Proteome Analyses of Strains ATCC 51142 and PCC 7822 of the Diazotrophic Cyanobacterium Cyanothece sp. under Culture Conditions Resulting in Enhanced H2 Production (original) (raw)
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2015
Cultures of the cyanobacterial genus Cyanothece have been shown to produce high levels of biohydrogen. These strains are diazotrophic and undergo pronounced diurnal cycles when grown under N 2-fixing conditions in light-dark cycles. We seek to better understand the way in which proteins respond to these diurnal changes, and we performed quantitative proteome analysis of Cyanothece sp. strains ATCC 51142 and PCC 7822 grown under 8 different nutritional conditions. Nitrogenase expression was limited to N 2-fixing conditions, and in the absence of glycerol, nitrogenase gene expression was linked to the dark period. However, glycerol induced expression of nitrogenase during part of the light period, together with cytochrome c oxidase (Cox), glycogen phosphorylase (Glp), and glycolytic and pentose phosphate pathway (PPP) enzymes. This indicated that nitrogenase expression in the light was facilitated via higher levels of respiration and glycogen breakdown. Key enzymes of the Calvin cycle were inhibited in Cyanothece ATCC 51142 in the presence of glycerol under H 2-producing conditions, suggesting a competition between these sources of carbon. However, in Cyanothece PCC 7822, the Calvin cycle still played a role in cofactor recycling during H 2 production. Our data comprise the first comprehensive profiling of proteome changes in Cyanothece PCC 7822 and allow an in-depth comparative analysis of major physiological and biochemical processes that influence H 2 production in both strains. Our results revealed many previously uncharacterized proteins that may play a role in nitrogenase activity and in other metabolic pathways and may provide suitable targets for genetic manipulation that would lead to improvement of large-scale H 2 production.
Applied and Environmental Microbiology, 2012
Cultures of the cyanobacterial genus Cyanothece have been shown to produce high 21 levels of biohydrogen. These strains are diazotrophic and undergo pronounced diurnal 22 cycles when grown under N 2 -fixing conditions in light-dark cycles. We seek to better 23 understand the way in which proteins respond to these diurnal changes and we 24 performed quantitative proteome analysis of Cyanothece ATCC 51142 and PCC 7822 25 grown under 8 different nutritional conditions. Nitrogenase expression was limited to N 2 -26 fixing conditions, and in the absence of glycerol, nitrogenase gene expression was 27 linked to the dark period. However, glycerol induced expression of nitrogenase during 28 part of the light period, together with cytochrome c oxidase (Cox), glycogen 29 phosphorylase (Glp), and glycolytic and pentose-phosphate pathway (PPP) enzymes. 30 This indicated that nitrogenase expression in the light was facilitated via higher 31 respiration and glycogen breakdown. Key enzymes of the Calvin cycle were inhibited in 32 Cyanothece ATCC 51142 in the presence of glycerol under H 2 producing conditions, 33 suggesting a competition between these sources of carbon. However, in Cyanothece 34 PCC 7822, the Calvin cycle still played a role in cofactor recycling during H 2 production. 35 Our data comprise the first comprehensive profiling of proteome changes in Cyanothece 36 PCC 7822, and allows an in-depth comparative analysis of major physiological and 37 biochemical processes that influence H 2 -production in both strains. Our results 38 revealed many previously uncharacterized proteins that may play a role in nitrogenase 39 activity and in other metabolic pathways and may provide suitable targets for genetic 40 manipulation that would lead to improvement of large scale H 2 production.
BMC genomics, 2014
Cyanothece sp. PCC 7822 is an excellent cyanobacterial model organism with great potential to be applied as a biocatalyst for the production of high value compounds. Like other unicellular diazotrophic cyanobacterial species, it has a tightly regulated metabolism synchronized to the light-dark cycle. Utilizing transcriptomic and proteomic methods, we quantified the relationships between transcription and translation underlying central and secondary metabolism in response to nitrogen free, 12 hour light and 12 hour dark conditions. By combining mass-spectrometry based proteomics and RNA-sequencing transcriptomics, we quantitatively measured a total of 6766 mRNAs and 1322 proteins at four time points across a 24 hour light-dark cycle. Photosynthesis, nitrogen fixation, and carbon storage relevant genes were expressed during the preceding light or dark period, concurrent with measured nitrogenase activity in the late light period. We describe many instances of disparity in peak mRNA an...
2011
Background: Unicellular cyanobacteria of the genus Cyanothece are recognized for their ability to execute nitrogen (N 2 )-fixation in the dark and photosynthesis in the light. An understanding of these mechanistic processes in an integrated systems context should provide insights into how Cyanothece might be optimized for specialized environments and/or industrial purposes. Systems-wide dynamic proteomic profiling with mass spectrometry (MS) analysis should reveal fundamental insights into the control and regulation of these functions. Results: To expand upon the current knowledge of protein expression patterns in Cyanothece ATCC51142, we performed quantitative proteomic analysis using partial ("unsaturated") metabolic labeling and high mass accuracy LC-MS analysis. This dynamic proteomic profiling identified 721 actively synthesized proteins with significant temporal changes in expression throughout the light-dark cycles, of which 425 proteins matched with previously characterized cycling transcripts. The remaining 296 proteins contained a cluster of proteins uniquely involved in DNA replication and repair, protein degradation, tRNA synthesis and modification, transport and binding, and regulatory functions. Functional classification of labeled proteins suggested that proteins involved in respiration and glycogen metabolism showed increased expression in the dark cycle together with nitrogenase, suggesting that N 2 -fixation is mediated by higher respiration and glycogen metabolism. Results indicated that Cyanothece ATCC51142 might utilize alternative pathways for carbon (C) and nitrogen (N) acquisition, particularly, aspartic acid and glutamate as substrates of C and N, respectively. Utilization of phosphoketolase (PHK) pathway for the conversion of xylulose-5P to pyruvate and acetyl-P likely constitutes an alternative strategy to compensate higher ATP and NADPH demand. Conclusion: This study provides a deeper systems level insight into how Cyanothece ATCC51142 modulates cellular functions to accommodate photosynthesis and N 2 -fixation within the single cell.
Journal of Phycology, 2009
Cyanothece sp. strain ATCC 51142 is a unicellular, diazotrophic cyanobacterium that demonstrates extensive metabolic periodicities of photosynthesis, respiration, and nitrogen fixation when grown under N 2 -fixing conditions. We have performed a global transcription analysis of this organism using 6 h light:dark (L:D) cycles in order to determine the response of the cell to these conditions and to differentiate between diurnal and circadian-regulated genes. In addition, we used a context-likelihood of relatedness (CLR) analysis with these data and those from 2 d L:D and L:D plus continuous light experiments to better differentiate between diurnal and circadian-regulated genes. Cyanothece sp. acclimated in several ways to growth under short L:D conditions. Nitrogen was fixed in every second dark period and only once in each 24 h period. Nitrogen fixation was strongly correlated to the energy status of the cells and glycogen breakdown, and high respiration rates were necessary to provide appropriate energy and anoxic conditions for this process. We conclude that glycogen breakdown is a key regulatory step within these complex processes. Our results demonstrated that the main metabolic genes involved in photosynthesis, respiration, nitrogen fixation, and central carbohydrate metabolism have strong (or total) circadian-regulated components. The short L:D cycles enable us to identify transcriptional differences among the family of psbA genes, as well as the differing patterns of the hup genes, which follow the same pattern as nitrogenase genes, relative to the hox genes, which displayed a diurnal, dark-dependent gene expression.
Journal of Bacteriology, 2008
We analyzed the metabolic rhythms and differential gene expression in the unicellular, diazotrophic cyanobacterium Cyanothece sp. strain ATCC 51142 under N2-fixing conditions after a shift from normal 12-h light-12-h dark cycles to continuous light. We found that the mRNA levels of ∼10% of the genes in the genome demonstrated circadian behavior during growth in free-running (continuous light) conditions. The genes for N2 fixation displayed a strong circadian behavior, whereas photosynthesis and respiration genes were not as tightly regulated. One of our main objectives was to determine the strategies used by these cells to perform N2 fixation under normal day-night conditions, as well as under the greater stress caused by continuous light. We determined that N2 fixation cycled in continuous light but with a lower N2 fixation activity. Glycogen degradation, respiration, and photosynthesis were also lower; nonetheless, O2 evolution was about 50% of the normal peak. We also demonstrate...
Plant Physiology, 2012
In order to accommodate the physiologically incompatible processes of photosynthesis and nitrogen fixation within the same cell, unicellular nitrogen-fixing cyanobacteria have to maintain a dynamic metabolic profile in the light as well as the dark phase of a diel cycle. The transition from the photosynthetic to the nitrogen-fixing phase is marked by the onset of various biochemical and regulatory responses, which prime the intracellular environment for nitrogenase activity. Cellular respiration plays an important role during this transition, quenching the oxygen generated by photosynthesis and by providing energy necessary for the process. Although the underlying principles of nitrogen fixation predict unicellular nitrogen-fixing cyanobacteria to function in a certain way, significant variations are observed in the diazotrophic behavior of these microbes. In an effort to elucidate the underlying differences and similarities that govern the nitrogen-fixing ability of unicellular dia...
Scientific Reports, 2015
To date, the proposed mechanisms of nitrogenase-driven photosynthetic H 2 production by the diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 have assumed that reductant and ATP requirements are derived solely from glycogen oxidation and cyclic-electron flow around photosystem I. Through genome-scale transcript and protein profiling, this study presents and tests a new hypothesis on the metabolic relationship between oxygenic photosynthesis and nitrogenasemediated H 2 production in Cyanothece 51142. Our results show that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized with nitrogenase expression and H 2 production. These findings provide a new and more complete view on the metabolic processes contributing to the energy budget of photosynthetic H 2 production and highlight the role of concurrent photocatalytic H 2 O oxidation as a participating process.