Project description:To identify novel phototransduction pathways in cyanobacteria, mutants defective for phytochrome-related proteins in Synechocystis sp. PCC 6803 that exhibit increased or decreased gene expression levels. were screened. Keywords: array-based
Project description:The direct photosynthetic production of polyhydroxyalkanoate in cyanobacteria was improved by increasing carbon flux to biosynthetic pathway and introducing enzyme with higher activity. To understand the global transcriptional changes under photoautotrophic PHA biosynthesis conditions, RNA-seq analysis was performed. Transcriptomes of recombinant Synechocystis sp. with different PHA-producing potential (three strains, two biological replicates for each strain) were analyzed.
Project description:Nitrogenases are the only enzymes able to ‘fix’ gaseous nitrogen into bioavailable ammonia and, hence, are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited, with electron transport to the iron (Fe)-nitrogenase having hardly been investigated. Here, we characterised the electron transfer pathway to the Fe-nitrogenase in Rhodobacter capsulatus via proteome analyses, genetic deletions, complementation studies and phylogenetics. Proteome analyses revealed an upregulation of four ferredoxins under nitrogen-fixing conditions reliant on the Fe-nitrogenase in a molybdenum nitrogenase knockout strain (nifD), compared to non-nitrogen-fixing conditions. Based on these findings, R. capsulatus strains with deletions of ferredoxin (fdx) and flavodoxin (fld, nifF) genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase. R. capsulatus deletion strains were characterised by monitoring diazotrophic growth and nitrogenase activity in vivo. Only deletion of fdxC or fdxN resulted in slower growth and reduced Fe-nitrogenase activity, whereas the double-deletion of both fdxC and fdxN abolished diazotrophic growth. Differences in the proteomes of ∆fdxC and ∆fdxN strains, in conjunction with differing plasmid complementation behaviours of fdxC and fdxN, indicate that the two Fds likely possess different roles and functions. These findings will guide future engineering of the electron transport systems to nitrogenase enzymes, with the aim of increased electron flux and product formation.
Project description:Substantial evidence has been accumulated about the molecular basis underlying halotolerance; however, insights into the regulatory networks for relevant genes and mechanisms of their interplay remain elusive. Here, we present a comprehensive transcriptome investigation, using RNA sequencing, of specific metabolic pathways and networks in a halotolerant cyanobacterium, Halothece sp. PCC7418, including the circadian rhythm profile. Dissecting the transcriptome presented the intracellular regulation of gene expressions, which was linked with ion homeostasis, protein homeostasis, biosynthesis of compatible solutes, and signal transduction, for adaptations to high-salinity environments. The efficient production and distribution of energy were also implicated in this acclimation process. Furthermore, we found that high-salinity environments had a dramatic effect on the global transcriptional expression regulated by the circadian clock. Our findings can provide a comprehensive transcriptome for elucidating the molecular mechanisms underlying halotolerance in cyanobacteria.
Project description:Cyanobacteria have developed an impressive array of proteins and pathways, each tailored for specific metabolic attributes, to execute photosynthesis and biological nitrogen (N2)-fixation. An understanding of these biologically incompatible processes provides important insights into how they can be optimized for renewable energy. To expand upon our current knowledge, we performed label-free quantitative proteomic analysis of the unicellular diazotrophic cyanobacterium Crocosphaera subtropica ATCC 51142 grown with and without nitrate under 12-hour light-dark cycles. Results showed significant shift in metabolic activities including photosynthesis, respiration, biological nitrogen fixation (BNF), and proteostasis to different growth conditions. We identified more than 20 nitrogenase enzymes which were among the most highly expressed proteins in the dark under nitrogen-fixing conditions, emphasizing their importance in BNF. Nitrogenase enzymes were not expressed under non nitrogen fixing conditions, suggesting a regulatory mechanism based on nitrogen availability. The synthesis of key respiratory enzymes and uptake hydrogenase (HupSL) synchronized with the synthesis of nitrogenase indicating a coordinated regulation of processes involved in energy production and BNF. Data suggests alternative pathways that cells utilize, such as oxidative pentose phosphate (OPP) and 2-oxoglutarate (2-OG) pathways, to produce ATP and support bioenergetic BNF. Data also indicates the important role of uptake hydrogenase for the removal of O2 to support BNF. Overall, this study expands upon our knowledge regarding molecular responses of Crocosphaera 51142 to nitrogen and light-dark phases, shedding light on potential applications and optimization for renewable energy.
Project description:The diazotrophic bacterium Rhodobacter capsulatus synthesizes a molybdenum nitrogenase and an alternative iron-only nitrogenase, enabling growth with molecular dinitrogen as sole nitrogen source. Regulation of nitrogen fixation was analyzed by proteome profiling of wild-type and mutant strains lacking the transcriptional regulators NifA, AnfA, and MopAB.
Project description:The direct photosynthetic production of polyhydroxyalkanoate in cyanobacteria was improved by increasing carbon flux to biosynthetic pathway and introducing enzyme with higher activity. To understand the global transcriptional changes under photoautotrophic PHA biosynthesis conditions, RNA-seq analysis was performed.
Project description:Molecular clocks are the basis for dating the divergence between lineages over macro-evolutionary timescales (~104-108 years). However, classical DNA-based clocks tick too slowly to inform us about the recent past. Here, we demonstrate that stochastic DNA methylation changes at a subset of cytosines in plant genomes possess a clock-like behavior. This ‘epimutation-clock’ is orders of magnitude faster than DNA-based clocks and enables phylogenetic explorations on a scale of years to centuries. We show experimentally that epimutation-clocks recapitulate known topologies and branching times of intra-species phylogenetic trees in the selfing plant A. thaliana and the clonal seagrass Z. marina, which represent the two primary modes of plant reproduction. This discovery will open new possibilities for high-resolution temporal studies of plant biodiversity.
Project description:Molecular clocks are the basis for dating the divergence between lineages over macro-evolutionary timescales (~104-108 years). However, classical DNA-based clocks tick too slowly to inform us about the recent past. Here, we demonstrate that stochastic DNA methylation changes at a subset of cytosines in plant genomes possess a clock-like behavior. This ‘epimutation-clock’ is orders of magnitude faster than DNA-based clocks and enables phylogenetic explorations on a scale of years to centuries. We show experimentally that epimutation-clocks recapitulate known topologies and branching times of intra-species phylogenetic trees in the selfing plant A. thaliana and the clonal seagrass Z. marina, which represent the two primary modes of plant reproduction. This discovery will open new possibilities for high-resolution temporal studies of plant biodiversity.