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

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

Anaerobically Grown <i>Escherichia coli</i> Has an Enhanced Mutation Rate and Distinct Mutational Spectra

<div><p>Oxidative stress is a major cause of mutation but little is known about how growth in the absence of oxygen impacts the rate and spectrum of mutations. We employed long-term mutation accumulation experiments to directly measure the rates and spectra of spontaneous mutation events in <i>Escherichia coli</i> populations propagated under aerobic and anaerobic conditions. To detect mutations, whole genome sequencing was coupled with methods of analysis sufficient to identify a broad range of mutational classes, including structural variants (SVs) generated by movement of repetitive elements. The anaerobically grown populations displayed a mutation rate nearly twice that of the aerobic populations, showed distinct asymmetric mutational strand biases, and greater insertion element activity. Consistent with mutation rate and spectra observations, genes for transposition and recombination repair associated with SVs were up-regulated during anaerobic growth. Together, these results define differences in mutational spectra affecting the evolution of facultative anaerobes.</p></div

Mutation rates of SV classes in aerobically and anaerobically grown REL4536.

<p>Shown are mean mutation rates per nucleotide per generation for A) SV classes (del, deletion; ins, insertion; med, mediated; inv, inversion; and trans, translocation; and B) IS elements. Error bars represent standard errors of the mean. * <i>p</i> < 0.05 by Mann-Whitney U-test.</p

Conditional BPS mutation rates per generation in aerobically and anaerobically grown REL4536.

<p>Mutation rates for a total of 147 BPS mutations normalized to the number of A, T, G or C base pairs in each genome. Error bars are standard errors of the mean. * <i>p</i> < 0.05 by Mann-Whitney U-test.</p

Mutation rates and spectra for aerobically and anaerobically grown REL4536.

<p>A) Genomic distribution of 124 and 158 mutations identified in the aerobically and anaerobically grown cells, respectively, mapped to the <i>E</i>. <i>coli</i> REL4536 genome. The outermost circle shows the genome organisation into replichores from the origin of replication, OriC, to the replication termination Ter sites. Arrows indicate the direction of replication for each replichore. The second circle shows the genome macrodomains (MDs), as defined previously [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006570#pgen.1006570.ref027" target="_blank">27</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006570#pgen.1006570.ref028" target="_blank">28</a>]. Coding sequences on the forward and reverse strands are shown on the third and fourth circles, respectively. BPSs, indels and SVs are shown on the fifth, sixth and seventh circles, respectively. Mutations in aerobic and anaerobic lineages are displayed in red and blue, respectively. The innermost circle displays the GC-skews, where green indicates an excess of G over C while purple indicates an excess of C over G. Detailed plots of cumulative mutation distribution against genome position are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006570#pgen.1006570.s013" target="_blank">S1 Fig</a>) Mean mutation rates per nucleotide per generation of 24 aerobic and anaerobic lineages. Error bars are standard errors of the mean. * <i>p</i> < 0.05 by Mann-Whitney U-test.</p

Genome-wide spontaneous mutation rates for <i>E</i>. <i>coli</i> grown aerobically and anaerobically.

<p>Genome-wide spontaneous mutation rates for <i>E</i>. <i>coli</i> grown aerobically and anaerobically.</p