Temperature dependence of the free-running period of the circadian clock.

<p>The data are based on at least two independent experiments. For 30°C, three independent experiments were averaged. Error bars show the SD. The temperature dependence, Q₁₀, of the period of the free-running circadian clock was calculated using a rate of 1 per period.</p

Chlorophyll content under 12h/12h dark/light regime in the presence and absence of O₂.

<p>Shown are the OD₆₈₀/OD₇₂₀ (green line, left axis), OD₇₂₀ (black line, right axis), dissolved O₂ concentration normalized to the air-saturated concentration (blue line, right axis) and growth rate normalized to the median (red circles, right axis). The inset on the right shows an enlargement of the time window from 288 h to 312 h in which sparging was stopped and started as indicated by the arrows. The blue line shows a gap where the dissolved oxygen concentration exceeded the measuring range of the probe. Dark periods are indicated by a dark bar.</p

Pearson’s correlation of growth rate with other culture parameters.

<p>Most parameters peak together with growth rate in the circadian cycle. Notably, relative chlorophyll content (OD₆₈₀/OD₇₂₀, light green circles), fluorescence emission from chlorophyll excitation (Ft_blue, blue line with crosses) and phycobilisome excitation (Ft_red, red diamonds) do not. The data shown here are of the same culture as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127715#pone.0127715.g001" target="_blank">Fig 1</a>. A value of 1 indicates a perfect positive linear correlation, 0 the absence of correlation and -1 perfect negative correlation. Note that the phase shift is obtained from sliding along the data points (i.e. growth/dilution cycles), which do not all represent the same time interval due to differences in growth rate. Shown further: pH (cyan squares), dissolved O₂ (green upward triangles) and dissolved CO₂ concentration (purple downward triangles).</p

On the Use of Metabolic Control Analysis in the Optimization of Cyanobacterial Biosolar Cell Factories

Oxygenic photosynthesis will have a key role in a sustainable future. It is therefore significant that this process can be engineered in organisms such as cyanobacteria to construct cell factories that catalyze the (sun)­light-driven conversion of CO₂ and water into products like ethanol, butanol, or other biofuels or lactic acid, a bioplastic precursor, and oxygen as a byproduct. It is of key importance to optimize such cell factories to maximal efficiency. This holds for their light-harvesting capabilities under, for example, circadian illumination in large-scale photobioreactors. However, this also holds for the “dark” reactions of photosynthesis, that is, the conversion of CO₂, NADPH, and ATP into a product. Here, we present an analysis, based on metabolic control theory, to estimate the optimal capacity for product formation with which such cyanobacterial cell factories have to be equipped. Engineered l-lactic acid producing <i>Synechocystis</i> sp. PCC6803 strains are used to identify the relation between production rate and enzymatic capacity. The analysis shows that the engineered cell factories for l-lactic acid are fully limited by the metabolic capacity of the product-forming pathway. We attribute this to the fact that currently available promoter systems in cyanobacteria lack the genetic capacity to a provide sufficient expression in single-gene doses

Relative amplitude of circadian oscillation.

<p>Shown are data from the free-running period following entrainment from the same culture as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127715#pone.0127715.g001" target="_blank">Fig 1</a>. Subjective ‘night’ in continuous light is indicated by striped bars. Each data point represents the average of a measured parameter in-between pump events. The time-weighted average of all data points after the first 24 h of continuous conditions (i.e. 84–162 h) was used to normalize each parameter to 1. All data points are plotted mid-cycle. Shown are growth rate (red circles), dissolved O₂ (dark green line) and dissolved CO₂ concentration (purple line), Ft_red (dark red line), Ft_blue (blue line), pH (cyan line) and the OD₆₈₀/OD₇₂₀ ratio (light green line).</p

Entrainment and free-running of a <i>Synechocystis</i> sp. PCC6803 culture in continuous culture in a photobioreactor.

<p>An unsynchronized culture was used to inoculate the photobioreactor approximately 140 h before entrainment, of which the last 12 h in continuous light are shown as the first 12 h. The culture was entrained by two periods of 12h/12h light/dark and subsequently subjected to continuous light. Dark periods are indicated by a grey background and solid dark bars. Subjective ‘night’ in continuous light is indicated by striped bars. Shown are the growth rate (red circles, left axis), the ratio of OD₆₈₀/OD₇₂₀ (green line, right axis) as measured by the photobioreactor, including the fit thereof (dashed line, right axis). Growth rate was calculated from the OD₇₂₀ measured by an integrated photocell in-between pump events using <i>μ</i> = (Δln(OD₇₂₀))/Δ<i>t</i>. Growth rate data points are plotted in the middle of each pump cycle.</p

Physiological analysis of the strains used in this study.

<p>Growth rates (hr⁻¹) for MG1655 (Wild type) and its quinone deletion mutants during exponential growth in Evan’s medium supplied with 50 mM glucose and 1% LB at 37°C under aerobic, anaerobic and anaerobic plus 50 mM fumarate conditions. The values represent the mean of measured values from biological triplicates with standard deviation.</p

Relationship between quinone concentrations and ArcA phosphorylation.

<p>Total ubiquinone content (nmoles/g), demethylmenaquinone content (nmoles/g), menaquinone content (nmoles/g) and ArcA phosphorylation (%) for MG1655 (Wild type) and its quinone mutants during exponential growth in Evan’s medium supplied with 50 mM glucose and 1% LB at 37°C under aerobic, anaerobic and anaerobic with 50 mM fumarate conditions. The amount of quinone (nmoles/g) is expressed in nanomoles per gram dry cell weight. The values represent the mean of measured values from biological triplicates with standard deviation. WT: K12-Wild type.</p

List of the strains used in this study.

<p>List of the strains used in this study.</p

Binding of Hydrogen-Citrate to Photoactive Yellow Protein Is Affected by the Structural Changes Related to Signaling State Formation

The tricarboxylic acid citric acid is a key intermediary metabolite in organisms from all domains of the tree of life. Surprisingly, this metabolite specifically interacts with the light-induced signaling state of the photoactive yellow protein (PYP), such that, at 30 mM, it retards recovery of this state to the stable ground state of the protein with up to 30%, in the range from pH 4.5 to pH 7. We have performed a detailed UV/vis spectroscopic study of the recovery of the signaling state of wild type (WT) PYP and two mutants, H108F and Δ25-PYP, derived from this protein, as a function of pH and the concentration of citric acid. This revealed that it is the dianionic form of citric acid that binds to the pB state of PYP. Its binding site is located in between the N-terminal cap and central β-sheet of PYP, which is accessible only in the signaling state of the protein. The obtained results show how changes in the distribution of subspecies of the signaling state of PYP influence the rate of ground state recovery