Diurnal rhythm of a unicellular diazotrophic cyanobacterium under mixotrophic conditions and elevated carbon dioxide

Mixotrophic cultivation of cyanobacteria in wastewaters with flue gas sparging has the potential to simultaneously sequester carbon content from gaseous and aqueous streams and convert to biomass and biofuels. Therefore, it was of interest to study the effect of mixotrophy and elevated CO2 on metabolism, morphology and rhythm of gene expression under diurnal cycles. We chose a diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 as a model, which is a known hydrogen producer with robust circadian rhythm. Cyanothece 51142 grows faster with nitrate and/or an additional carbon source in the growth medium and at 3 % CO2. Intracellular glycogen contents undergo diurnal oscillations with greater accumulation under mixotrophy. While glycogen is exhausted by midnight under autotrophic conditions, significant amounts remain unutilized accompanied by a prolonged upregulation of nifH gene under mixotrophy. This possibly supports nitrogen fixation for longer periods thereby leading to better growth. To gain insights into the influence of mixotrophy and elevated CO2 on circadian rhythm, transcription of core clock genes kaiA, kaiB1 and kaiC1, the input pathway, cikA, output pathway, rpaA and representatives of key metabolic pathways was analyzed. Clock genes’ transcripts were lower under mixotrophy suggesting a dampening effect exerted by an external carbon source such as glycerol. Nevertheless, the genes of the clock and important metabolic pathways show diurnal oscillations in expression under mixotrophic and autotrophic growth at ambient and elevated CO2, respectively. Taken together, the results indicate segregation of light and dark associated reactions even under mixotrophy and provide important insights for further applications

Historisches Jahrbuch

Background The over consumption of fossil fuels has led to growing concerns over climate change and global warming. Increasing research activities have been carried out towards alternative viable biofuel sources. Of several different biofuel platforms, cyanobacteria possess great potential, for their ability to accumulate biomass tens of times faster than traditional oilseed crops. The cyanobacterium Cyanothece sp. ATCC 51142 has recently attracted lots of research interest as a model organism for such research. Cyanothece can perform efficiently both photosynthesis and nitrogen fixation within the same cell, and has been recently shown to produce biohydrogen--a byproduct of nitrogen fixation--at very high rates of several folds higher than previously described hydrogen-producing photosynthetic microbes. Since the key enzyme for nitrogen fixation is very sensitive to oxygen produced by photosynthesis, Cyanothece employs a sophisticated temporal separation scheme, where nitrogen fixation occurs at night and photosynthesis at day. At the core of this temporal separation scheme is a robust clocking mechanism, which so far has not been thoroughly studied. Understanding how this circadian clock interacts with and harmonizes global transcription of key cellular processes is one of the keys to realize the inherent potential of this organism. Results In this paper, we employ several state of the art bioinformatics techniques for studying the core circadian clock in Cyanothece sp. ATCC 51142, and its interactions with other key cellular processes. We employ comparative genomics techniques to map the circadian clock genes and genetic interactions from another cyanobacterial species, namely Synechococcus elongatus PCC 7942, of which the circadian clock has been much more thoroughly investigated. Using time series gene expression data for Cyanothece, we employ gene regulatory network reconstruction techniques to learn this network de novo, and compare the reconstructed network against the interactions currently reported in the literature. Next, we build a computational model of the interactions between the core clock and other cellular processes, and show how this model can predict the behaviour of the system under changing environmental conditions. The constructed models significantly advance our understanding of the Cyanothece circadian clock functional mechanisms

Rhythmic oscillations in KaiC1 phosphorylation and ATP/ADP ratio in nitrogen-fixing cyanobacterium <i>Cyanothece</i> sp. ATCC 51142

<p>Cyanobacterial circadian clock composed of the Kai oscillator has been unraveled in the model strain <i>Synechococcus elongatus</i> PCC 7942. Recent studies with nitrogen-fixing <i>Cyanothece</i> sp. ATCC 51142 show rhythmic oscillations in the cellular program even in continuous light albeit with a cycle time of ~11 h. In the present study, we investigate correlation between cellular rhythms, KaiC1 phosphorylation cycle, ATP/ADP ratio, and the redox state of plastoquinone pool in <i>Cyanothece</i>. KaiC1 phosphorylation cycle of <i>Cyanothece</i> was similar to that of <i>Synechococcus</i> under diurnal cycles. However, under continuous light, the cycle time was shorter (11 h), in agreement with physiological and gene expression studies. Interestingly, the ATP/ADP ratio also oscillates with an 11 h period, peaking concomitantly with the respiratory burst. We propose a mathematical model with C/N ratio as a probable signal regulating the clock in continuous light and emphasize the existence of a single timing mechanism regardless of the cycle time.</p

STRENGTH ANALYSIS OF 400 MM OVERLAPPED STEEL ADHESIVE DOUBLE LAP JOINTS REINFORCED BY RIVET

This is the age of technological revolutions. For newer mechanisms, advancement in bonding procedures is desperately needed. The most famous bonding type is Adhesive bonding. Double lap joints are considered as a good category joint in many applications. This paper explains the investigation on double lap hybrid joint. This was done for the vertical loaded MS Steel specimens. Overlapping length was kept constant and equal to 400 mm and two other factors viz. Number of rivets and adhesive composition (Resin to Hardner ratio) were varied. Finite Element analysis is done by using Ansys to see the nature of changes in the joint. Further validation by Mechanical testing is done by using Universal Testing Machine for Max. Stresses induced. Load- Stress relation obtained by Ansys results is approximately closer to experimental results

Heat map and hierarchical clustering of cyclic genes.

<p>Heat map was generated by performing hierarchical clustering for 1202 genes, which show 11h periodicity in oscillations and satisfy both Sinusoidal and Fourier Transform based models. Genes that peak under photosynthesis or respiration based metabolism are marked notionally as dawn peaking and dusk peaking, respectively. Normalized gene expression value, log₂ (fold change) is plotted as per the scale bar shown below the x-axis.</p

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

<div><p>Unicellular diazotrophic cyanobacteria such as <i>Cyanothece</i> sp. ATCC 51142 (henceforth <i>Cyanothece</i>), 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 <i>Synechococcus elongates</i> 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 <i>Cyanothece</i> 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.</p></div

Heat map and hierarchical clustering of cyclic genes.

<p>Heat map was generated by performing hierarchical clustering for 1202 genes, which show 11h periodicity in oscillations and satisfy both Sinusoidal and Fourier Transform based models. Genes that peak under photosynthesis or respiration based metabolism are marked notionally as dawn peaking and dusk peaking, respectively. Normalized gene expression value, log₂ (fold change) is plotted as per the scale bar shown below the x-axis.</p

Assessment of overrepresentation of Gene Ontology terms among the gene clusters of <i>Cyanothece</i> 51142^a.

<p>^aGenes were clustered using the K-means algorithms. Genes of <i>Cyanothece</i> 51142 that satisfy the 1.5-fold change filter in the gene expression data under LL (9 time points) (present study) and LD (12 time points) (data from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125148#pone.0125148.ref019" target="_blank">19</a>]) were subjected to K-means clustering using K = 40 clusters.</p><p>^bOverrepresentation of Gene Ontology terms was performed using Biological Networks Gene Ontology tool (BiNGO) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125148#pone.0125148.ref034" target="_blank">34</a>].</p><p>Assessment of overrepresentation of Gene Ontology terms among the gene clusters of <i>Cyanothece</i> 51142<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125148#t002fn001" target="_blank">^a</a>.</p

Comparison of oscillatory behavior of cyclic genes in nitrogen fixing (<i>Cyanothece</i> 51142) and non-nitrogen fixing organism (<i>Synechococcus</i> 8942).

<p>Cyclic genes were selected for <i>Cyanothece</i> 51142 and their homologs in non-nitrogen fixing <i>Synechococcus</i> 7942 (data from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125148#pone.0125148.ref032" target="_blank">32</a>]). Panels (a) and (b) show the genes that are involved in day metabolism, such as carbon concentrating mechanism (CCM) and ATP synthase complex, peaking in the dawn or at the onset of photosynthetic phase. Panels (c)-(f) show the genes that peak during night metabolism, such as glycogen degradation, central carbon metabolism, hydrogenase and housekeeping gene, all peaking at dusk or at the onset of respiration phase or at subjective night. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125148#pone.0125148.g004" target="_blank">Fig 4</a> for details.</p