Project Report

David I. WestenbergMicrobial Diversity Course, 1994

Circadian Rhythms in Cyanobacteria

Abstract

The goal of this research project has been to study circadian rhythms in two cyanobacterialspecies Synechococcus .sp. strain PCC7942 and Anabaena strain PCC712O. In nature, thecyanobacteria, which are dependent on light for growth, might be expected to adapt to thenormal light/dark cycles by altering their cellular composition in a cyclical manner. Such apattern has been observed for the single cellular cyanobacteria Gleotheca andSynechococcus. In Synechococcus this phenomenon has been shown to be functioning atthe level of gene expression. The first part of this project has been directed at determiningthe extent to which this process may occur. The approach was to synchronize continuousand batch cultures of Synechococcus strain PCC7942 and search for cyclical patterns ofprotein expression or turnover. This was determined by removing samples over a 36 hourperiod, separating whole cell extracts on an SDS-PAGE gel and examining for appearanceor disappearance of protein bands over time. The second part of the project was todetermine if heterocyst differention in the filamentous cyanobacterium, Anabaena strainPCC7I2O, is influenced by circadian rhythms. The approach for the second part of theproject required adapting cells to a light dark cycle and removing samples at dawn and atdusk. The cells were then transfered to nitrogen free medium and incubated in continuouslight. The timing and pattern of heterocyst differentiation was then observed.

Introduction.

The cyanobacteria are a oup of oxygenic phototrophic bacteria which are capableof fixing molecular nitrogen”. As phototrophic organisms, the cyanobacteria aredependent on light energy for growth. Due to this light dependency, their growth is subjectto the natural cycle of light and dark periods. One might expect, based on their lifestyle,that the cyanobacteria might have evolved a mechanism for regulating gene expression suchthat light period and dark period specific enzymes would be expressed at the appropriatetime in the light/dark cycle. Such a mechanism may be similar to the well studied biologicalclocks responsible for the circadian rhythms which have been observed in eukaryoticorganism4. Therefore, the cyanobacteria may provide an excellent model system forstudying the nature of biological clocks.

For the unicellular cyanobacteria, separation of light and dark period specificenzymes takes on a whole new meaning. For these organisms it is necessary to separatethe process of oxygenic photosynthesis and the oxygen sensitive process of dinitrogenfixation. It has been known for several years that the unicellular cyanobacteria Gleothecaand Synechococcus restrict the process of dinitrogen fixation to the dark period5’6’9.Later work demonstrated that if light/dark phased cells are subjected to constant light

conditions, nitrogen fixation activity occurs only during the times at which the cells wouldhave experienced darkness. This cycling of nitrogenase activity was shown to persist forup to 4 days. These experiments gave the first hint that cyanobacteria may containbiological clocks.

By definition, a tn.ie biological clock must meet 3 criteria. First, a biological clockmust be entrainable. In other words, it should be possible to set the clock at differentperiods of the day and maintain the same periodicity of the cycle. Secondly, the periodicitymust be persistent under constant conditions: Finally, the periodicity must be temperaturecompensable. Recent work by Kondo et al demonstrated that the unicellularcyanobacterium Synechococcus does indeed have a biological clock7’8. Using a luciferase(luxA) gene fusion to the psbAl gene, which codes for a subunit of photosystem II, theywere able to demonstrate a periodicity of gene expression which can be entrained, whichpersists for up to 6 days and which is temperature compensated.

In Part I of this research project the goal is to determine how many proteins areexpressed in a cyclical manner and potentially under the control of the biological clock.This research project was set up to begin to answer this question by visualizing total cellproteins from cells harvested at different times during a 36 hour constant light period afterbeing entrained to a 12 hour light/12 hour dark cycle.

Part II of this research project is directed toward determining if heterocystformation in the filanientous cyanobacterium Anabaena strain PCC7 120 is influenced bycircadian rhythms. The approach for Part II is to transfer cells grown on nitrogen richmedium which have been phased to a light/dark cycle, transfer them to nitrogen freemedium and examine the timing, frequency and pattern of heterocyst formation.

The results of both experiments are incomplete and further experimentation will berequired to answer these questions.

Part I - Circadian Rhythms in Synechococcus strain R-2 (PCC7942).

Materials and Methods.

Synechococcus strain R-2 was obtained form Dr. John Waterbwy of the WoodsHole Oceanographic Institute. Growth was in a minimal medium described by Bustos andGolden3. The composition was as follows: NaNO3 (1.5 gIl); K2NPO4 (0.039 gIl);MgSO4’7H20(0.075 gIl); Na2CO3(0.02 gIl); CaC122H20(0.027 gIl); EDTA (0.001 gIl);FeNH4Citrate (0.012 gIl); SLIO (micronutrients) (1 ml).

Continuous culture was attempted using a 250 ml chemostat as diagramed in Figure1. The vessel was filled with 250 ml of the medium described above and innoculated with10 ml of a mid-log phase culture of PCC7942. The medium maintained at 30°C and wasstirred continuously and aerated with an aquarium pump. Fresh medium was continuouslysupplied at a rate of approximately 17 mI/hour for a diulution rate of 8 hours. Effluent wascollected in a sterile 2 1 flask. Illumination was from a 75W incadescent light bulb. For thebatch culture, 50 ml of the above described medium was innoculated with 5 ml of astationary phase culture and incubated at room temperature at a distance of 20 cm from a75 W incadescent light bulb.

Results and Discussion.The object of this experiment was to obtain a steady-state culture of Synechococcus

strain R-2. The culture could then be phased to a 12 hour dark/12 hour light cycle. Thephased culture would then be subjected to continuous light and samples collected every 2hours. Cells from each time point would then be pelleted, resuspended in sample buffer,boiled and the total cellular protein separated on a 5-25% gradient SDS-PAGE gel.

Unfortunately, a steady-state culture could not be obtained and the experimentcould not be completed as designed. Initially, due to pump faillure, the flow rate could notbe maintained. Once a continuous flow rate was obtained, the culture became washed out,apparently due to the inability of the organism to maintain a doubling time of 8 hours.Attempts to reduce the flow rate resulted in discontinuous flow. Possible causes for thelow growth rate of strain PCC7942 include insufficient aeration and insufficientillumination. If this experiment was to be repeated, it could be improved by using asparging stone for aeration and by using a stronger light source for illumination. Using abank of fluorescent lights placed near the chemostat would be preferred. It would also beadvisable to use a peristaltic pump which can produce a consistent, slow flow rate.

Due to the failure of the cheniostat, a similar experiment was attempted with abatch culture. A 50 ml late log culture was placed in the dark for 12 hours to phase thecells. The culture was then exposed to continuous light. Samples (0.5 ml) were removedevery 2 hours and the cells were pelleted and frozen. After 36 hours, samples wereresuspended in 25 ul H20 and 8 ul of 4X sample buffer. After boiling for 2 mm., 5 ul wereapplied to a 9% SDS-PAGE gel to separate total cellular protein. The stained gel is shownas Figure 2. Insufficient protein was loaded on the gel and the pattern of protein was notreadily apparent. Due to time constraints, a straight percent gel had to be used. If thisexperiment was repeated, it would be better to separate the proteins on a gradient gel asoriginally planned.

Conclusions.Although it is not apparent from the photocopy of Figure 2, there appear to be

fluctuations in some of the bands, indicating that if sufficient protein had been loaded it mayhave been possible to discern rhythmic patterns of protein expression. These experimentswould have provided a crude analysis of the number of proteins which are subjected tocircadian rhythms but would not have been sensitive enough to identi’ proteins which arenot expressed at high levels. However, if successful, they would have provided a startingpoint for a more complete survey. Later experiments might have included 2-D gel analysisof total cellular protein at different time points in the clock cycle.

Part II - Influnce of light/dark cycles on heterocyst differentiation.

Anabaena strain PCC7 120 was obtained from Dr. Robert Haselkorn of theUniversity of Chicago. Growth of this strain was on K&M medium at 23° with gentle butconstant shaking. The cells were phased to a 10 hour dark! 14 hour light cycle.

For each experiment, 5 ml were removed at the appropriate time and filtered on ascintered glass filter by gravity flow. The cells were then washed 2 times with 2 volumes of

nitrogen free K&M medium and resuspended in 5 ml of nitrogen free medium. Washedcells were spotted onto agar‘1squares” and allowed to sit for 5 mm. Excess liquid wasremoved and the agar “square” was covered with a 20mm x 60mm glass coverslip. Thecover slip was sealed on 3 sides with vaspar and the space surrounding the agar “square”

was filled half-way with nitrogen free K&M medium and the last side was sealed.Individual filaments were located and their location recored. The growth and developmentof 4-6 filaments were followed at 3 hour intervals over a period of up to 48 hours.

Agar “squares” were prepared by aufoclaving 1.6% nitrogen-free agar in 1420 anddiluting 1:1 with sterile nitrogen free K&M medium. Diluted agar was used to cover asterile glass microscope slide and excess agar was drawn off so that a layer ofapproximatedly 1mm remained. After solidification, agar was cut away from each sideleaving a rectangle of approximately 1cm x 1.5 cm (Figure 3)

Results and Discussion.Initial experiments were performed to veri& that Anabaena strain PCC712O would

survive and develop heterocysts under the experimental conditions. Using Difco Agarproved to be unsatisfactory with no heterocyst differentiation within 48 hours, likely due tothe presence of a contaminating source of nitrogen (Figure 4a). NF Agar from Oxoid,Washed Difco agar from John Waterbury (“Super Agar”) and agarose were tried asalternatives. The washed agar from John Waterbury gave the best results with healthylooking cells and well spaced heterocysts within 36 hours (Figure 4b) followed by agarose(Figure 4c) but the NP Agar from Oxoid was unsatisfactory. Further experiments wereperformed using washed Difco agar.

The process of heterocyst differentiation has been well studied at the structural aswell as molecular level2”1. To determine if heterocyst development is influenced by lightdark cycles, cells were first phased to a 10 hour darkll4 hour light cycle in nitrogencontaining medium. At dusk (7:30 pm) and at dawn (7:30 am) 5 ml samples weretransfered to nitrogen free medium and spotted onto agar slides as described in Materialand Methods. Growth and heterocyst development were followed every 3 hours for 48hours and at random intervals after that point. The cells prepared from both time pointsappeared to grow at similar rates (Figures 5 and 6) but the dusk cells never developedheterocysts or obvious pro-heterocysts, whereas the dawn samples developed obvious proheterocysts (Figure 6) within 24 hours. Some of the proheterocysts appeared as pairs(Figure 6). The pro-heterocysts developed into heterocysts within 48 hours and one cell ofthe double pro-heterocysts regressed (Figure 6). Blown up photos of the dusk and dawnfilaments at t=3 6 are shown as Figure 7. Unfortunately, the lack of heterocyst developmentin the dusk sample precludes the comparison of heterocyst development relative to the lightdark cycle unless the lack of heterocyst development in the dusk sample is a phenotyperesulting from the light dark cycle.

After removal of the dusk and dawn samples, the culture was inclubated at constantlight for 36 hours. Samples were removed at what would have been dusk and what wouldhave been dawn and treated in identical fashion as the original samples. The results areshown as Figure 8 a and b. The pictures are incomplete due to time limits but the oppositepattern of heterocyst development was observed - heterocysts developed in the dusksample but not in the dawn sample. The pattern and timing of the appearance of pro-

heterocysts and heterocysts was similar to that observed for the dawn samples in the firstpart of the experiment.

The culture was then entrained to a new light dark cycle for 36 hours (2 darkcycles) and the first part of the experiment was repeated. Unfortunately, photographs arenot available for this experiment. This time, both samples developed pro-heterocysts andheterocysts at similar times, with similar spacing except that the dusk sample showed agreater occurance of double heterocysts. Both samples contained double pro-heterocystsat a similar frequency (20-2%) but whereas iii the dawn samples one pro-heterocyst withinthe pair regressed, in the dusk sample regression did not occur.

ConclusionsThe results of these experiments are inconclusive. The inconsistency of heterocyst

differentiation makes it difficult to make any conclusions. However, the fact, thatindependent of the time at which cells were transferred to N-free medium, heterocystdevelopment occured with the same timing and spacing. The lone exception was thedevelopment of double heterocysts in cells transferred at dusk in the final experiment. Amore rigorous examination of heterocyst development relative to light/dark cycles wouldbe required to answer this question.

References.

1. Mains, D.G. and N.G. Can. 1981. Heterocyst Differentiation and Cell Division in theCyanobaeteriumAnaebaena cylindrica: Effect of High Light Intensity. 1. Cell. Sd 49:341-352.2. Buikema, WI and R Haselkorn. 1993. Molecular Genetics of Cyanobacterial DevelopmentAnn. Rev. Plant Pliysiol. Plant Mol. Biol. 44:33-52.3. Bustos, S.A. and S.S. Golden. Expression of the psbDll Gene in Synechococcus sp. StrainPCC7942 Requires Sequences Downstream of the Transcription Start Site. 3. Bacteriol. 173:7525-7533.4. Dunlap, J.C. 1990. Closely Watched Cloth: Molecular Analysis of Cirdinn Rhythms inNeurospora and Drosophila Trends Genet 6:159-165.5. Grobbelar, N., Huang, T.C., Liii, ELY. and T.J. Chow. 1986. Dinitrogen-fixung EndogenousRhythm in Synechococcus RF-1. FEMS Microbiol Left. 37:173-177.6. Leon, C., Kumazawa, S. and A. Mitsui. 1986. Cyclic Appearance of Aerobic NitrogenaseActivity during Synchronous Growth of Unicellular Cyanobacteria. Curr. Microbiol. 13:149-153.7. Kondo, T. and !vL Ishiura. 1994. Circadian Rhythms of Cyanobacteria: Monitoring theBiological Clocks of Individual Colonies by Bioluminescence. 3. Bacteriol. 176:1881-1885.8. Kondo, T., Strayer, CA, Kulkarni, RD., Taylor, W. Lshiura, 1st, Golden, S.S. and C.H. Johnson.1993. Circadian Rhythms in Prokaiyotes: Luciferase as a Reporter of Circadian Gene Expression inCyanobacteria. Proc. Nail. Acad. Sd. USA 90:5672-5676.9. Mitsui, A., Kumazawa, S., Takabashi, A., Ikemoto, H., Cao, S. and T. Arai. 1986. Strategy byWhich Nitrogen-fixing Unicellular Cyanobacteria Grow Photoautotrophically. Nature 323:720-722.10. Waterbury, LB. 1992. The Cyanobacteria - Isolation, purification and Identification. In Balows, Ael a! (eds). The Prokaryotes. Springer-Verlag, New York.11. Wilcox; 1st, Mitchison, G.J. and RJ. Smith. 1973. Pattern Formation in the Blue-Green Alga,Anabaena 1. Basic Mechanisms. 3. Cell. Sci. 12:707-723.

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