Project description:To identify transcriptional adaptations associated with increased alkane production in Nostoc punctiforme, we performed comparative transcriptomic analysis of an alkane overproduction strain. RNA-seq data identified a large number of highly up-regulated genes in the overproduction strain potentially involved in rRNA processing, mycosporine-glycine production, and synthesis of non-ribosomal peptides including nostopeptolide A. Other up-regulated genes encoding helical carotenoid proteins, stress-induced proteins, and those for microviridin synthesis were also up-regulated. The presence of several up-regulated genes or operons on multi-copy plasmids resulted in reduced alkane production, indicating possible targets for mutagenesis that might limit lipid droplet and alkane production.
Project description:Background: High-energy-density biofuels are typically derived from the fatty acid biosynthesis pathway, thus establishing free fatty acids (FFAs) as important fuel precursors. FFA production using photosynthetic microorganisms like cyanobacteria allows for direct conversion of carbon dioxide into fuel precursors. Recent studies investigating cyanobacterial FFA production have demonstrated the potential of this process, yet FFA production was also shown to have negative physiological effects on the cyanobacterial host, ultimately limiting high yields of FFAs. Results: Cyanobacterial FFA production was shown to generate reactive oxygen species (ROS) and lead to increased cell membrane permeability. To identify genetic targets that may mitigate these toxic effects, RNA-seq analysis was used to investigate the host response of Synechococcus elongatus PCC 7942. Stress response, nitrogen metabolism, photosynthesis, and protein folding genes were up-regulated during FFA production while genes involved in carbon and hydrogen metabolisms were down-regulated. Select genes were targeted for mutagenesis to confirm their role in mitigating FFA toxicity. Gene knockout of two porins and the overexpression of ROS-degrading proteins and hypothetical proteins reduced the toxic effects of FFA production, allowing for improved growth, physiology, and FFA production. Comparative transcriptomics, analyzing gene expression changes associated with FFA production and other stress conditions, identified additional key genes involved in cyanobacterial stress response. Conclusions: A total of 15 gene targets were identified to reduce the toxic effects of FFA production. While single-gene targeted mutagenesis led to minor increases in FFA production, the combination of these targeted mutations may yield additional improvement, advancing the development of high-energy-density fuels derived from cyanobacteria.
Project description:Previous molecular and mechanistic studies have identified several principles of prokaryotic transcription, but less is known about the global transcriptional architecture of bacterial genomes. Here we perform a comprehensive study of a cyanobacterial transcriptome, that of Synechococcus elongatus PCC 7942, generated by combining three high-resolution data sets: RNA sequencing, tiling expression microarrays, and RNA polymerase chromatin immunoprecipitation (ChIP) sequencing. We report absolute transcript levels, operon identification, and high-resolution mapping of 5' and 3' ends of transcripts. We identify several interesting features at promoters, within transcripts and in terminators relating to transcription initiation, elongation, and termination. Furthermore, we identify many putative non-coding transcripts. We provide a global analysis of a cyanobacterial transcriptome. Our results uncover insights that reinforce and extend the current views of bacterial transcription.
Project description:Previous molecular and mechanistic studies have identified several principles of prokaryotic transcription, but less is known about the global transcriptional architecture of bacterial genomes. Here we perform a comprehensive study of a cyanobacterial transcriptome, that of Synechococcus elongatus PCC 7942, generated by combining three high-resolution data sets: RNA sequencing, tiling expression microarrays, and RNA polymerase chromatin immunoprecipitation (ChIP) sequencing. We report absolute transcript levels, operon identification, and high-resolution mapping of 5' and 3' ends of transcripts. We identify several interesting features at promoters, within transcripts and in terminators relating to transcription initiation, elongation, and termination. Furthermore, we identify many putative non-coding transcripts. We provide a global analysis of a cyanobacterial transcriptome. Our results uncover insights that reinforce and extend the current views of bacterial transcription. RNA Sequencing of the cyanobacterium Synechococcus elongatus PCC 7942 RNA polymerase ChIP Sequencing of the cyanobacterium Synechococcus elongatus PCC 7942 Tiling Microarray of the cyanobacterium Synechococcus elongatus PCC 7942
Project description:Despite a significant increase in genomic data, our knowledge of gene functions and their transcriptional responses to environmental stimuli remains limited. Here, we use the model keystone species Daphnia pulex to study environmental responses of genes in the context of their gene family history to better understand the relationship between genome structure and gene function in response to environmental stimuli. Daphnia were exposed to five different treatments, each consisting of a diet supplemented with one of five cyanobacterial species, and a control treatment consisting of a diet of only green algae. Differential gene expression profiles of Daphnia exposed to each of these five cyanobacterial species showed that genes with known functions are more likely to be shared by different expression profiles whereas genes specific to the lineage of Daphnia are more likely to be unique to a given expression profile. Furthermore, while only a small number of non-lineage specific genes was conserved across treatment type, there was a high degree of overlap in expression profiles at the functional level. The conservation of functional responses across the different cyanobacterial treatments can be attributed to the treatment specific expression of different paralogous genes within the same gene family. Comparison with available gene expression data in the literature suggests differences in nutritional composition in diets with cyanobacterial species compared to diets of green algae as a primary driver for cyanobacterial effects on Daphnia. We conclude that conserved functional responses in Daphnia across different cyanobacterial treatments are mediated through alternate regulation of paralogous gene families.
Project description:Despite a significant increase in genomic data, our knowledge of gene functions and their transcriptional responses to environmental stimuli remains limited. Here, we use the model keystone species Daphnia pulex to study environmental responses of genes in the context of their gene family history to better understand the relationship between genome structure and gene function in response to environmental stimuli. Daphnia were exposed to five different treatments, each consisting of a diet supplemented with one of five cyanobacterial species, and a control treatment consisting of a diet of only green algae. Differential gene expression profiles of Daphnia exposed to each of these five cyanobacterial species showed that genes with known functions are more likely to be shared by different expression profiles whereas genes specific to the lineage of Daphnia are more likely to be unique to a given expression profile. Furthermore, while only a small number of non-lineage specific genes was conserved across treatment type, there was a high degree of overlap in expression profiles at the functional level. The conservation of functional responses across the different cyanobacterial treatments can be attributed to the treatment specific expression of different paralogous genes within the same gene family. Comparison with available gene expression data in the literature suggests differences in nutritional composition in diets with cyanobacterial species compared to diets of green algae as a primary driver for cyanobacterial effects on Daphnia. We conclude that conserved functional responses in Daphnia across different cyanobacterial treatments are mediated through alternate regulation of paralogous gene families. Whole transcriptome dual color arrays were used to discover differentially expressed genes following sub-lethal exposure to five cyanobacteria in D. pulex. RNA was isolated from eight independent and concurrently replicated exposures of Daphnia to control and five cyanobacteria conditions. RNA was hybridized to microarrays using a standard, control vs. treated design that included dye swaps. Cyanobacteria were Anabaena (ANA), Aphanizomenon (Aph), Cylindrospermopsis (Cyl), Nodularia (Nod) and Oscillatoria (Osl).