both organisms. Our data suggest antisense transcription for three quarters of all genes. A direct comparison revealed very little conservation in the use of TSS and the nature of non-coding transcripts between both strains. We conclude that the major transcriptional output from these highly streamlined genomes consists of antisense RNA and that a hitherto unrecognized high degree of variability exists in the transcriptional architecture of these rapidly evolving prokaryotes. Furthermore, we detected extremely short 5M-bM-^@M-^Y untranslated regions with a median length of only 27 nt and 29 nt for MED4 and MIT9313, respectively, and noticed the absence of the Shine-Dalgarno motif, which is suggestive of alternative mechanisms for the initiation of translation. For 8 % of all protein-coding genes, the median length to the initiator codon is 10 nt or shorter, suggesting that leaderless translation and ribosomal protein S1-dependent translation constitute the primary avenues for protein synthesis in Prochlorococcus. Prochlorococcus cells were either grown under standard growth conditions or various stress conditions (darkness, high light, iron or nitrogen depletion, cold or heat stress). Stress conditions were subsequently pooled and hybridized on one microarray.

Project description:We compared changes induced by the addition of 100 nM and 5 mM glucose on the proteome and metabolome complements in Synechococcus sp. strains WH8102, WH7803, and BL107 and Prochlorococcus sp. strains MED4, SS120, and MIT9313, grown either under standard light conditions or in darkness. Our results suggested that glucose is metabolized by these cyanobacteria, using primarily the oxidative pentoses and Calvin pathways, while no proof was found for the involvement of the Entner-Doudoroff pathway in this process. We observed differences in the effects of glucose availability, both between genera and between Prochlorococcus MED4 and SS120 strains, which might be related to their specific adaptations to the environment. We found evidence for fermentation in Prochlorococcus sp. strain SS120 and Synechococcus sp. strain WH8102 after 5 mM glucose addition. Our results additionally suggested that marine cyanobacteria can detect nanomolar glucose concentrations in the environment and that glucose might be used to sustain metabolism under darkness. Furthermore, the KaiB and KaiC proteins were also affected in Synechococcus sp. WH8102, pointing to a direct link between glucose assimilation and circadian rhythms in marine cyanobacteria. In conclusion, our study provides a wide overview on the metabolic effects induced by glucose availability in representative strains of the diverse marine picocyanobacteria, providing further evidence for the importance of mixotrophy in marine picocyanobacteria. The Prochlorococcus sp. strain PCC 9511 is genetically identical to MED4IMPORTANCE Glucose uptake by marine picocyanobacteria has been previously described and strongly suggests they are mixotrophic organisms (capable of using energy from the sun to make organic matter, but also to directly use organic matter from the environment when available). However, a detailed analysis of the effects of glucose addition on the proteome and metabolome of these microorganisms had not been carried out. Here, we analyzed three Prochlorococcus sp. and three Synechococcus sp. strains which were representative of several marine picocyanobacterial clades. We observed differential features in the effects of glucose availability, depending on both the genus and strain; our study illuminated the strategies utilized by these organisms to metabolize glucose and showed unexpected links to other pathways, such as circadian regulation. Furthermore, we found glucose addition had profound effects in the microbiome, favoring the growth of coexisting heterotrophic bacteria.

Project description:Background: Prokaryotes have relatively small genomes, densely-packed and apparently dominated by protein-encoding sequences. However, data now generated by high throughput RNA sequencing (RNA-seq) reveal surprisingly more-complex transcriptomes with many previously unrecognized and unanticipated non-coding small and antisense transcripts. To date, such studies have investigated primarily Bacteria. Here, we report the transcripts present in Thermococcus kodakarensis, a model hyperthermophilic Archaeon, synthesized under different growth and metabolic conditions. Results: cDNA libraries, generated from RNA preparations isolated from cells growing in media with sulfur or pyruvate, with sulfur to stationary phase, and growing with pyruvate but with sulfur added 20 min before RNA isolation, have been deep-sequenced. The results identify >2,700 sites of transcription initiation, establish a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements in T. kodakarensis. Primary transcription start sites (TSS) are identified upstream of 1,254 annotated genes, including ~78 % of those predicted by promoter locations, and an additional 644 primary TSS and their promoters have been identified within genes. Most of the mRNAs have a 5'-untranslated region (5'-UTR) between 10 and 50 nt long (median length = 16 nt), ~20 % have 5'-UTRs from 50 to 300 nt long, ~14 % are leaderless with 5'-UTRs ≤8 nt, and ~50% contain a consensus ribosome binding sequence. The results also identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA. The data confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 snoRNAs predicted to be encoded in the T. kodakarensis genome. Two transcripts, putatively identified as riboswitches, were present in RNA preparations isolated from growing but not from stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the data obtained also provide a semi-quantitative overview of global operon expression. They document substantial differences in gene expression under different physiological conditions and are consistent with previous observations of substrate-dependent specific gene expression. Many previously unrecognized and unanticipated small RNAs have been identified, some with relative low GC contents (≤50%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile. Conclusion: We have identified >2,700 TSS that include almost all of the primary sites of transcription initiation upstream of annotated genes, and also many secondary sites, sites within genes and sites resulting in antisense transcripts. The T. kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the results reveal the presence of many non-coding RNAs and predict extensive RNA-based regulation in T. kodakarensis.

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Comparative genome-wide identification of transcriptional start sites in two environmentally relevant cyanobacteria reveals unexpected diversity in the transcriptome architecture

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