12 research outputs found
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
No full textFor 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
Dynamics and genetic diversification of Escherichia coli during experimental adaptation to an anaerobic environment
No full textBackground Many bacteria are facultative anaerobes, and can proliferate in both anoxic and oxic environments. Under anaerobic conditions, fermentation is the primary means of energy generation in contrast to respiration. Furthermore, the rates and spectra of spontaneous mutations that arise during anaerobic growth differ to those under aerobic growth. A long-term selection experiment was undertaken to investigate the genetic changes that underpin how the facultative anaerobe, Escherichia coli, adapts to anaerobic environments. Methods Twenty-one populations of E. coli REL4536, an aerobically evolved 10,000th generation descendent of the E. coli B strain, REL606, were established from a clonal ancestral culture. These were serially sub-cultured for 2,000 generations in a defined minimal glucose medium in strict aerobic and strict anaerobic environments, as well as in a treatment that fluctuated between the two environments. The competitive fitness of the evolving lineages was assessed at approximately 0, 1,000 and 2,000 generations, in both the environment of selection and the alternative environment. Whole genome re-sequencing was performed on random colonies from all lineages after 2,000-generations. Mutations were identified relative to the ancestral genome, and based on the extent of parallelism, traits that were likely to have contributed towards adaptation were inferred. Results There were increases in fitness relative to the ancestor among anaerobically evolved lineages when tested in the anaerobic environment, but no increases were found in the aerobic environment. For lineages that had evolved under the fluctuating regime, relative fitness increased significantly in the anaerobic environment, but did not increase in the aerobic environment. The aerobically-evolved lineages did not increase in fitness when tested in either the aerobic or anaerobic environments. The strictly anaerobic lineages adapted more rapidly to the anaerobic environment than did the fluctuating lineages. Two main strategies appeared to predominate during adaptation to the anaerobic environment: modification of energy generation pathways, and inactivation of non-essential functions. Fermentation pathways appeared to alter through selection for mutations in genes such as nadR, adhE, dcuS/R, and pflB. Mutations were frequently identified in genes for presumably dispensable functions such as toxin-antitoxin systems, prophages, virulence and amino acid transport. Adaptation of the fluctuating lineages to the anaerobic environments involved mutations affecting traits similar to those observed in the anaerobically evolved lineages. Discussion There appeared to be strong selective pressure for activities that conferred cell yield advantages during anaerobic growth, which include restoring activities that had previously been inactivated under long-term continuous aerobic evolution of the ancestor
Dynamics and genetic diversification of Escherichia coli during experimental adaptation to an anaerobic environment
No full textBackground. Many bacteria are facultative anaerobes, and can proliferate in both anoxic and oxic environments. Under anaerobic conditions, fermentation is the primary means of energy generation in contrast to respiration. Furthermore, the rates and spectra of spontaneous mutations that arise during anaerobic growth differ to those
under aerobic growth. A long-term selection experiment was undertaken to investigate the genetic changes that underpin how the facultative anaerobe, Escherichia coli, adapts to anaerobic environments.
Methods. Twenty-one populations of E. coli REL4536, an aerobically evolved 10,000th generation descendent of the E. coli B strain, REL606, were established from a clonal ancestral culture. These were serially sub-cultured for 2,000 generations in a defined minimal glucose medium in strict aerobic and strict anaerobic environments, as well as in a treatment that fluctuated between the two environments. The competitive
fitness of the evolving lineages was assessed at approximately 0, 1,000 and 2,000 generations, in both the environment of selection and the alternative environment. Whole genome re-sequencing was performed on random colonies from all lineages after 2,000-generations. Mutations were identified relative to the ancestral genome, and based on the extent of parallelism, traits that were likely to have contributed towards adaptation were inferred.
Results. There were increases in fitness relative to the ancestor among anaerobically evolved lineages when tested in the anaerobic environment, but no increases were found in the aerobic environment. For lineages that had evolved under the fluctuating regime, relative fitness increased significantly in the anaerobic environment, but did not increase in the aerobic environment. The aerobically-evolved lineages did not increase in fitness when tested in either the aerobic or anaerobic environments. The strictly anaerobic lineages adapted more rapidly to the anaerobic environment than did the fluctuating lineages. Two main strategies appeared to predominate during adaptation to the anaerobic environment: modification of energy generation pathways, and inactivation of non-essential functions. Fermentation pathways appeared to alter through selection for mutations in genes such as nadR, adhE, dcuS/R, and pflB. Mutations were frequently identified in genes for presumably dispensable functions such as toxin-antitoxin systems, prophages, virulence and amino acid transport. Adaptation of the fluctuating lineages to the anaerobic environments involved mutations affecting traits similar to those observed in the anaerobically evolved lineages.
Discussion. There appeared to be strong selective pressure for activities that conferred cell yield advantages during anaerobic growth, which include restoring activities that had previously been inactivated under long-term continuous aerobic evolution of the ancestor
Anaerobically grown Escherichia coli has an enhanced mutation rate and and distinct mutational spectra
No full textFor 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 mutation molecular spectrum of organisms grown in aerobic as compared to anaerobic environments is not well understood at the whole genome level. We directly measured the spontaneous mutation rates and spectra of a model facultative anaerobe, Escherichia coli strain REL4536, grown aerobically and anaerobically in long-term mutation accumulation experiments. Whole genome analyses revealed that the spontaneous mutation rate of the anaerobically grown cultures, 4.14 × 10-10 mutations per nucleotide per generation, was greater than that of the aerobically grown cultures, at 2.50 × 10-10 mutations per nucleotide per generation. Different base pair substitutions (BPSs) biases under aerobic and anaerobic growth conditions were observed. In particular, G T transversions, commonly associated with oxidative DNA damage, were more abundant on the clockwise replichore in aerobically grown cells, while C T mutations and A C mutations were more prevalent on the clockwise and counter-clockwise replichores, respectively, of anaerobically grown cells. Structural variations (SVs) were over two-fold more abundant in anaerobically grown cells as compared to aerobically grown cells, where IS150 transposition occurred almost 10 times more frequently. The frequency and spectrum of spontaneous mutations during anaerobic growth provides valuable insights into factors affecting mutations during anaerobic metabolism in facultative anaerobes
Anaerobically Grown Escherichia coli Has an Enhanced Mutation Rate and Distinct Mutational Spectra.
No full textOxidative 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 Escherichia coli 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
Genome-wide spontaneous mutation rates for <i>E</i>. <i>coli</i> grown aerobically and anaerobically.
No full textGenome-wide spontaneous mutation rates for E. coli grown aerobically and anaerobically.</p
Mutation rates and spectra for aerobically and anaerobically grown REL4536.
No full textA) Genomic distribution of 124 and 158 mutations identified in the aerobically and anaerobically grown cells, respectively, mapped to the E. coli 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 [27, 28]. 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 S1 Fig) Mean mutation rates per nucleotide per generation of 24 aerobic and anaerobic lineages. Error bars are standard errors of the mean. * p < 0.05 by Mann-Whitney U-test.</p
Mutation rates of SV classes in aerobically and anaerobically grown REL4536.
No full textShown 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. * p < 0.05 by Mann-Whitney U-test.</p
Anaerobically Grown <i>Escherichia coli</i> Has an Enhanced Mutation Rate and Distinct Mutational Spectra - Fig 3
No full textFrequencies of BPSs in the clockwise and counter-clockwise replichores of A) aerobic and B) anaerobic lineages. Expected values account for the uneven replichore sizes for REL4536 (clockwise replichore is 2.06 Mb, whereas the counterclockwise replichore is 2.54 Mb), and the assumption of equal mutation rates on the leading and lagging strand. * p 2-test.</p
Conditional BPS mutation rates per generation in aerobically and anaerobically grown REL4536.
No full textMutation 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. * p < 0.05 by Mann-Whitney U-test.</p
