Project description:<p>Diazotrophic cyanobacteria have a pivotal role in nitrogen fixation and soil fertility in paddy ecosystems, yet their responses to soil acidity stress (SAS) remain elusive. This study investigated the physiological and metabolic mechanisms underlying acid tolerance in diazotrophic cyanobacteria by comparing an acid-resistant strain (Nostoc sp. AT-23S) and an acid-sensitive strain (Nostoc sp. AS-61S) under acidic (pH 4.68) and neutral (pH 7.0) soil conditions. The results demonstrated that AT-23S maintained cytoplasmic pH homeostasis, sustained high levels of photosynthetic efficiency and nitrogenase activity, and exhibited enhanced synthesis of tightly bound extracellular polysaccharides (TB-EPS) under acid stress. Metabolomic analysis revealed significant up-regulation of gamma-aminobutyric acid (GABA) and 1-pyrroline-5-carboxylic acid (P5C) in AT-23S under acid stress; in contrast, AS-61S failed to maintain pH homeostasis, showed severe oxidative stress, and down-regulated GABA and P5C synthesis. The GABA was primarily originated from the putrescine degradation pathway, as confirmed by elevated diamine oxidase (DAO) activity and putrescine utilization rate. These findings demonstrate that the accumulation of GABA and P5C may be critical adaptive mechanism for acid tolerance in diazotrophic cyanobacteria, providing novel insights into their survival in acidic paddy soils.</p>
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: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: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:Threonine, an essential amino acid for translation, exhibits toxicity towards slow-growing cyanobacteria. This prompted us to perform transcriptomic and proteomic analyses using Microcystis aeruginosa cells treated with threonine, revealing a noteworthy upregulation of thrC, annotated as pyridoxal 5’-phosphate (PLP)-dependent threonine synthase. PyMoL-based structural prediction and in vitro biochemical assays unveiled the moonlighting functions of ThrC in both threonine biosynthesis and deamination. The production of α-ketobutyrate from threonine, both in vivo and in vitro, provided strong support for the role of ThrC as a threonine deaminase. The kinetics of ThrC as a threonine synthase using O-phospho-homoserine were consistent with the Michaelis-Menten kinetics of ThrC in Thermus thermophilus, albeit with a slightly lower Vmax value. However, the in vitro deaminase activity of ThrC, exhibiting sigmoidal kinetics, suggests the potential production of α-ketobutyrate under threonine oversupply conditions in slow-growing cyanobacteria. The phylogenetic lineage of our ThrC is positioned distinctively apart from canonical threonine synthase and threonine deaminase. Gene duplication and subsequent divergence of thrC, followed by gene deletion, could have contributed to the possession of moonlighting ThrC protein in aquatic bacteria including cyanobacterial lineages. Metabolomic data revealed that the presence of threonine and α-ketobutyrate disrupted the balance of amino acids (alanine and methionine) and DNA biosynthetic pathways via feedback inhibition. Interestingly, the addition of exogenous alanine or methionine could alleviate threonine toxicity. Our data revealed that threonine does not uniformly support the growth of freshwater cyanobacteria to the same extent, but rather can be toxic to certain groups within this bacterial family.
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: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.