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CyanobacteriaHow to survive the night

By: Gerrit-Jan SchuttenSupervisor: Milou Schuurmans

Klaas Jan Hellingwerf

How to get most out of your day

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IntroductionCyanobacteria or blue-green algae are photorophic microorganisms, they gather energy form sunlight to grow and fix CO2 into biomass.Some also have the ability to fixate nitrogen (N2) into ammonia (NH4

+).There photosynthesis looks a lot like those found in plants, as unlike many other bacteria they have 2 photosystems. Cyanobacteria are thought to be partly responsible for changing the earths atmosphere in an oxidizing one, and thus forcing a change for life on earth.

One of the more important thinks for lifeforms is to be able to store energy/materials for times when they are scarce, like at night.One of the main problems for surviving the night is that the main energy source, “the sun” is missing. To counter this, energy is stored as the glucose polymer, glycogen. Glycogen is formed around the enzyme glycogenin, to which the abundant glucose that is produced during the day is attached. The glucose is linked in chains that can have multiple side chains.

The enzyme responsible for turning glycogen back into glucose (1-phosphate) during the night is glycogen phosphorylase. It is not yet fully know how exactly this enzyme is regulated in cyanobacteria, but in mammals cAMP and hormones play a role in Glycogen regulation1. Glycogen phosphorylase can only cut chains, the branches have to be cut by special debranching proteins.

However day an night rhythms change, and so does the required amount of glycogen to survive the night. To handle these changes, many cyanobacteria have a circadian clock2. This clock is based on 3 proteins the KaiA, KaiB and KaiC that interact with each other and regulate expression patterns, with this clock they are able to anticipate the day and night time, so they can properly prepare. One of the proteins regulated by this clock is GlgX (Slr0237), which is a debranching protein3 4.

Mass screening with yeast 2 hybrid assays revealed that there is a possible binding (interaction) between PixD (aka Slr1694) and glycogen phosphorylase5. This could indicate an alternative/redundant regulation of glycogen. In this research this hypothesis is confirmed, by studying PixD knockout mutants.

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In this research all strains are derivatives of the Synechocystis (PCC 6803), one of the most studied cyanobacteria and were provided by Stanford.Synechocystis can grow both autotrophically or heterotrophically in the absence of light. It was isolated from a freshwater lake in 1968 and is easily transformed by exogenous DNA. These strains have however already been in labs for so long that some have evolved slightly different.

WT3 A glucose tolerant strain

WT4 The parent strain on of ΔpixD

ΔpixD The pixD knockout mutant

The Synechocystis used have a relative slow growth rate.

PixD has a Blue Light Using FAD (BLUF) domain6, which is involved in photosyntaxis.

Blast, PixDBlast, PixDFigure 1: pixD's BLUF domain (image from blast).

The energy captured by this BLUF domain changes the conformation of the protein. PixD has no known enzymatic or kinase like activities.Synechocystis can move towards light gradients (phosyntaxis), however the ΔpixD mutant moves away from the light (negative photosyntaxis)6.

In this research these strains are grown in chemostats, and it was studied how a change in the day/night period, would affect glycogen production and storage. Glycogen levels where determined by glycogen assays.

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Materials and methods

ChemostatsThe cultures were grown in a flat backed chemostat with a diameter of around 10 cm for equal light distribution. The day night period started at 4 hours light, and was changed over weekends, by increasing the day length by 2 hours. The light for Synechocystis ΔpixD and its parent strain WT4, was provided by fluorescent lamps 30~60 µE (less when the cultures were diluted, and was kept stable from 12h on). WT3 was grown on LED light. All three cultures were run in duplicate.

Because of the weekly changes to the day/night rhythm, the cultures in the chemostats were never really stable. After the 10 hours of light samples were taken, the chemostats were reset. After this break the chemostats were put on normal air instead of nitrogen. The settings to get the chemostats “healthy” were as followed:

Volume: ~ 1.9 litersFlowrate: ~ 10.5 ml/hour of BG-11 Medium, ΔpixD with kanamycineDilution rate: ~ 0.0055 hours-1

Light intensity: ~ 30~60 µE (The chemostats that were reset stayed around 60µE)

Airflow: ~ 30 l/hourCO2: ~ 2% (rest normal air (on just nitrogen they died))Temperature ~ 30 °C

SamplingThe OD750 and the pH was measured using small samples (30 ml bottles), taken during the day night period switch. 2 mL sample was isolated, and centrifuged for 2 min at ~500 g1 ml supernatant was used for a HPLC analysisThe pellet was frozen (using liquid nitrogen) and stored for the glycogen assay.Every friday samples were taken each hour, for the duration of a light or dark cycle, the sampling during this time could not be fully compensated by the flowrate, but it could recover during the weekend. Because the OD750 was very low in some cases, larger samples were spun down (4-8 ml), to form a combined pellet.

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HPLC metabolic/fermentation product analysisHPLC was used to determine any metabolic/fermentation products secreted into the medium. 100 µl 35% PCA and 55 µl 7 M KOH was added to the supernatant, which was then filtered using 0.45 µm PTFe membrane filters and stored at 4 °C for max a week, before being injected into the HPLC.The column used was a Rezex column (for Sugars, Organic Acids and Alcohols)

Glycogen assayAn enzymatic glycogen assay7 8 9 was used to determine the amount of glycogen in the cells. Because there is no known way to easily measure glycogen levels inside cells, glycogen is first hydrolyzed by amyloglucosidase to glucose. For each chemostat the glycogen assay was done in duplicate (so a total of 4 measurements per culture).

Day 1:200 µl 5.35M KOH was added to the (frozen) pellet and hydrolyzed for 90 min at 100 °C using screwcaps. To precipitate the glycogen 600 µl of absolute ethanol was added to the cooled samples, and the samples were put on ice for 1-2 hours. The pellet was centrifuged for 5 min at max speed (>10k g) and washed twice with ethanol. The pellet was resuspended in 300 µl 200mM PH 5.2 acetate buffer and split in half. 10µl amyloglucosidase in 200 mM pH 5.2 acetate buffer, was added to half of the sample and incubated overnight at 57 °C under constant agitation.

Day 2:The samples were centrifuged for 3 min, at 5000 g. The glucose was determined by adding 20 µl of supernatant to 200 µl glucose oxidase mix (0,1 mg/ml D-glucose oxidase, 0,05 mg/ml peroxidase, 0,5mg/ml ABTS in 0,5 M Tris-HLC pH 7). Which was incubated for 1 hour at 37 °C under constant agitation. The glucose was then indirectly measured by using Glucose oxidase to create hydrogen peroxide. Which in turn was used by peroxidase to oxidize ABTS, which changes color. This color change was measured at 418 nm.

Glucose oxidase: glucose + 02 -> glucono delta-lactone + HOOH

figure 2: the reaction of glucose oxidase

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Peroxidase: HOOH + ABTSreduced -> ABTSoxidized+ 2 H2O

figure 3: the reaction of peroxidase10

Before the samples of the 12 hours of light period, the measurements were done by a normal photospectometer, after the 12 hour sample a 96Well plate reader was used (Different mixtures were used as the photospectrometer requires ~1ml samples).

Glycogen calculationThe molarity of calibration curve is the same as the 320 µl sample in acetate, which contained all the glycogen from the cell sample. To normalize everything was converted as if the sample taken, had an OD750 of 1.To convert from OD750 to dry weight, it was assumed that 1 liter of OD750 3 (against MQ, and 1 cm sample) = 1 gram of dry weight.

OD418CalibrationCurve

×320 .10−6× 1OD750×SamplesTaken

×10−3×1500 = mmol glucose per gram of dry-weight

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Results & ConclusionsThe cultures were unstable, the first 2 weekends the CO2 tanks went empty and the pH of the medium went up to high levels (pH levels are in the supplementary data), this resulted in worthless data from the 4 & 6 hours light periods.

Absorbtion

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Figure 4: the OD750 of the chemostats, reflecting the general health of the cultures, the light period is changed over the weekend, during which no measurements were taken and varies from 4 to 10 hours of light per day.

You can clearly see that the chemostats are never truly stable, and that they like to crash.

HPLC:We anticipated that without O2 in the airflow, we would find fermentation products during the night cycle, as there would most likely be to few oxygen to support normal respiration. However the HPLC column for metabolites and fermentation products, showed no results, indicating that there is no significant amount of fermentation products in the medium. This was the case for both with and without O2.

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Glycogen Assay:

Figure 5: Results glycogen assay, 8 hours light and 16 hours dark (indicated by the black vertical bars), and 10 hours light and 14 hours dark, during the weekend there were no measurements.

As you can see the error bars are very large, but in most cases there is an indication that glycogen is produced during the day, and used during the night, as expected. You can also see that during the day, the cells produce more then they use during the night. It's also clear that one week is apparently not enough for glycogen levels to stabilize as they should in a continuous culture, because glycogen stocks continue to increase.

It appears that the ΔpixD mutant produces significantly less glycogen, and has far less stored, as well.

If you add the data from figure 4 and 5 you can see that chemostat 5 has a lower OD750 then chemostat 6, but has higher glycogen levels, this might be that due to the lower concentration, they have more light per cell.

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Because of the decline in cell numbers and the crashes, the chemostats were reset, the glycogen assays from 12 hour light and longer were done in a 96well plate reader instead of done by hand. Some weeks are not shown as its more of the same, those results can be found in the supplementary data.

Absorbtion

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Figure 6: the OD750 of the chemostats, reflecting the general health of the cultures in; 12, 14, 16 and 18 hours of light, the period is changed over the weekend, during which no measurements were taken.

Because they were freshly inoculated the cultures started out at a relative low OD750, they all seem to grow well except for chemostat 8, which declines after a while.

Figure 7: Results glycogen assay, 12 hours light and dark (indicated by the black vertical bars), WT 3 was also introduced here.

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When the samples of the 12 hour light period in figure 7 where taken, chemostats 5-8, still had a low concentration, and the introduction of the day night period caused the OD750 to decrease a little further. Still the values are not that different compared to those of 8 and 10 hour light (figure 5).

The hourly samples taken during the night of a 12 hours light and dark cycle in figure 8, clearly show that glycogen is used up gradually during the night.This might indicate that the debranching enzyme (Slr0237) is not the rate limiting step, though there is not enough evidence to definitively draw that conclusion.

Figure 8: Results glycogen assay, 12 hours light and dark, 1 hour time samples all taken the same day.

Figure 9: Results glycogen assay, 16 hours light and 8 hours dark.

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The 16 hours of light period had the highest measured glycogen levels, the large peak on the last day is probably caused by the use of a different glucose oxidase mix.

If you ignore the glycogen stocks, and only look at the difference between the start and end of the day, you can see how much glycogen is produced.

Gross glycogen production

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Figure 10: Gross glycogen production, determined by the difference between the start and end of the day, using the results from the glycogen assay over the period of 1 week per point. The vertical black line indicates new chemostats.

The measurement errors are to big and there are to many unfavorable factors to draw any definitive conclusions, but it looks like the top glycogen production is at 16 hours of light for all cultures.

This could indicate some kind of regulation, as on continuous light there is no glycogen storage, and glycogen production isn't just increased by a longer daytime. Though it might just be that the night time is to short for ample degrading enzymes to be produced/activated.

The glycogen usage and net production (figures 11 and 12) follow similar trends, again the cumulative experimental errors are to big to use this as definitive evidence on its own.

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Glycogen usage

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Figure 11: Gross glycogen usage, determined by the difference between the start and end of the night, using the results from the glycogen assay over the period of 1 week per point. The vertical black line indicates new chemostats.

Net glycogen production

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Figure 12: Gross glycogen usage, determined by the difference between the start and end of the night, using the results from the glycogen assay over the period of 1 week per point. The vertical black line indicates new chemostats.

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DiscussionThe glycogen usage is also increased at 16 hours of light, as can be seen in figure 12. This is a little surprising because the nights get shorter, Of course due to the extra production there is more to spend, though there doesn't appear to be any real increase in OD750 of the chemostat at this point. But since the chemostats weren't stable this isn't the most accurate measurement of growth rates.

Unstable cultures:Either due to lack of oxygen during the night when the cultures were run on nitrogen, the harmful aluminum connectors, CO2 limitation (due to running out of gas) or a to short day period, it was troublesome keeping the chemostats healthy, especially the ΔpixD mutant had trouble maintaining its concentration.

It takes longer then a week for the cells to become a stable culture in the chemostat. This can be seen both in the OD750 and the increasing glycogen stocks. It is unknown what the effect of the current glycogen stocks on the glycogen production/usage is. The long adapting periods might be explained due to the circadian clock that has a longer adaption period and has almost no change after 3 days2. If the experiment is done again, it should include longer adaption periods. This should also better reveal what the max storage is per day/night period.

Measurement inaccuracies:The glycogen assay was kind of unstable and didn't always produce repeatable results, this is partly due to that the ABTS is not stable and goes back into it's non colored reduced form10 (see figure 3). Though this rate is to slow for it to change the conclusion drawn from the results, as can also be seen when this effect was studied (see supplementary data) and in the many time points taken by the 96 well plate reader. It is said that the peroxides may damage the enzymes11, which would be limiting the reaction, though I doubt this is significant.

The biggest error is probably introduced during the washing with ethanol,because this is the main spot were the glycogen could be discarded.The results would probably have become a lot more accurate if there was a safer way to remove the KOH from the samples (see materials and methods).

The difference in calibration curves (see supplementary data) are somewhat conflicting, as the time curves show that the reactions are glucose limited and not enzyme or ABTS limited (as it can still reach higher OD values) and yet the glucose all came from the same stock solution, and don't appear to have any obvious dilution errors. These calibration curves have a large impact in the

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current results. The ingredients for the assay were very old, though this shouldn't have any significant impact.

Since the molar weight of glucose is ~180.16 this means that at its peak at ~2.7 mmol (see figure 9), the total glycogen would be 48% of the total weight of the cell. Which is very unlikely, in comparison, the human liver has around 10%1, and this is a specialized and much larger cell.

In any case a better alternative should be found to determine the exact amounts of glycogen.

Differences WT3 and WT4:WT3 is always higher in glycogen storage then WT4, it should be investigated if this is due to the LED vs fluorescent light, or because they have genetic differences. To determine this, both should be grown under the same conditions, and if there are still significant differences a micro-array might be useful.

Lower glycogen levels for ΔpixD:The consistent lower concentrations of glycogen in the ΔpixD mutant confirms that PixD --| glycogen phosphorylase12 .

The running theory:During the night glycogen phosphorylase is actively breaking down glycogen into glucose 1-phosphate (which is then converted to glucose 6-phosphate by phosphoglucomutase, and from there it enters the well known glucose pathways)

Figure 13: Glycogen phosphorylase breaks glycogen down to Glucose 1-phosphate.

In the absence of blue light (during the night) 5 PixE (aka slr1693) and 10 PixD form a complex13. During the day blue light results in the breakage of this complex and releases free PixD dimer. Free PixD dimers then bind and deactivate glycogen phosphorylase, resulting in reduced glycogen breakdown.

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figure 14: Proposed model of glycogen phosphorylase regulation.

Note that there are more ways glycogen is regulated.

Testing this Theory:For the proposed theory to work most PixD must be in this superstructure during the night. It should be researched if the levels of PixD, PixE & Glycogen phosphorylase are in the right ratios, or that there is a surplus of some in the following relationship PixE>PixD>Glycogen phosphorylase. It should also be determined in what form they naturally occur in the cell: mono, dimer or superstructure.

Repeating the experiment with a ΔpixE mutant should ensure PixD is always in dimer, and so there should always be a lot of glycogen as the glycogen phosphorylase should always be inactive. And a double pixD/pixE knockout, should have similar results as the ΔpixD mutant.

Growing without blue light should have similar results as the ΔpixD mutant, as the superstructure should remain intact during the day, and thus glycogen phosphorylase will remain active.

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It would also be reassuring if the mechanism would work in vitro.An enzymatic assay using purified glycogen phosphorylase and glycogen,with or without added purified PixD should indicate the effect of PixD on the glycogen phosphorylase activity. Later PixE could also be added to the mix.

To confirm the mechanism used in earlier research14 a new pixD mutant, that is modified so that it doesn't respond and remains in its super structure even under blue light should be made. For instance by replacing Trp91 with something else like leucine or alanine or something, that wont interfere too much with the folding, but will hinder either the light absorption, or its push effect.

Usage of glycogen:Gradual glycogen consumption, might indicate that it is for instance limited by the concentrations of glycogen phosphorylase. Though the fact that higher usage of glycogen during shorter nights, contradicts this (unless more phosphorylase is produced). Still, it might be interesting to determine the total activity of glycogen phosphorylase per cell.

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References1. Biochemistry, 6e. at <http://bcs.whfreeman.com/biochem6/default.asp?

s=&n=&i=&v=&o=&ns=0&uid=0&rau=0>2. Kucho, K. et al. Global Analysis of Circadian Expression in the

Cyanobacterium Synechocystis sp. Strain PCC 6803. J Bacteriol 187, 2190-2199 (2005).

3. Suzuki, E. et al. Role of the GlgX protein in glycogen metabolism of the cyanobacterium, Synechococcus elongatus PCC 7942. Biochimica et Biophysica Acta (BBA) - General Subjects 1770, 763-773 (2007).

4. Osanai, T. et al. Positive Regulation of Sugar Catabolic Pathways in the Cyanobacterium Synechocystis sp. PCC 6803 by the Group 2 σ Factor SigE. Journal of Biological Chemistry 280, 30653 -30659 (2005).

5. Sato, S. et al. A Large-scale Protein–protein Interaction Analysis in Synechocystis sp. PCC6803. DNA Res 14, 207-216 (2007).

6. Okajima, K. et al. Biochemical and Functional Characterization of BLUF-Type Flavin-Binding Proteins of Two Species of Cyanobacteria. J Biochem 137, 741-750 (2005).

7. REAGENT COMPOSITION AND PROCESS FOR THE DETERMINATION OF GLUCOSE - Patent 3721607. at <http://www.freepatentsonline.com/3721607.html>

8. Ernst, A., Kirschenlohr, H., Diez, J. & Böger, P. Glycogen content and nitrogenase activity in Anabaena variabilis. Archives of Microbiology 140, 120-125 (1984).

9. Parrou, J.L. & François, J. A Simplified Procedure for a Rapid and Reliable Assay of both Glycogen and Trehalose in Whole Yeast Cells. Analytical Biochemistry 248, 186-188 (1997).

10. Kadnikova, E.N. & Kostic, N.M. Oxidation of ABTS by hydrogen peroxide catalyzed by horseradish peroxidase encapsulated into sol-gel glass.: Effects of glass matrix on reactivity. Journal of Molecular Catalysis B: Enzymatic 18, 39-48 (2002).

11. Bateman, R.C. & Evans, J.A. Using the Glucose Oxidase/Peroxidase System in Enzyme Kinetics. Journal of Chemical Education 72, A240 (1995).

12. Hoff, W.D., Horst, M.A., Nudel, C.B. & Hellingwerf, K.J. Prokaryotic Phototaxis. Chemotaxis 25-49 (2009).at <http://dx.doi.org/10.1007/978-1-60761-198-1_2>

13. Yuan, H. & Bauer, C.E. PixE promotes dark oligomerization of the BLUF photoreceptor PixD. Proceedings of the National Academy of Sciences 105, 11715-11719 (2008).

14. Yuan, H. et al. Crystal Structures of the Synechocystis Photoreceptor Slr1694 Reveal Distinct Structural States Related to Signaling†,‡. Biochemistry 45, 12687-12694 (2006).

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Not available online:I. Thesis: Influence of photorepiodicity on algal growth kinetics - J. G.

Loogman- 1982

EXTRA STUFF

A handy linking DB is found here: http://string.embl.de/

http://string-db.org/newstring_cgi/show_network_section.pl?identifier=8897%208219%208220%208221&additional_network_nodes=10&chemicalmode=-1&input_query_species=1148&interactive=yes&internal_call=1&limit=10&minprotchem=0&network_depth=1&network_flavor=confidence&previous_network_size=34&required_score=400&sessionId=BlG3JkLEwYZi&targetmode=proteins&userId=MAg0VGbSgZ0W

Apparently its not 100% up-to-date, as the link between PixD en glycogen phosphorylase is missing. Still tools like this can be very usefull in finding related proteins. Though metabolites always are a spoil sport in these linkages.Kegg (http://www.genome.jp/kegg/pathway.html), might be useful for those.

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Information about the 3D linking:

The models I used to make the 3D images are:

Viewer: Swiss-PdbViewer DeepView 4.1 http://spdbv.vital-it.ch/ or http://www.expasy.org/spdbv/(This program also added the missing parts of amino acids from the X-ray crystallography models)

Binding calculated by Hex 6.1 (http://hex.loria.fr/):Default settings... (Shape only mode (also has shape + electrostatics mode, which gave the same results (as in same position) but less negative values)Algoritm: Global: Fourier correlation of spherical harmonics (basically turn both ligand and receptor, and make em bump into each other (both are considered rigid structures (i think)) (3 billion attempts))

The origin of the pdb files:PixD (Slr1694) - pdb file is determined by X-ray crystallography: http://www.pdb.org/pdb/explore/explore.do?structureId=2HFN & http://www.pdb.org/pdb/explore/explore.do?structureId=2HFO

Glycogen phosphorylase - pdb file is computer generated (based on a protein with 50% homology) http://swissmodel.expasy.org/repository/?pid=smr03&uid=&token=&zid=async&mid=e14cb2d653caf4fddd5d7e48bd93226a_1&query_1_input=P73511Note that this model is crap, since it lacks the active modification, that is present in other glycogen phosphorylases.

An other tool I used was a website:http://vakser.bioinformatics.ku.edu/resources/gramm/grammx/though this one gave no values back on how favorable each of the bindings was, so I could not compare. Also the top few looked different than the ones made by Hex.

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