Effects of aerobic and anaerobic environments on bacterial mutation rates and mutation spectra assessed by whole genome analyses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand

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For organisms that are exposed to different environments, the rates and types of spontaneous mutations that arise in each environment can vary, and potentially impact the direction of evolution as a whole. Oxidative stress is a major cause of mutation, but the effect of oxygen availability on the mutation rates and spectra of organisms grown in aerobic as compared to anaerobic environments is not well understood at the whole genome level. To investigate the mutation rates and spectra of a facultative anaerobic bacterium grown under strictly aerobic or anaerobic conditions, 24 mutation accumulation lineages, derived from Escherichia coli REL4536, were established and propagated through 180 and 144 single-colony population bottlenecks, respectively. Spontaneous mutation rates of 2.50 × 10-10 and 4.14 × 10-10 mutations per nucleotide per generation were obtained for aerobically and anaerobically grown cells, respectively. Mutations in the aerobic environment were significantly biased towards G  T mutations and IS186 transposition, while C  A, T  G, A  C mutations, gross chromosomal rearrangements (GCRs) and IS150 transposition were significantly more prevalent under anaerobic conditions. Transcriptional profiling, via RNAseq, of REL4536 grown under aerobic and anaerobic environments revealed that repair genes, especially those involved in the repair of GCRs, were generally up-regulated in the anaerobic environment, consistent with findings that mutation rates, especially for GCRs, are higher in the anaerobic environment. GCRs have long been thought to play an important role in the evolutionary process, though their contributions to the process have not been specifically defined. SbcCD, an exonuclease, is involved in the repair of DNA secondary structures, and is thought to help prevent the occurrence of GCRs. Transcriptome analyses showed that in E. coli, sbcC was up-regulated during growth in an anaerobic environment, as compared to an aerobic environment. To investigate the impact of GCRs on adaptive evolution, an E. coli REL4536 strain with disrupted sbcC was constructed and evolved under anaerobic conditions for 1,000 generations in glucose-limited media in 14 parallel populations. Mutations that arose during evolution were determined by whole genome re-sequencing of selected clones, and evolved sbcC mutant strains displayed more GCRs and enhanced population-level fitness on average. Together, these results suggest that GCRs may play an important role in the rate of adaptation.
Mutation, Bacterial genetics, Molecular evolution, Gross chromosomal rearrangements (GCR)