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Periodic Variation of Mutation Rates in Bacterial Genomes Associated With Replication Timing

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Periodic Variation of Mutation Rates in Bacterial Genomes Associated With Replication Timing

Marcus M Dillon et al. mBio.

Abstract

The causes and consequences of spatiotemporal variation in mutation rates remain to be explored in nearly all organisms. Here we examine relationships between local mutation rates and replication timing in three bacterial species whose genomes have multiple chromosomes: Vibrio fischeri, Vibrio cholerae, and Burkholderia cenocepacia Following five mutation accumulation experiments with these bacteria conducted in the near absence of natural selection, the genomes of clones from each lineage were sequenced and analyzed to identify variation in mutation rates and spectra. In lineages lacking mismatch repair, base substitution mutation rates vary in a mirrored wave-like pattern on opposing replichores of the large chromosomes of V. fischeri and V. cholerae, where concurrently replicated regions experience similar base substitution mutation rates. The base substitution mutation rates on the small chromosome are less variable in both species but occur at similar rates to those in the concurrently replicated regions of the large chromosome. Neither nucleotide composition nor frequency of nucleotide motifs differed among regions experiencing high and low base substitution rates, which along with the inferred ~800-kb wave period suggests that the source of the periodicity is not sequence specific but rather a systematic process related to the cell cycle. These results support the notion that base substitution mutation rates are likely to vary systematically across many bacterial genomes, which exposes certain genes to elevated deleterious mutational load.IMPORTANCE That mutation rates vary within bacterial genomes is well known, but the detailed study of these biases has been made possible only recently with contemporary sequencing methods. We applied these methods to understand how bacterial genomes with multiple chromosomes, like those of Vibrio and Burkholderia, might experience heterogeneous mutation rates because of their unusual replication and the greater genetic diversity found on smaller chromosomes. This study captured thousands of mutations and revealed wave-like rate variation that is synchronized with replication timing and not explained by sequence context. The scale of this rate variation over hundreds of kilobases of DNA strongly suggests that a temporally regulated cellular process may generate wave-like variation in mutation risk. These findings add to our understanding of how mutation risk is distributed across bacterial and likely also eukaryotic genomes, owing to their highly conserved replication and repair machinery.

Keywords: Vibrio cholerae; Vibrio fischeri; genome organization; mutation rate; periodicity.

Figures

FIG 1
FIG 1
Patterns of base substitution mutation (bpsm) rates at various size intervals extending clockwise from the origin of replication (oriC) in MMR-deficient mutation accumulation lineages of V. fischeri (A) and V. cholerae (B) on chromosome 1. bpsm rates are calculated as the number of mutations observed within each interval divided by the product of the total number of sites analyzed within that interval across all lines and the number of generations of mutation accumulation. The two intervals that meet at the terminus of replication (dotted red line) on each replichore are shorter than the interval length for that analysis, because the size of chromosome 1 is never exactly divisible by the interval length.
FIG 2
FIG 2
Relationship between base substitution mutation (bpsm) rates in 100-kb intervals on the right replichore with concurrently replicated 100-kb intervals on the left replichore in MMR-deficient Vibrio fischeri (A) and Vibrio cholerae (B). Both linear regressions are significant on chr1 (V. fischeri, F = 10.98, df = 13, P = 0.0060, r2 = 0.46; V. cholerae, F = 6.76, df = 13, P = 0.0221, r2 = 0.34) but not on chr2 (V. fischeri, F = 0.02, df = 6, P = 0.8910, r2 = 0.03 × 10−1; V. cholerae, F = 0.06, df = 4, P = 0.8140, r2 = 0.02).
FIG 3
FIG 3
Patterns of base substitution mutation (bpsm) rates in 100-kb intervals extending clockwise from the origin of replication (oriCI) on chromosome 1 (chr1) and patterns of bpsm rates of concurrently replicated 100-kb intervals on chromosome 2 (chr2) for MMR-deficient Vibrio fischeri (A) and Vibrio cholerae (B). Patterns of bpsm rates on chr2 appear to map to those of concurrently replicated regions on chr1 in both species, but the linear regressions between concurrently replicated intervals are not significant on chr1 and chr2 in either V. fischeri or V. cholerae (V. fischeri, F = 0.62, df = 14, P = 0.4442, r2 = 0.04; V. cholerae, F = 0.07, df = 10, P = 0.7941, r2 = 0.01).
FIG 4
FIG 4
Wavelet power spectrum and resultant reconstruction of the patterns of base substitution mutation (bpsm) rates in 100-kb intervals extending clockwise from the oriCI region of chromosome 1 (A and B, V. fischeri; E and F, V. cholerae) and the oriCII region of chromosome 2 (C and D, V. fischeri; G and H, V. cholerae) using the MMR-deficient mutation accumulation lineages. Wavelet power analyses follow an interval color key (A, C, E, and G), where colors code for the power values at each interval in the genome for all possible wave periods, from dark blue (low power) to dark red (high power). White contour lines denote a significance cutoff of 0.1. Reconstructed series were generated using only the wave periods whose average power was significant over the entire interval (B, D, F, and H).
FIG 5
FIG 5
Patterns of base substitution mutation (bpsm) rates in 100-kb intervals extending clockwise from the origin of replication (oriC) on chromosome 1 (chr1) and concurrently replicated intervals of chromosome 2 (chr2) for WT (MMR+) Vibrio fischeri (A), Vibrio cholerae (B), and Burkholderia cenocepacia (C). B. cenocepacia also has a third chromosome, which is not shown. These visual patterns are not statistically significant, perhaps owing to low sample size: (linear regression; V. fischeri wild type, F = 0.16, df = 14, P = 0.7001, r2 = 0.01; V. cholerae wild type, F = 2.72, df = 10, P = 0.1300, r2 = 0.21; B. cenocepia wild type, F = 0.32, df = 30, P = 0.5760, r2 = 0.01).
FIG 6
FIG 6
Hypothesized model of the relationship between replication timing, ribonucleotide reductase (RNR) activity, and the resulting availability of dNTPs per active replication fork. The model is fit to the V. cholerae genome with two chromosomes (chr1 and -2): one of 3.0 Mb and one of 1.1 Mb. RNR activity follows a wave that rises after the firing of the origin of chr1 and then steadily declines until additional origins fire. The chr2 origin should fire after ~950 kb of replication on each replichore of chr1 to ensure termination synchrony between chromosomes, stimulating a second wave of RNR activity. The right axis uses arbitrary relative units (dNTPs/fork) to depict how RNR activity is expected to increase dNTP pools to a maximum level (2.0) that is diluted by the number of concurrent, active forks. (A and B) Under slow growth, RNR activity rises and then falls to the baseline required to maintain synthesis. (C and D) Faster growth requires a second round of replication. Note that further rounds of overlapping replication do not significantly alter predicted dNTPs/fork, the hypothesized driver of mutation rate variability.

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