Light-driven chloride transport kinetics of halorhodopsin

Despite growing interest in light-driven ion pumps for use in optogenetics, current estimates of their transport rates span two orders of magnitude due to challenges in measuring slow transport processes and determining protein concentration and/or orientation in membranes in vitro. In this study, we report, to our knowledge, the first direct quantitative measurement of light-driven Cl transport rates of the anion pump halorohodopsin from Natronomonas pharaonis (NpHR). We used light-interfaced voltage clamp measurements on NpHR-expressing oocytes to obtain a transport rate of 219 (± 98) Cl /protein/s for a photon flux of 630 photons/protein/s. The measurement is consistent with the literature-reported quantum efficiency of ∼30% for NpHR, i.e., 0.3 isomerizations per photon absorbed. To reconcile our measurements with an earlier-reported 20 ms rate-limiting step, or 35 turnovers/protein/s, we conducted, to our knowledge, novel consecutive single-turnover flash experiments that demonstrate that under continuous illumination, NpHR bypasses this step in the photocycle

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

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

Day peaking oscillatory genes of <i>Cyanothece</i> 51142.

<p>Genes were selected based on 1.5-fold change cutoff and clustered using the gene expression data under continuous light (LL) (present study) and light/dark (LD) condition [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125148#pone.0125148.ref019" target="_blank">19</a>]. Panels (a) and (b) show the essential genes that are involved in the organization of Phycobilisomes and Phycobiliproteins; panels (c) and (d) show the genes that participate in biogenesis, repair and assembly of Photosystem II. Panels (e) and (f) refer to the genes that are involved in functioning of Photosystem I, peaking during the mid-day or mid of the photosynthetic phase. Panel (g)-(h) represent genes that participate in the electron transport chain and panels (i)-(k) represent the genes that play an important role in carbon fixation, glycogen synthesis and central carbon metabolism. Panels (l)-(n) show the genes that are involved in cell division, ribosome recycling, RNA polymerase sigma factor, where all up-regulate at dawn or during the onset or middle of the photosynthetic phase to support cell growth and maintenance. Grey and White filled and continuously white filled boxes in the X-axis, indicate the culture growth conditions of LD and LL, respectively.</p

Sinusoidal model fitting to cyclic genes and estimation of model parameters.

<p>A: Results shown for <i>nifK</i> gene (cce_0561) as a representative under continuous light with the observed expression profile (●), linear term of the model (grey straight line, a + b*t from Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125148#pone.0125148.e001" target="_blank">1</a>)), data points after subtracting the linear term (○) and sinusoidal fit (grey sinusoidal curve). B: Histogram showing periodicity of oscillation for n = 2152 genes, deemed to be cyclic by sinusoidal model with a cut-off of 25 percent residual percent energy as a measure of goodness of fit.</p

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