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The first step in monitoring the transcriptional effect of each damaging agent on S. solfataricus was to determine a level of damage that would stress the cells into a damage response, but not prove fatal, allowing them to repair themselves.

Increasing concentrations of the damaging agents were added to exponentially growing cells and their subsequent growth monitored. The damaging agents chosen were Mitomycin C, Methyl methane sulfonate, Phleomycin and Hydrogen peroxide.

4.2.1 Mitomycin C (MMC)

Mitomycin C is a chemotherapy drug used as an anti-cancer antibiotic. It is a naturally occurring compound produced by a number of Streptomyces species. To exert a cytotoxic effect MMC requires reductive activation. MMC generates covalent inter- strand cross-links, and in doing so inhibits DNA synthesis which leads to cell death (Iyer and Szybalski 1963). Although the type of damage produced by MMC is different from that produced by UV irradiation both types of damage cause a stalling of replication and so may elicit a similar damage response.

MMC was added to growing cultures of S. solfataricus, optical density readings were taken at 600 nm and the growth monitored for the next 16 hours, see Figure 4.1. It was assumed that an increase in OD600 readings indicated that the cells were growing, however an increase in OD600 can also be observed if the cells are increasing in size, rather than in number (Samson, Obita et al. 2008). It was assumed in the following experiments that the increasing OD600 indicated a growing culture, however cells were not checked by microscopy.

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 2 4 6 8 10 12 14 16

Growth Curve of S. solfatarius After Addition of Mitomycin C

Control 1 µM 2 µM 20 µM 40 µM OD 600 Time (hrs)

Cells treated with higher concentrations of MMC grew more quickly than cells treated with lower concentrations. To determine if this trend continued, higher concentrations of MMC (100, 200 and 400 µM) were added to fresh cultures of cells and their growth measured by reading the optical density at 600 nm, see figure 4.2.

0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0 2 4 6 8 10

Growth Curve of S. solfatarius After Addition of Mitomycin C

Control 40 µM 100 µM 200 µM OD 600 Time (hrs)

The cells were treated with higher concentrations of MMC. The limited effect of

Figure 4.2 Growth curves of S . solfataricus after addition of increasing concentrations of Mitomycin C

Initial concentrations of MMC (1, 2, 20, 40 µM) had little effect on the cells; higher concentrations (100 and 200 µM) were added to determine if there was any effect.

Figure 4.1 Growth curves of S . solfataricus after addition of increasing concentrations of Mitomycin C

MMC to final concentrations of 1, 2, 20 and 40 µM was added to growing cells. The two higher concentrations of MMC appeared to have less effect on the cells growththan the two lower concentrations.

to determine whether the MMC was being destroyed by the low pH and/or high temperature conditions of the Sulfolobus growth media. Three E. coli cultures were grown to an OD600 of 0.4, one was kept as a control, one had 40 µM MMC added, the third culture also had 40 µM MMC added, however the MMC had been added to

Sulfolobus media and incubated at 80 °C for 30 minutes prior to addition, see Figure 4.3. 0.4 0.6 0.8 1 1.2 1.4 0 20 40 60 80 100 120 140

Growth Curve of Escherichia coli

After Addition of Mitomycin C

Control MMC

MMC + Heat and pH

OD 600

Time (min)

The growth of the control culture, and the culture containing the pH/heat treated MMC was very similar. The initial lag in growth of the culture containing the pH/heated treated MMC maybe due to lowering of the pH of the culture on addition of the MMC in Sulfolobus media, rather that any effect of the MMC. The growth of the culture containing the normal MMC was greatly reduced, showing that the MMC was effective. It appears that MMC is destroyed by either the low pH and/or the high temperature of the Sulfolobus media and so has little effect on the growth of the cells. It was decided that MMC to a final concentration of 2 µM would be used to damage the S. solfataricus cells and test the expression of two of the nine genes to determine if there was any effect on gene expression, this is discussed in section 4.3.1.

Figure 4.3 Graph showing growth of E.coli after addition of 40 µM Mitomycin C (normal and heat/pH treated) The high temperature, low pH conditions of the S u l f o l o b u s

media appear to be having an inhibitory or destructive effect on the MMC, as the growth of cells is not effect by the MMC that has been heat and pH treated before addition.

4.2.2 Methyl methane sulfonate (MMS)

Methyl methane sulfonate is an alkylating agent and a carcinogen. Alkylating agents attach small alkyl groups to DNA bases, which results in the DNA being targeted for degradation by repair enzymes, and fragmented as the repair enzymes attempt to replace the alkylated bases. Alkylated bases prevent DNA synthesis and transcription (Valenti, Napoli et al. 2006).

MMS was added to growing cultures of S. solfataricus, optical density readings were taken at 600 nm to monitor the growth for the next 10 hours, see Figure 4.4.

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 5 10 15 20 25

Growth Curve of S. solfataricus After Addition of Methyl Methane Sulfonate

Control 100 µM 200 µM 300 µM 400 µM OD 600 Time (hrs)

MMS had little effect on the growth of the cells. It was decided that MMS to a final concentration of 300 µM would be used to damage the S. solfataricus cells in subsequent experiments, discussed in section 4.3.2.

4.2.3 Phleomycin

Phleomycin is a copper-containing antibiotic of the Bleomycin family. It is a naturally occurring compound and was isolated from a mutant strain of Streptomyces verticillus. It acts on membrane bound DNA affecting the integrity of the cell wall. It

Figure 4.4 Growth curves of S . solfataricus after addition of Methyl methane sulfonate

MMS to final concentrations of 100, 200, 300, and 400 µM was added to growing cultures of S. solfataricus. The MMS appeared to have little effect on the growth of the cells.

breaks, leading to the arrest of DNA synthesis, and the fragmentation of the DNA (Reiter, Milewskiy et al. 1972).

Phleomycin was added to growing cultures of S. solfataricus, optical density readings were taken at 600 nm to monitor the growth for the next 10 hours, see Figure 4.5.

0.2 0.3 0.4 0.5 0.6 0.7 0 2 4 6 8 10

Growth Curve of S. solfataricus After Addition of Phleomycin Control 100 µM 200 µM 500 µM OD 600 Time (hrs)

Addition of phleomycin caused a lag in the initial cell growth that was more pronounced the higher the concentration. It was decided that phleomycin to a final concentration of 200 µM would be used in subsequent experiments to damage the S. solfataricus cells, discussed in section 4.3.3.

4.2.4 Hydrogen peroxide

Hydrogen peroxide is a natural by-product of oxygen metabolism. In the cell it is normally converted to water by catalase or peroxidases because it is a reactive oxygen species (ROS) and is toxic to the cell. It also has the potential, if it reacts with iron, to produce even more damaging hydroxyl radicals (Valko, Morris et al. 2005). Cells encounter hydrogen peroxide all the time, however its potentially damaging effects are kept in check by a plethora of enzymes (such as catalase and superoxide

Figure 4.5 Growth curves of S . s o l f a t a r i c u s after addition of phleomycin

Increasing concentrations of

phleomycin (100, 200, 500 µM) caused increasing lag in the initial growth of the cultures.

the cell must act to stop damage by ROS. Hydrogen peroxide was added to growing cultures of S. solfataricus, optical density readings were taken at 600 nm to monitor the growth for the next 10 hours, see Figure 4.6.

0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0 2 4 6 8 10

Growth Curve of S. solfataricus After

Addition of Hydrogen Peroxide

Control 1 µM 2 µM 5 µM 10 µM OD 600 Time (hrs)

Hydrogen peroxide elicited the strongest effect of any of the damaging agents, with as little as 5 µM having a dramatic effect on cell growth. It was decided that hydrogen peroxide to a final concentration of 1 µM would be used in subsequent experiments to damage the S. solfataricus cells, discussed in section 4.3.4.