CSEN

Biology and physiology of the cell cycle regulation

Due to more than 40 years of continuous research of the cell cycle of algae the know how obtained is represented by unique and very extensive range of outputs in both basic and applied research. (For more details see CV , OUTPUTS, Zachleder V. and pages of Milada Vítová and Kateřina Bišová).

The topics studied recently are: (i) the role of environmental factors like light intensity and temperature or different compounds, (selen, rare earth elements) on the cell cycle progression; (ii) study of the molecular mechanisms responsible for regulation of algal cell cycle, progression into mitosis and response to DNA damage; (iii) development of processes allowing usage of waste CO2 and waste waters for algal biomass production, with controlled synthesis of large amounts of starch and lipids for a various applications including bioethanol and biofuels production.

Outputs of basic research in 2006-2011

 

 

 

 

Diagrams showing the types of the cell cycle phases and their ordering in a classical cell cycle model and those found in the algae Scenedesmus and Chlamydomonas dividing into two (A) and four (B) daughter cells.

Classical cell cycle model after Howard and Pelc (1953), Scenedesmus cell cycle model after Šetlík and Zachleder (1984) (Zachleder V.), Chlamydomonas cell cycle model after Zachleder and van den Ende (1992). Individual stripes indicate the sequence of cell cycle phases during which growth and reproductive processes leading to duplication of reproductive structures take place. Whereas only one sequence of events leading to duplication of cell structures occurs during the cell cycle of cells dividing into two daughter cells (panel A), two, partially overlapped sequences of growth and reproductive events occur within one cycle in cell dividing into four daughter cells (panel B). Two stripes (panel B) illustrates simultaneous course of different phases from two consecutively started sequences of growth and reproductive events. Schematic pictures of cells indicate their size changes during the cell cycle and the black spots inside illustrate the size and number of nuclei. Large black spots indicate doubling of DNA. G1 - the phase during which the threshold size of the cell is attained. It can be called a precommitment period because it is terminated by attainment of the commitment point. C.P. - the state of the cell cycle at which the cell becomes committed to triggering and terminating the sequence of processes leading to the duplication of reproductive structures. pS - the prereplication phase between the commitment point and the start of the round of DNA replication. The processes required for the initiation of DNA replication are assumed to happen during this phase. S ‑ the phase during which DNA replication takes place. G2 ‑ the phase between the termination of DNA replication and the start of mitosis. Processes leading to the initiation of mitosis are assumed to take place. M - the phase during which nuclear division occurs. G3 ‑ the phase between nuclear division and cell division. The processes leading to cellular division are assumed to take place during this phase. C ‑ the phase during which the cleavage of cells occurs.

Publications in peer reviewed journals

1. Cepák, V., Přibyl, P., and Vítová, M.: The effect of light color on the nucleocytoplasmic and chloroplast cycle of the green chlorococcal alga Scenedesmus obliquus. Folia Microbiol 51: 342-348, 2006.
2. Oldenhof, H., Zachleder V., van den Ende, H.: Blue- and red-light regulation of the cell cycle in Chlamydomonas reinhardtii (Chlorophyta). Eur J Phycol 41: 313–320, 2007.
3. Oldenhof, H., Zachleder V., van den Ende, H.: Phase resetting of the mammalian circadian clock by DNA damage. Folia Microbiol 51: 53-60, 2007.
4. Oldenhof, H., Zachleder V., van den Ende, H.: The cell cycle of Chlamydomonas reinhardtii: the role of the commitment point. Folia Microbiol 52: 53-60, 2007.
5. Cepák, V., Přibyl, P., Vítová, M., Zachleder V.: The nucleocytosolic and chloroplast cycle in the green alga Scenedesmus obliquus (Chlorophyceae, Chlorococcales) grown under various temperatures. Phycologia 46: 263-269, 2007.
6. Čížková, M., Pichová, A., Vítová, M., Hlavová, M., Hendrychová, J., Umysová, D., Gálová, E., Ševčovičová, A., Zachleder V., Umen, J.G., Bišová, K.: CDKA and CDKB kinases from Chlamydomonas reinhardtii are able to complement cdc28 temperature-sensitive mutants of Saccharomyces cerevisiae.Protoplasma 232: 183-191 2008.
7. Vítová, M., Hendrychová, J., Čížková, M., Cepák, V., Umen, J.G., Zachleder V., Bišová, K.: Accumulation, activity and localization of cell cycle regulatory proteins and the chloroplast division protein FtsZ in the alga Scenedesmus quadricauda under inhibition of nuclear DNA replication. Plant Cell Physiol 49: 1805-1817, 2008.
8.  Pribyl, P., Cepák, V.,Zachleder V.: Cytoskeletal alterations in interphase cells of the green alga Spirogyra decimina in response to heavy metals exposure: II. The effect of aluminium, nickel and copper. Toxicol in Vitro 22: 1160-1168, 2008.
9.  Umysová, D., Vítová, M., Doušková, I., Bišová, K., Hlavová, M., Čížková, M., Doucha, J., Machát, J., Zachleder V.: Bioaccumulation and toxicity of selenium compounds in the green alga Scenedesmus quadricauda. BMC Plant Biol 9: 58-74, 2009.
10.  Vítová, M., Bišová, K., Hlavová, M., Zachleder V., Rucki, M., Čížková, M.: Glutathione peroxidase activity in the selenium-treated alga Scenedesmus quadricauda. Aquatic Toxicol. 102, 87–94, 2011.
11.  Vítová, M., Bišová, K., Umysová, D., Hlavová, M., Kawano, S., Zachleder V., Čížková, M.: Chlamydomonas reinhardtii: Duration of its cell cycle and phases at growth rates affected by light intensity. Planta 233: 75–86, 2011.
12.  Vítová, M., Bišová, K., Umysová, D., Hlavová, M., Kawano, S., Zachleder V., Čížková, M.: Chlamydomonas reinhardtii: Duration of its cell cycle and phases at growth rates affected by temperature. Planta, DOI: 10.1007/s00425-011-1427-7, 2011.
13.  Hlavová, M., Čížková, M., Vítová, M., Bišová, K., Zachleder V.: DNA damage during G2 phase does not affect cell cycle progression of the green alga Scenedesmus quadricauda. PLoS ONE 6(5) e19623, 2011.

A B

A: Duration of the cell cycle and its phases in synchronized populations of the alga Chlamydomonas reinhardtii grown at different temperatures and constant mean light intensity 150 μmol m-2 s-1. Curves 1: duration of the cell cycle (from the beginning of illumination to release of daughter cells); curves 2: duration of pre-commitment phase (from the beginning of illumination to attaining the first commitment to cell division); curves 3: duration of post‑commitment phase (from the first commitment point to release of daughter cells); curves 4: growth rate expressed in doubling per hour. Vítová et al. Planta, DOI: 10.1007/s00425-011-1427-7, 2011.

B: Duration of the cell cycle and its phases in synchronized populations of the alga Chlamydomonas reinhardtii grown at different light intensities, temperature 30 °C. Curve 1: duration of the cell cycle (from the beginning of illumination to the time of daughter cells release): illumination regime: solid circles – alternation of light and dark periods, open circles - continuous illumination; curve 2: duration of pre-commitment phase (from the beginning of illumination to attaining the first commitment to cell division); curve 3: duration of post‑commitment phase (from the first commitment point to the time of daughter cells release); curve 4: growth rate expressed in doubling per hour.
Vítová et al. Planta 233: 75–86, 2011.

Grants

1. MŠMT ME 104 (2006-2007): Study of the answer reparation-defect strains of the alga Chlamydomonas reinhardtii to DNA damage in relation to regulation of cell cycle. Principal investigator Zachleder, V.
2. GAČR 204/06/0102 (2006-2008): Regulation of mitosis by plant specific cyclin-dependent kinasis type B in green alga Chlamydomonas reinhardtii.Principal investigator Bišová, K.
3. GAAV A500200614 (2006-2010): Regulation of mitosis by kinase WEE1 using the green alga Chlamydomonas reinhardtii as a model organisms.Principal investigator Bišová, K.
4. GAAV A600200701 (2007-2009): Výzkum akumulace selenu a jeho forem v průběhu buněčného cyklu zelených řas. Principal investigator Vítová, M.
5. GAČR 204/09/0111 (2009-2011): Příprava teplotně sensitivních mutantů buněčného cykluu zelené řasy Chlamydomonas reinhardtii. Principal investigator Bišová, K.
6. GAČR 525/09/0102 (2009-2011): Bioaccumulation of rare earth lements by green algae, effect on physiology anf cell cycle. Principal investigator Vítová, M.
7. GAAV M200200904 (2009-2012): Role of endogeneous timers in regulationships between growth and cell division of algae. Principal investigator Zachleder, V. GRANTS
8. GAČR P501/10/P258 (2010-2012): Role of phosphatases in regulation of CDK in G2/M phase of cell cycle Chlamydomonas reinhardtii. Principal investigator Čížková, M.

Outputs of applied research in 2006-2011

 

Changes in biomass and starch content (A) and starch yield per biomass (B) in cultures of Chlorella. The cultures were grown in an outdoor scale up thin layer photobioreactor in complete mineral medium for 120 h and then in sulphur limiting medium (–S). Dark periods (nights) are marked by black stripes and separated by vertical solid lines

 

Changes in biomass and starch content (A) and starch yield per biomass (B) in cultures of Chlorella. The cultures were grown in an outdoor scale up thin layer photobioreactor in complete mineral medium for 120 h and then in sulphur limiting medium (–S). Dark periods (nights) are marked by black stripes and separated by vertical solid lines.

Brányiková et al. Biotechnol. Bioeng. 108, 766-776, 2011.
Fluorescence microphotographs of Parachlorella kessleri to treated to produce increased amount of lipid reserves
Red color: autofluorescence of chlorophyll Yellow color: vesicules of lipid reserves

 

Electron microscopic photomicrographs of daughter (A) and mother cells (B) of the alga Chlorellagrown in complete nutrient medium (A,B) and the cells grown in the presence of cycloheximide (1 mg/L) (C) and in nutrient medium, where sulfates were omitted (D).

Nu nucleus, St starch granules, Bars = 20 μm

 

Brányiková et al. Biotechnol. Bioeng. 108, 766-776, 2011.

Publications in peer reviewed journals

1) Lívanský, K., Doucha, J., Hu, H., Li, Y.: CO2 partial pressure - pH relationships in the medium and relevance to CO2 mass balance in outdoor open thin-layer Arthrospira (Spirulina) cultures. Arch Hydrobiol 165, 365-381, 2006.

2) Doucha, J., Lívanský, K.: Productivity, CO2/O2 exchange and hydraulics in outdoor open high density microalgal (Chlorella sp.) photobioreactors operated in a Middle and Southern European climate. J Appl Phycol 18, 811-826, 2006.

3) Skřivan, M., Šimáně, J., Dlouhá, G., Doucha, J.: Effect of dietary sodium selenite, Se-enriched yeast and Se-enriched Chlorella on egg Se concentration, physical parameters of eggs and laying hen production. Czech J Anim Sci 51, 163-167, 2006.

4) Doucha, J., Livansky, K.: Influence of processing parameters on disintegration of Chlorella cells in various types of homogenizers. Appl Microbiol Biotechnol 81, 431-440, 2008.

5) Trávníček, J., Racek, J., Trefil, L., Rodinová, H., Kroupová, V., Illek, J., Doucha, J., Písek, L.: Activity of glutathione peroxidase (GSH-Px) in the blood of ewes and their lambs receiving the selenium-enriched unicellular alga Chlorela. Czech J Anim Sci 53, 292–298, 2008.

6) Doucha, J., Lívanský , K.: Outdoor open thin-layer microalgal photobioreactor: potential productivity. J Appl Phycol 21, 111-117, 2009.

7) Doucha, J., Lívanský, K., Kotrbáček, V., Zachleder V.: Production of Chlorella biomass enriched by selenium and its use in animal nutrition: a review.Appl Microbiol Biotechnol 83, 1001–1008, 2009.

8) Douskova, I., Doucha, J., Livansky, K., Machat, J., Novak, P., Umysova, D., Zachleder V., Vitova, M.: Simultaneous flue gas bioremediation and reduction of microalgal biomass production costs. Appl Microbiol Biotechnol 82, 179–185, 2009.

9) Doušková, I., Kaštánek, F., Maléterová, Y., Kaštánek, P., Doucha, J.,Zachleder V.: Utilization of distillery stillage for energy generation and concurrent production of valuable microalgal biomass in the sequence: Biogas-cogeneration-microalgae-products. Energy Conver Mngm 51, 606–611,2010.

10) Kaštánek, F., Šabata, S., Šolcová, O., Maléterová, Y., Kaštánek, P., Brányiková, I., Kuthan, K., Zachleder V.: In-field experimental verificatiom of cultuvation of microalgae Chlorella sp. using the flue gas from a cogeneration unit as a source of carbon dioxide. Waste Mngm. Res 28, 961-966, 2010.

11) Skřivan, M., Skřivanová, V., Dlouhá, G., Brányiková, I., Zachleder V., Vítová, M.: The use of selenium-enriched alga Scenedesmus quadricauda in a chicken diet. Czech J Anim Sci 55, 565–571, 2010.

12) Doucha, J., Lívanský, K.: Production of high-density Chlorella culture grown in fermenters. J Appl Phycol DOI 10.1007s10811-010-9643-2, 2011.

13) Brányiková, I., Maršálková, B., Doucha, J., Brányik, T., Bišová, K., Zachleder V., Vítová, V.: Microalgae - novel highly-efficient starch producers.Biotechnol. Bioeng. 108, 766-776, 2011.

14) Bumbak, F., Cook, S., Zachleder V., Hauser, S., and Kovar, K.: Best practices in heterotrophic high cell density microalgal processes: achievements, potential and possible limitations. Appl Microbiol Biotechnol 91, 31-36, 2011

Fulltext papers from oral presentations

1) Douskova, I., Machat, J., Umysova, D., Vitova, M., Doucha, J. , Zachleder V.: Scenedesmus quadricauda - a promising microorganism for selenium-enriched algal biomass production. In 5th Symposium for European Freshwater Sciences (sefs-5) (Palermo, Italy), pp. 197-201, 2007.

2) Umysova, D., Douskova, I., Vitova, M., Machat, J., Doucha, J., Zachleder V.: Toxicity and accumulation of selenium in Scenedesmus quadricauda. In5th Symposium for European Freshwater Sciences (sefs-5) (Palermo, Italy), pp. 261-263, 2007.

3) Douskova I., Doucha J., Umysova D., Vitova M., Zachleder V.: (2008): Microalgae – A promising source of starch for bioethanol production,Polysaccharides IV, Praha, pp. 14-17, 2008.

4) Doušková, H., Doucha, J., Machat, J., Novák, P., Umysová, D., Vítová, M., Zachleder V.: Microalgae as a means for converting flue gas CO2 into biomass with a high content of starch.– In: „Bioenergy: Challenges and Opportunities“, International Conference and Exhibition on Bioenergy, , Guimaraes, Portugal, pp. 191-196, 2008.

5) Doušková, I., Doucha, J., Novák, P., Umysová, D., Vítová, M., Zachleder V.: CO2 removal from municipal waste incinerator flue gas and biomass production by microalgae. - In: „Biotechnology 2008“, Part 4 – Environmental Biotechnology, České Budějovice, pp. 35-37, 2008.

6) Novák, P., Bernát, P., Doucha, J., Zachleder V., Doušková, I., Straka, F.: Biofuels from incinerator of municipal waste. In Aprochem 2008, Milovy , Waste Forum, pp. 3225-3232. In Czech, 2008. (in Czech)

7) Umysová, D., Hlavová, M., Vítová, M., Doušková, I., Bišová, K., Zachleder V.: Selection of selenium resistant strains of the green alga Scenedesmus quadricauda. - In: „Biotechnology 2008“, Part 2 – Plant Biotechnology, České Budějovice, pp. 273-275, 2008.

8) Vítová, M., Umysová, D., Doušková, I., Rucki, M., Zachleder V.: Aktivity of thioredoxin reductase in wild and selenium-resistant strains of the algaScenedesmus quadricauda grown in the presence of inorganic selenium compounds. - In: „Biotechnology 2008“, Part 4 – Environmental Biotechnology, České Budějovice, pp. 205-207, 2008.

9) Novák, P., Bernát, P., Doucha, J., Zachleder V., Brányiková, I., Straka, F., Brányik, T., Maršálková, B.: Biofuels of the 2nd. generation from gas waste of incinerator. All for Power. 5, 79-84, 2011. (in Czech)

Patents

1) Doušková I., Hlavová M., Umysová D., Vítová M.; Zachleder V.: Industrial strain Scenedesmus quadricauda SeIV of green chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. (in Czech). Cz. Patent 300861-2009.

2) Doušková I.; Hlavová M.; Umysová D.; Vítová M.; Zachleder V.: Industrial strain Scenedesmus quadricauda SeVI of green chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. (in Czech). Cz. Patent-300809-2009.

3) Doušková I., Hlavová M., Umysová D., Vítová M.; Zachleder V.: Industrial strain Scenedesmus quadricauda SeIV+VI of green chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. (in Czech). Cz. Patent 300808-2009.

4) Doucha J., Lívanský K., Zachleder V.: Production strain P13/1998 of unicellular alga Chlorella vulgaris BEIJ. (in Czech). PV 2009-214 (Z6452), 2009.

5) Brányik, T., Maršálková, B., Melzoch, K., Brányiková, I., Novák, P., Doucha, J., Zachleder V.:  Zařízení na pěstování a komplexní zpracování řasové biomasy bohaté na polysacharidy na výrobu bioetanolu a výživového/krmivového doplňku PV 2011-CZ 21678 U1, 2011.

Grants

1) EUREKA MŠMT OE 221 Biofix (2006-2009): Utilization of carbon dioxide in flue gases in production cultures of algae. Co-investigator Zachleder, V.

2) EUREKA MŠMT OE 09025 Alganol (2009-2012): Production biofuels from algae with high content of strach and lipids using carbon dioxide from flue gas a source of carbon. Co-investigator Zachleder, V. GRANTS

3) FP 7 AquaFuels (2010-2011): Algae and aquatic biomass for a sustainable production of 2nd generation biofuels. Members of consortium EBB and society EABA Zachleder, V.; Doucha J., Bišová, K. GRANTS, Barter agreement

4) GAČR  P503/10/1270 (2010-2012): Adhesion of microalgae on solid surfaces. Co-investigator Zachleder, V. GRANTS

5) CREST Japan Science and Technology Agency (2011-2013): Creation of Basic Technology for Improved Bioenergy Production through Functional Analysis and Regulation of Algae and Other Aquatic Microorganisms. Foreign adviser Zachleder, V. GRANTS

6) INTERREG BioRee: Austria-Czech research of utilisation of rare earth elements (REEs) in biomedicine and thein recyclation by microorganisms Principal investigator Vítová, M. GRANTS

Methods

1. Synchronization of algal cultures

Zachleder V., Šetlík I.(1990): Timing of events in overlapping cell reproductive sequences and their mutual interactions in the alga Scenedesmus quadricauda. Journal of Cell Science 97: 631-638.

2. Characterization of cell cycle progress:

2a Analysis of RNA and DNA content and timing of DNA replication

Zachleder V. (1986): Optimization of nucleic acids assay in green and blue-green algae: Extraction procedures and the light-activated reaction for DNA. Arch. Hydrobiol. (Suppl. 67) Algolog. Stud. 36: 313-328.

2b Assessment of commitment curves

Zachleder V., Schläfli O., Boschetti A. (1997): Growth-controlled oscillation in activity of histone H1 kinase during the cell cycle of Chlamydomonas reinhardtii(Chlorophyta). Journal of Phycology 33: 673-681.

Vítová M., Zachleder V. (2005): Points of commitment to reproductive events as a tool for analysis of the cell cycle in synchronous cultures of algae. Folia Microbiologica: in press.

2c Determination of number of nuclei by means of DNA staining by SYBR Green I or DAPI, nuclear division curves

Zachleder V. (1986): Nuclei stained by SYBR Green I (left) and by DAPI (right) in Scenedesmus quadricauda

Vítová M., Hendrychová J., Cepák V., Zachleder V. (2005): Visualization of DNA containing structures in Chlamydomonas reinhardtii and other species of Chlorophyta, Rhodophyta and Cyanophyta using SYBR Green I dye. Folia Microbiologica: in press.

Zachleder V., Cepák V.(1987): Visualization of DNA containing structures by fluorochrome DAPI in those algal cells which are not freely permeable to the dye. Arch. Hydrobiol. (Suppl. 78) Algolog. Stud. 47: 157-168.

3. Cyclin-dependent kinase activity assay in vitro

Bišová K., Vítová M., Zachleder V. (2000): The activity of total histone H1 kinases is related to growth and commitment points while the p13(suc1)-bound kinase activity relates to mitoses in the alga Scenedesmus quadricauda. Plant physiology and biochemistry 38: 755-764.

4. Single CDK complex activity assay in-gel

5. Nuclear transformation of Chlamydomonas using glass beads

Pacanovská M.(2003): Construction of plasmid with E.coli recA+ gene to increase frequention of homologous recombination in Chlamydomonas reinhardtii. MSc. thesis. Comenius University, the Faculty of Natural Sciences, Bratislava, Slovak Republic.

Slaninová M., Fritsche L., Pacanovská M., Fuseková M., Treuner G., Vlček D., Mages W.(2004): New approaches to improve homologous recombination in Chlamydomonas reinhardtii. In book of European Environmental Mutagen Society (Maastricht, the Netherlands).

6. Fluorescence microscopy (Olympus fluorescent microscope BX51 with attached CCD camera, Nomarski (DIC) and phase contrast)

Vítová M., Hendrychová J., Cepák V., Zachleder V. (2005): Visualization of DNA containing structures in Chlamydomonas reinhardtii and other species of Chlorophyta, Rhodophyta and Cyanophyta using SYBR Green I dye. Folia Microbiologica: in press.

Hendrychová J., Vítová M., Bišová K., Wiche G., Zachleder V. (2002): Plectin-like proteins are present in cells of Chlamydomonas eugametos(Volvocales). Folia Microbiologica 47: 535-539.

 

 

 

 

 

 

 

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