YARROWIA LIPOLYTICA CELL CYCLE STUDY

Agnieszka Mituła¹, Wojciech Barszczewski², Małgorzata Robak^2
1 Institute of Biochemistry and Molecular Biology, University of Wrocław, Poland
² Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

Flow cytometry (FC) was used for measuring ploidity and number of cells  at each phase of the cell cycle  of four Yarrowia lipolytica yeast strains. Synchronization at G0/G1-phase was partially achieved by carbon source starvation, followed by glucose addition. Approximately 10 000 cells were analyzed by measuring the fluorescence of propidium iodine (PI) bound to DNA after 0, 1, 2, 4, 6 hours of glucose addition to the starved cells population. Estimation of cells percentage at each cycle phase was achieved by Cylchred and WinMDI analysis of obtained data. However complete synchronization was not obtained for any of the strains, the generation time representing time interval between two maxima of cells at S phase was determined. The shortest generation time was noted for Y. lipolytica AWG7 (1h).
Comparison of histograms of all tested strains with a haploid type strain proved their haploidy.

Key words: flow cytometry, cell cycle, yeasts, Yarrowia lipolytica.

INTRODUCTION

Flow cytometry (FC) is a technique, which permits rapid, optical analysis of individual cells (rates of 100 to 100 000 cells per second) and is often used in cytology, immunology, hematology, microbiology and biotechnology [5,6]. Typical commercially available flow cytometer permits collection of different types of data for each cell (e.g. size, protein, DNA and  lipid content, antigenic properties, enzyme activity etc.) [11,20]. Finally, allows a multidimensional characterization of a given population.

DNA represents one of the most important cell component. Its content changes during cell cycle [18,24], either as a result of mutation [1], or genetic engineering modification [8,16]. So, the quantity of DNA may reflect physiological condition of the cell.

Yarrowia lipolytica is a species of nonconventional and dimorphic yeasts, which have an important biotechnological potential [15,22]. The analysis of this species cell cycle has not been extensively investigated. Only a few reports have been available [9,17].

The aim was to apply FC for strains ploidity determination, counting  number of cells at different phases of cell cycle during growth of four Y. lipolytica yeast strains used for citric acid production.

MATERIALS AND METHODS

Three strains of Y. lipolytica A-101, A-101.1.31 and AWG7, different in citric acid synthesis and acetate utilization ability, came from the collection of Department of Biotechnology and Food Microbiology of Wrocław University of Environmental and Life Sciences [21]. Two types of strains were a kind gift from INRA (Y. lipolytica W29) and  TUD (Y. lipolytica H222-S4). All strains were maintained at YM-slants and stored at 4°C. Freshly growing yeasts (YM-slants, 24h at 28°C) were used for further analysis, which encompassed: primary culture, starvation (synchronization of cells division) and growth at 28°C for 1, 2, 4 and 6 hours (Fig. 1).

Primary cultures were prepared in 10 mL of MMT (Minimal Medium with Thiamine) with 1% glucose (24h, 28°C, 200 rpm). For starvation and synchronization of cell division 1 mL of primary cultures (approx. 1,5 x 10⁸ cells/mL) was  added to 10 mL of MMT without glucose. Consumption of residual carbon source was performed by growth for 24h at 28°C, with shaking (200 rpm) and starvation by maintaining  cultures for 24h at 4°C. The second step for all cultures was done in triplicated samples (see Fig. 1). Finally, after 48h of carbon  source starvation 1 mL samples of each culture were taken (time 0, t₀) and the growth of yeasts was induced by glucose supplementation to final concentration of 1%. Next, samples were collected at 1, 2, 4 and 6 hours (t₁, t₂, t₄, t₆ respectively) after the addition of glucose.

Cells were collected by centrifugation (5 min at 8000 rpm) and suspended in 1mL of 70% ice-cold ethanol for membrane destabilization. Permeabilized yeast cells were washed twice with PBS and were subjected to RNA digestion (RNase A: 1 ľL, 10 mg/mL), staining  with propidium iodine (2.5 µL, 2 mM) and subsequently directly analyzed on flow cytometer (Becton Dicinson FACScalibur) with argon laser (488 nm). Collected data were analyzed using Cylchred and WinMDI 2.8 software (http://www.ucl.ac.uk/wibr/services/docs/cellcyc.pdf, http://www.cardiff.ac.uk/medicine/haematology/cytonetuk/documents/software.htm).

RESULTS

FC-analyses were  successfully done and histograms of DNA content (as maximum PI fluorescence) for four strains of Y. lipolytica: A-101; A-101.1.31; AWG7 and W29 ura3.302 were presented in Figures 2-5. For Y. lipolytica A-101 detailed analysis of cell cycle phases by Cylchred software was shown in Figure 6.

In our study, the maximum level of yeast cells in G1/0 phase after glucose starvation was 30%  and 48% of population for Y. lipolytica AWG7 and A-101, respectively (t₀, Table 1). For Y. lipolytica W29 and A-101.1.31 it was 40%. Surprisingly as many as 44% of Y. lipolytica AWG7 cells in starved population were in S phase of cell cycle. For other strains it ranged from 25 to 41%. Also, cells in G2/M phase were present,  at 13–35%. The smallest number of cells at G2/M phase was observed for Y. lipolytica A-101 (13%). The number of Sub-G1 cells (apoptotic and damaged) varied from 0.2 to 10%, respectively for Y. lipolytica AWG7 and A-101.

One hour after glucose supplementation Y. lipolytica strains A-101 and A-101.1.31 showed higher number of cells in G1/0 phase (t₁, Table 1). For Y. lipolytica AWG7 the G1/0 cells percentage decrease after glucose addition, and at t₁ achieved only 7% of population. Those strains, having significant cell number in S phase at t₀ (44%), at t₁ shown as many as 65% of cells in that cycle phase. It was the maximum level of cells at S phase for all tested strains and time intervals. The number of cells at G2/M phase reached from 8 to 27% and at Sub-G1 from 0.4 to 4.5%.

Two hours after addition of carbon source into the cultures of Y. lipolytica A-101.1.31 and W29 ura3.302 strains, the number of cells at G1/0 phase decreased and at S phase increased compared with t₁. For example this decrease concerned approximately 16% of Y. lipolytica A-101.1.31 population. For Y. lipolytica A-101 and  AWG7 another cells behavior took place. For the first of mentioned strains the increase of up to 64% of cells in G1/0 was observed (t₂, Table 1). The level of cells at S phase varied from 25 to 50%, at G2/M from 10 to 33% and at Sub-G1 from 0.8–3%.

Four hours after glucose supplementation for all tested strains (except Y. lipolytica AWG7) a dropdown of cells  in G1/0 phase was observed. The most important decrease was noted for Y. lipolytica A-101 (about 30% in comparison to t₂). At this time the maximum number of cells at S-phase was achieved for Y. lipolytica W29 (44%). For the rest of the strains it was around 33%. The number of cells at G2/M was similar, about 30%. Sub-G1 population was more considerable, and ranged from 0.3 to 6%. The last value was observed for Y. lipolytica A-101.

Six hours after glucose supplementation, the number of cells in G1/0 phase changed differently (decreased or increased), and achieved 27-41% of population. For all tested strains, number of cells in S-phase was similar to the amount at t₄ (except Y. lipolytica AWG7), and varied from 31 to 49%. For Y. lipolytica AWG7, 62%  of cells was at S phase. Number of cells at G2/M phase was from 11 to 32%. The latter value was measured for Y. lipolytica A-101.1.31. Sub-G1 number of cells at t₆ slightly decreased. It was from 0.2 to 3.5% of population.

The generation time was determined from those results, representing time interval between two maxima of cells at S phase, clearly shown in Fig. 6. The shortest generation time was noted for Y. lipolytica AWG7 (1h), and the longest for Y. lipolyticaW29 (6h).

Interestingly, the fast growing Y. lipolytica AWG7 presented the smallest number of apoptotic cells (<0.5%), while the maximal number of those cells was detected for Y. lipolytica A-101 (from 3 to 10%).

Comparison of fluorescence intensity for two type strains Y. lipolytica W29, Y. lipolytica H222-S4 and family of Y. lipolytica A-101 allowed determination of their ploidity. The strains Y. lipolytica H222-S4 and Y. lipolytica W29 are known as haploids, so Y. lipolytica A-101 family clones are haploids also (Fig. 7).

DISCUSSION

DNA is the most commonly stained cellular constituent in flow cytometry analysis [26]. The DNA-specific dyes (Hoechst 33342, SYTOX Green and DAPI) have been used for the analysis of microorganisms [7,20,24,25]. Stains, such as ethidium bromide and propidium iodide (PI), are highly fluorescent but unfortunately they bind not only to DNA, but also to RNA [23]. PI intercalates between base pairs of the nucleic acids giving maximum absorption at 536 nm and emission at 617 nm (orange-red fluorescence). Usage of those dyes for measuring cell's DNA content compels to use ribonucleases prior to staining [4,13]. In our study ribonuclease A (MacheryNagel) has been used for RNA degradation.

FC methodology allows the determination of DNA content in cells. Wine S. cerevisiae strains are diploids, aneuploids or polyploids [19]. Bradbure et al.[3] studied the ploidity of 45 commercial strains by comparison of their relative PI fluorescence with type strain fluorescence. Relative fluorescence for S. cerevisiae S288C (diploid type strain) was 1.0, for haploids control clones it was 0.46–0.51. For 40 tested wine strains relative fluorescence value was 0.80–1.06  and for 5 of them 1.24–1.76. According to authors interpretation, the first group contained diploids and the second aneuploids. According to our results, Y. lipolytica A-101 strains family is haploid, showing the same relative fluorescence as two type strains described in references as haploids [2,12].

The cell cycle can be described as a series of distinct biochemical and morphological events, which occur in a reproducing cell. The distinct events within the cell cycle process can be categorized into five main phases. Cells starting from resting state (G0) will proceed into the cell cycle by successive entering G1, S, G2  (known as Interphase), followed by M phase (Mitosis). The use of a DNA binding dye in flow cytometry resolves the cell cycle into only 3 steps which are G0+G1, S and G2+M. During cell transition from G1 to M, chromosomes are duplicated as the result of DNA replication in phase S. Cells in the M phase and G2 have the same DNA content.

Carbon source starvation is one of methods leading to cell cycle synchronization in G0/G1 phase. During growth the yeasts accumulate metabolites and in rich medium cells were in all phases of the cells cycle, while glucose starvation should stop them at G0/G1 or  sometimes at S stage. Kron and Gow [14] described that carbon source starvation arrests the cycle before the budding. In this study, cell cycle synchronization was not complete after 48h yeasts starvation. At any time of growing population, cells at G0/G1 phase did not predominate. Yeast cells  distribution in particular phases of the cycle was as follows:  31–58% in G0/G1; 17–41.5% in S, and 13.5–39.5% in G2/M. A possible explanation of this could be too long starvation period allowing usage of dead cell debris as the carbon source for growth of the next generation. Density of inoculum could be also important for effective cell cycle synchronization. For Y. lipolytica researchers obtained complete synchronization of cell cycle in  G0/G1 by 12h nitrogen source limitation/starvation [9,17]. Other methodology of yeasts cells growth synchronization involves chemicals as alpha pheromone, rapamycine, vorcamine, compactin, lovastatin [1,10,13].  Stöver et al. [24] described the arrest of 95% of Candida albicans cells in G0/G1 by addition of 100 mg/L of 8-hydroxyquinoline, a zinc chelator.

Despite the problem with growth synchronization, FC-analyses were successfully done for Y. lipolytica yeasts. Presented methodology may be applied for cell cycle study, genetic stability, and viability monitoring in bioreactor processes during citric acid or erythritol biosynthesis.

CONCLUSIONS

1. Flow cytometry could be applied for Y. lipolytica cell cycle study.

2. In G1-phase, strains of the family of Y. lipolytica A-101 (A-101.1.31, A-101.1.31.K1, AWG7) had the same fluorescence intensity as Y. lipolytica H222-S4 and Y. lipolytica W29 known as haploid.

3. The percentage of apoptotic cells was measured during the growth of all strains. Y. lipolytica A-101 showed the highest level of these cells.

4. Synchronization of Y. lipolytica cells growth in G1-phase was not achieved by glucose starvation.

ACKNOWLEDGMENTS

We wish to thank to prof. Gerold Barth and dr Stephan Mauesberger from Technical University of Dresden (TUD, Germany) and prof. Jean-Marc Nicaud from National Institute of Agronomic Research (INRA, Grignon, France) for kind providing yeast strains. We thank mgr Iwona Zbyryt from Department of Epizootiology and Veterinary Administration with Clinic of Infectious Diseases (UP, Wrocław) for technical assistance during FC analysis.

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Accepted for print: 12.05.2010

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