Regulation of PSII function in Cyanothece sp. ATCC 51142 during a light-dark cycle.

Cyanobacteria, as well as green algae and higher plants, have highly conserved photosynthetic machinery. Cyanothece sp. ATCC 51142 is a unicellular, aerobic, diazotrophic cyanobacterium that fixes N2 in the dark. In Cyanothece, the psbA gene family is composed of five members, encoding different isoforms of the D1 protein. A new D1 protein has been postulated in the literature, which blocks PSII during the night and allows the fixation of nitrogen. We present data showing changes in PSII function in cells grown in cycles alternating between 12 h of light and dark, respectively, at Cyanothece sp. ATCC 51142. Cyanothece sp. ATCC 51142 uses intrinsic mechanisms to protect its nitrogenase activity in a two-stage process. In Stage I, immediately after the onset of darkness, the cells lose photosynthetic activity in a reversible process, probably by dissociation of water oxidation complex from photosystem II via a mechanism that does not require de novo protein synthesis. In Stage II, a more severe disruption of photosystem II function occurs is in part protein synthesis dependent and it could be a functional signature of the presence of sentinel D1 in a limited number of reaction centers still active or not yet inactivated by the mechanism described in Stage I. This process of inhibition uses light as a triggering signal for both the inhibition of photosynthetic activity and recovery when light returns. The intrinsic mechanism of photosynthetic inactivation during darkness with the interplay of the two mechanisms requires further studies.

Dinitrogenase-Driven Photobiological Hydrogen Production Combats Oxidative Stress in Cyanothece sp. Strain ATCC 51142.

Photobiologically synthesized hydrogen (H2) gas is carbon neutral to produce and clean to combust, making it an ideal biofuel. Cyanothece sp. strain ATCC 51142 is a cyanobacterium capable of performing simultaneous oxygenic photosynthesis and H2 production, a highly perplexing phenomenon because H2 evolving enzymes are O2 sensitive. We employed a system-level in vivo chemoproteomic profiling approach to explore the cellular dynamics of protein thiol redox and how thiol redox mediates the function of the dinitrogenase NifHDK, an enzyme complex capable of aerobic hydrogenase activity. We found that NifHDK responds to intracellular redox conditions and may act as an emergency electron valve to prevent harmful reactive oxygen species formation in concert with other cell strategies for maintaining redox homeostasis. These results provide new insight into cellular redox dynamics useful for advancing photolytic bioenergy technology and reveal a new understanding for the biological function of NifHDK. IMPORTANCE: Here, we demonstrate that high levels of hydrogen synthesis can be induced as a protection mechanism against oxidative stress via the dinitrogenase enzyme complex in Cyanothece sp. strain ATCC 51142. This is a previously unknown feature of cyanobacterial dinitrogenase, and we anticipate that it may represent a strategy to exploit cyanobacteria for efficient and scalable hydrogen production. We utilized a chemoproteomic approach to capture the in situ dynamics of reductant partitioning within the cell, revealing proteins and reactive thiols that may be involved in redox sensing and signaling. Additionally, this method is widely applicable across biological systems to achieve a greater understanding of how cells navigate their environment and how redox chemistry can be utilized to alter metabolism and achieve homeostasis.

Coupling of Cellular Processes and Their Coordinated Oscillations under Continuous Light in Cyanothece sp. ATCC 51142, a Diazotrophic Unicellular Cyanobacterium.

Unicellular diazotrophic cyanobacteria such as Cyanothece sp. ATCC 51142 (henceforth Cyanothece), temporally separate the oxygen sensitive nitrogen fixation from oxygen evolving photosynthesis not only under diurnal cycles (LD) but also in continuous light (LL). However, recent reports demonstrate that the oscillations in LL occur with a shorter cycle time of ~11 h. We find that indeed, majority of the genes oscillate in LL with this cycle time. Genes that are upregulated at a particular time of day under diurnal cycle also get upregulated at an equivalent metabolic phase under LL suggesting tight coupling of various cellular events with each other and with the cell's metabolic status. A number of metabolic processes get upregulated in a coordinated fashion during the respiratory phase under LL including glycogen degradation, glycolysis, oxidative pentose phosphate pathway, and tricarboxylic acid cycle. These precede nitrogen fixation apparently to ensure sufficient energy and anoxic environment needed for the nitrogenase enzyme. Photosynthetic phase sees upregulation of photosystem II, carbonate transport, carbon concentrating mechanism, RuBisCO, glycogen synthesis and light harvesting antenna pigment biosynthesis. In Synechococcus elongates PCC 7942, a non-nitrogen fixing cyanobacteria, expression of a relatively smaller fraction of genes oscillates under LL condition with the major periodicity being 24 h. In contrast, the entire cellular machinery of Cyanothece orchestrates coordinated oscillation in anticipation of the ensuing metabolic phase in both LD and LL. These results may have important implications in understanding the timing of various cellular events and in engineering cyanobacteria for biofuel production.

Transcriptomic and proteomic dynamics in the metabolism of a diazotrophic cyanobacterium, Cyanothece sp. PCC 7822 during a diurnal light-dark cycle.

BACKGROUND: 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. RESULTS: 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 and protein abundances, and strong correlation of light dependent expression of both antisense and CRISPR-related gene expression. The proteins for nitrogenase and the pentose phosphate pathway were highest in the dark, whereas those for glycolysis and the TCA cycle were more prominent in the light. Interestingly, one copy of the psbA gene encoding the photosystem II (PSII) reaction center protein D1 (psbA4) was highly upregulated only in the dark. This protein likely cannot catalyze O2 evolution and so may be used by the cell to keep PSII intact during N2 fixation. The CRISPR elements were found exclusively at the ends of the large plasmid and we speculate that their presence is crucial to the maintenance of this plasmid. CONCLUSIONS: This investigation of parallel transcriptional and translational activity within Cyanothece sp. PCC 7822 provided quantitative information on expression levels of metabolic pathways relevant to engineering efforts. The identification of expression patterns for both mRNA and protein affords a basis for improving biofuel production in this strain and for further genetic manipulations. Expression analysis of the genes encoded on the 6 plasmids provided insight into the possible acquisition and maintenance of some of these extra-chromosomal elements.

Sustained H(2) production driven by photosynthetic water splitting in a unicellular cyanobacterium.

UNLABELLED: The relationship between dinitrogenase-driven H(2) production and oxygenic photosynthesis was investigated in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142, using a novel custom-built photobioreactor equipped with advanced process control. Continuously illuminated nitrogen-deprived cells evolved H(2) at rates up to 400 µmol ⋅ mg Chl(-1) ⋅ h(-1) in parallel with uninterrupted photosynthetic O(2) production. Notably, sustained coproduction of H(2) and O(2) occurred over 100 h in the presence of CO(2), with both gases displaying inverse oscillations which eventually dampened toward stable rates of 125 and 90 µmol ⋅ mg Chl(-1) ⋅ h(-1), respectively. Oscillations were not observed when CO(2) was omitted, and instead H(2) and O(2) evolution rates were positively correlated. The sustainability of the process was further supported by stable chlorophyll content, maintenance of baseline protein and carbohydrate levels, and an enhanced capacity for linear electron transport as measured by chlorophyll fluorescence throughout the experiment. In situ light saturation analyses of H(2) production displayed a strong dose dependence and lack of O(2) inhibition. Inactivation of photosystem II had substantial long-term effects but did not affect short-term H(2) production, indicating that the process is also supported by photosystem I activity and oxidation of endogenous glycogen. However, mass balance calculations suggest that carbohydrate consumption in the light may, at best, account for no more than 50% of the reductant required for the corresponding H(2) production over that period. Collectively, our results demonstrate that uninterrupted H(2) production in unicellular cyanobacteria can be fueled by water photolysis without the detrimental effects of O(2) and have important implications for sustainable production of biofuels. IMPORTANCE: The study provides an important insight into the photophysiology of light-driven H(2) production by the nitrogen-fixing cyanobacterium Cyanothece sp. strain ATCC 51142. This work is also of significance for biotechnology, supporting the feasibility of "direct biophotolysis." The sustainability of the process, highlighted by prolonged gas evolution with no clear sign of significant decay or apparent photodamage, provides a foundation for the future development of an effective, renewable, and economically efficient bio-H(2) production process.

Genome-scale modeling of light-driven reductant partitioning and carbon fluxes in diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142.

Genome-scale metabolic models have proven useful for answering fundamental questions about metabolic capabilities of a variety of microorganisms, as well as informing their metabolic engineering. However, only a few models are available for oxygenic photosynthetic microorganisms, particularly in cyanobacteria in which photosynthetic and respiratory electron transport chains (ETC) share components. We addressed the complexity of cyanobacterial ETC by developing a genome-scale model for the diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142. The resulting metabolic reconstruction, iCce806, consists of 806 genes associated with 667 metabolic reactions and includes a detailed representation of the ETC and a biomass equation based on experimental measurements. Both computational and experimental approaches were used to investigate light-driven metabolism in Cyanothece sp. ATCC 51142, with a particular focus on reductant production and partitioning within the ETC. The simulation results suggest that growth and metabolic flux distributions are substantially impacted by the relative amounts of light going into the individual photosystems. When growth is limited by the flux through photosystem I, terminal respiratory oxidases are predicted to be an important mechanism for removing excess reductant. Similarly, under photosystem II flux limitation, excess electron carriers must be removed via cyclic electron transport. Furthermore, in silico calculations were in good quantitative agreement with the measured growth rates whereas predictions of reaction usage were qualitatively consistent with protein and mRNA expression data, which we used to further improve the resolution of intracellular flux values.

Dynamic proteome analysis of Cyanothece sp. ATCC 51142 under constant light.

Understanding the dynamic nature of protein abundances provides insights into protein turnover not readily apparent from conventional, static mass spectrometry measurements. This level of data is particularly informative when surveying protein abundances in biological systems subjected to large perturbations or alterations in environment such as cyanobacteria. Our current analysis expands upon conventional proteomic approaches in cyanobacteria by measuring dynamic changes of the proteome using a (13)C(15)N-l-leucine metabolic labeling in Cyanothece ATCC51142. Metabolically labeled Cyanothece ATCC51142 cells grown under nitrogen-sufficient conditions in continuous light were monitored longitudinally for isotope incorporation over a 48 h period, revealing 414 proteins with dynamic changes in abundances. In particular, proteins involved in carbon fixation, pentose phosphate pathway, cellular protection, redox regulation, protein folding, assembly, and degradation showed higher levels of isotope incorporation, suggesting that these biochemical pathways are important for growth under continuous light. Calculation of relative isotope abundances (RIA) values allowed the measurement of actual active protein synthesis over time for different biochemical pathways under high light exposure. Overall results demonstrated the utility of "non-steady state" pulsed metabolic labeling for systems-wide dynamic quantification of the proteome in Cyanothece ATCC51142 that can also be applied to other cyanobacteria.

Mixotrophic and photoheterotrophic metabolism in Cyanothece sp. ATCC 51142 under continuous light.

The unicellular diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 (Cyanothece 51142) is able to grow aerobically under nitrogen-fixing conditions with alternating light-dark cycles or continuous illumination. This study investigated the effects of carbon and nitrogen sources on Cyanothece 51142 metabolism via (13)C-assisted metabolite analysis and biochemical measurements. Under continuous light (50 mumol photons m(-2) s(-1)) and nitrogen-fixing conditions, we found that glycerol addition promoted aerobic biomass growth (by twofold) and nitrogenase-dependent hydrogen production [up to 25 mumol H(2) (mg chlorophyll)( -1) h(-1)], but strongly reduced phototrophic CO(2) utilization. Under nitrogen-sufficient conditions, Cyanothece 51142 was able to metabolize glycerol photoheterotrophically, and the activity of light-dependent reactions (e.g. oxygen evolution) was not significantly reduced. In contrast, Synechocystis sp. PCC 6803 showed apparent mixotrophic metabolism under similar growth conditions. Isotopomer analysis also detected that Cyanothece 51142 was able to fix CO(2) via anaplerotic pathways, and to take up glucose and pyruvate for mixotrophic biomass synthesis.

Metabolic rhythms of the cyanobacterium Cyanothece sp. ATCC 51142 correlate with modeled dynamics of circadian clock.

These experiments aim to reveal the dynamic features that occur during the metabolism of the unicellular, nitrogen fixing cyanobacterium Cyanothece sp. when exposed to diverse circadian forcing patterns (LD 16:8, LD 12:12, LD 8:16, LD 6:6). The chlorophyll concentration grew rapidly from subjective morning when first illuminated to around noon, then remained stable from later in the afternoon and throughout the night. The optical density measured at 735 nm was stable during the morning chlorophyll accumulation, then increased in the early afternoon toward a peak, followed at dusk by a rapid decline toward the late night steady state. The authors propose that these dynamics largely reflect accumulation and subsequent consumption of glycogen granules. This hypothesis is consistent with the sharp peak of respiration that coincides with the putative hydrocarbon catabolism. In the long-day regimen (LD 16:8), these events may mark the transition from the aerobic photosynthetic metabolism to microaerobic nitrogen metabolism that occurs at dusk, and thus cannot be triggered by the darkness that comes later. Rather, control is likely to originate in the circadian clock signaling an approaching night. To explore the dynamics of the link between respiration and circadian oscillations, the authors extrapolated an earlier model of the KaiABC oscillator from Synechococcus elongatus to Cyanothece sp. The measured peak of respiratory activity at dusk correlated strongly in its timing and time width with the modeled peak in accumulation of the KaiB(4) complex, which marks the late afternoon phase of the circadian clock. The authors propose a hypothesis that high levels of KaiB(4) (or of its Cyanothece sp. analog) trigger the glycogen catabolism that is reflected in the experiments in the respiratory peak. The degree of the correlation between the modeled KaiB(4) dynamics and the dynamics of experimentally measured peaks of respiratory activity was further tested during the half-circadian regimen (LD 6:6). The model predicted an irregular pattern of the KaiABC oscillator, quite unlike mechanical or electrical clock pacemakers that are strongly damped when driven at double their endogenous frequency. This highly unusual dynamic pattern was confirmed experimentally, supporting strongly the validity of the circadian model and of the proposed direct link to respiration.

Effect of continuous light on diurnal rhythms in Cyanothece sp. ATCC 51142.

BACKGROUND: Life on earth is strongly affected by alternating day and night cycles. Accordingly, many organisms have evolved an internal timekeeping system with a period of approximately 24 hours. Cyanobacteria are the only known prokaryotes with robust rhythms under control of a central clock. Numerous studies have been conducted to elucidate components of the circadian clock and to identify circadian-controlled genes. However, the complex interactions between endogenous circadian rhythms and external cues are currently not well understood, and a direct and mathematical based comparison between light-mediated and circadian-controlled gene expression is still outstanding. Therefore, we combined and analyzed data from two independent microarray experiments, previously performed under alternating light-dark and continuous light conditions in Cyanothece sp. ATCC 51142, and sought to classify light responsive and circadian controlled genes. RESULTS: Fourier Score-based methods together with random permutations and False Discovery Rates were used to identify genes with oscillatory expression patterns, and an angular distance based criterion was applied to recognize transient behaviors in gene expression under constant light conditions. Compared to previously reported mathematical approaches, the combination of these methods also facilitated the detection of modified amplitudes and phase-shifts of gene expression. Our analysis showed that the majority of diurnally regulated genes, essentially those genes that are maximally expressed during the middle of the light and dark period, are in fact light responsive. In contrast, most of the circadian controlled genes are up-regulated during the beginning of the dark or subjective dark, and are greatly enriched for genes associated with energy metabolism. Many of the circadian controlled and light responsive genes are found in gene clusters within the Cyanothece sp. ATCC 51142 genome. Interestingly, in addition to cyclic expression patterns with a period of 24 hours, we also found several genes that oscillate with an ultradian period of 12 hours, a novel finding among cyanobacteria. CONCLUSION: We demonstrate that a combination of different analytical methods significantly improved the identification of cyclic and transient gene expression in Cyanothece sp. ATCC 51142. Our analyses provide an adaptable and novel analytical tool to study gene expression in a variety of organisms with diurnal, circadian and ultradian behavior.