Single base substitutions can be classified into 12 possible substitution types. The rates for each of these were calculated for cells grown aerobically and anaerobically, with mutation rates normalized to account for the nucleotide content of the genome and the predominant mutations in both environments were G Æ A or C Æ T transitions
(Figure 4.2). As spontaneous deamination of cytosine nucleotide bases into uracil
bases, or of 5-methylcytosine nucleotide bases into thymine bases, are frequently occurring DNA lesions known to cause G Æ A or C Æ T transitions respectively, these results were expected (13, 19, 247). As can be seen in Figure 4.2b,G Æ A and C Æ T
transitions appear to the predominant types of BPSs in aerobically grown cells per unit time. As the aerobically grown cells of this study spent more time in DNA replication, it stands to reason that the DNA of aerobically grown cells spent more time in a single- stranded state. Thus, these findings are in agreement with previous observations that ssDNA is particularly susceptible to cytosine deamination (248). On the other hand, A Æ G and T Æ C transitions, possibly caused by the spontaneous deamination of
98 adenine nucleotide bases into hypoxanthine or by the tautomerization of thymine bases (13, 19, 247), respectively, were less common in both environments (Figure 4.2).
Overall, these findings are consistent with mutational data from previous studies reporting a spontaneous mutation bias towards increasing the A:T content of bacterial genomes (29, 249, 250). In short, these studies imply that the nucleotide content of bacteria are not as affected by mutational biases as previously thought but rather, selection pressures that the bacteria may have been subject to over time are more likely causes for any observed variations in nucleotide content.
ROS generated during aerobic respiration, are also known to induce specific mutation types. For instance, the GO lesion is a typical DNA lesion caused by the oxidation of guanine nucleotide bases to 8-oxo-guanine nucleotide bases, and can ultimately lead to G Æ T and C Æ A transversions. In keeping with this expectation, G Æ T transversion mutation rates per generation were 5.4-fold greater (Figure 4.2a) in aerobically grown
cells, as compared to anaerobically grown cells(Mann-Whitney U = 230.0, p = 0.042).
Surprisingly, C Æ A transversion rates per generation were approximately eight-fold greater (Figure 4.2a) in anaerobically grown cells, as compared to aerobically grown
cells (Mann-Whitney U = 225.0, p = 0.011). A similar pattern of G Æ T versus C Æ A
asymmetry was also observed in mutation rates calculated per day (Figure 4.2b). These
results were most unexpected and suggest the presence of a strand bias in the types of BPSs that arose under aerobic and anaerobic conditions and will be investigated further in section 4.2.2.1.2.
More curious, however, was the presence of G Æ T and C Æ A transversions in anaerobic lineages. These findings suggest that there may be some oxidative damage occurring during growth under the anaerobic conditions in this study and that the repair systems that are normally induced in response to this specific kind of damage are not being efficiently induced. While this may imply that the growth conditions in the anaerobic conditions are not truly anaerobic, Sakai et al. (2006) have also observed G Æ T and C Æ A transversions in the anaerobic cells of their study (28). In fact, they reported almost equivalent rates of G Æ T and C Æ A transversions in aerobically and anaerobically grown cells (28). While they did not give much thought to these mutations, they concluded that their anaerobically grown cells were not experiencing any oxidative damage as evidenced by the lack of overall G Æ T and C Æ A
99 transversions in mutator cells (28). While ROS can also be produced as a byproduct of certain biological mechanisms, such as part of the inflammatory response or intracellular signalling (19), there is a strong likelihood that G Æ T and C Æ A transversions are spontaneously arising under anaerobic conditions, but not because of exposure to ROS. While no specific mechanism underlying this BPS spectrum is apparent, it may be associated with pH, as acids are generated as fermentation end- products during growth under anaerobic conditions. Alternatively, the slower growth rate, and the resultant physiological condition of the cells under anaerobic growth may have created specific mutagenic pressures resulting in the observed BPS spectrum.
Mutation rates per generation for T Æ G transversions (Mann-Whitney U = 217.5,
p = 0.032 ) and A Æ C transversions (Mann-Whitney U = 223.0, p = 0.021) were at
least two-fold greater in anaerobically grown E. coli as compared to aerobically grown E. coli (Figure 4.2a). Per day, T Æ G and A Æ C transversions occurred at rates that
were not statistically significantly different between aerobically and anaerobically grown E. coli (Figure 4.2b). Therefore, it is possible that T Æ G and A Æ C
transversion mutations occur independently of DNA replication. As oxidation of the guanine nucleotide can result in T Æ G or A Æ C transversions (19), these results were unexpected. Maybe these spontaneously arising transversion mutations are not being repaired efficiently under the anaerobic conditions of this study, allowing for their accumulation in anaerobic lineages. Or maybe, perhaps there is not enough oxidative stress under the aerobic growth conditions of this study, accounting for the fewer oxidative damage related mutations observed in aerobically grown cells. Or alternatively, these results raise the possibility of there being causative agents, other than ROS, that lead to T Æ G or A Æ C transversions, especially for anaerobically grown cells. To obtain a better understanding of these results, the expression values of various genes involved in repairing DNA damage under aerobic and anaerobic conditions were analysed and will be discussed in section 4.2.3.1.1.
100 Figure 4.2. Mutation rates of single base substitutions in aerobically and anaerobically grown E. coli. Shown are a) mean mutation rates per genome per generation and b) mean mutation rates per genome per day of growth. Error bars represent standard error of the mean. Asterisk denotes a significant difference between the aerobic and anaerobic mutation rates (p < 0.05).