Department of Ecology and Evolutionary Biology, University of Arizona, Tucson;
Department of Biology, University of Utah, Salt Lake City
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Abstract |
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Introduction |
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Rates of replication errors, which underlie many spontaneous mutations, also vary among sites. Error rates can be affected by the specific bases adjacent to a base; for example, when the nucleotide adjacent to a thymidine is changed from a guanosine to an adenosine, T G errors decrease more than 30-fold, and T
A errors increase more than 10-fold (Kunkel and Soni 1988
). In addition, there are differences in the rates of errors originating on the leading and lagging strands. In a mismatch-repairdeficient strain of E. coli, G·C
A·T transitions were four times more frequent, and A·T
G·C transitions were two times less frequent, on the leading strand (Fijalkowska et al. 1998
; Maliszewska-Tkaczyk et al. 2000
), and errors attributable to misincorporations of dTTP were four times less frequent on the leading strand in the mammalian in vitro system (Roberts et al. 1994
).
Rates of point mutations determined from sequence comparisons, as with those determined experimentally, depend on the particular type of mutation (Li, Wu, and Luo 1984
), the adjacent nucleotides (Blake, Hess, and Nicholson-Tuell 1992
), and the strand location (Lobry 1996
; Francino and Ochman 1999
). An additional factor influencing mutation rates, as revealed from comparisons of homologous genes of E. coli and S. enterica, is the distance from the replication origin: for genes of similar codon usage bias, those genes farthest from the origin have approximately twofold higher synonymous substitution rates than those nearest to it (Sharp et al. 1989
). This difference may be solely because of the chromosomal position; for example, rates of postreplication recombinational repair may be higher for sequences closer to the origin because these sequences are more likely to be replicated and in higher copy numbers (Sharp et al. 1989
). However, because different sets of genes are being compared, the inherent properties of the genes themselves, such as base composition and nearest-neighbor frequencies, might also contribute to variation in the mutation rates with chromosomal position.
To test how chromosomal location affects point mutation rates, we assayed mutation rates of lacZ alleles inserted at several positions on the chromosome. By assaying mutations that reside at identical positions within identical genes, but at different genomic locations, we were able to remove any confounding effects that neighboring bases or local base composition may have on mutation rates. We found that mutation rates do vary with chromosomal position, but do not increase with distance from the origin of replication, indicating that other factors underlie the observed trend in synonymous substitution frequencies.
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Materials and Methods |
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Mutation Assays
For each strain, four estimates of lacZ reversion rates were derived from batches of 1570 cultures. To make estimates statistically independent, each batch was derived from a different colony, but all cultures within a batch were from the same colony. Cultures were grown in 20 ml of M9 minimal glycerol media (Miller 1992
) supplemented with 300 µM amino acids when appropriate. Each culture was inoculated with 2.0 µl of an overnight culture grown in Luria-Bertani (LB) medium. (Inocula of this size rarely contain Lac revertants and allow cultures to reach the stationary phase before sampling.) Cultures were incubated at 37°C for 24 h under moderate shaking.
Serial dilutions of one to three cultures per batch were plated on minimal glycerol (0.2%) plates to obtain total cell counts. To enumerate revertants, cultures were centrifuged for 20 min at 3000 g, then cells were resuspended in 0.4 ml of M9 salts and plated onto minimal lactose plates (0.2%) supplemented with 100 µM IPTG, 20 µg/ml X-gal, and 0.3 mM amino acids. Blue colonies visible after 3-day incubation at 37°C were scored as revertants.
To reduce the survival of nonrevertants, minimal lactose plates were pretreated overnight with 1010 scavengers (S. enterica serovar Typhimurium strain 14028s), i.e., Lac- cells added to consume residual nutrients. Scavenger cells from an overnight culture were resuspended in 1/10 volume M9 salt solution, and 100 µl of the suspension was spread onto each plate.
To test for differences in overall mutation rates among the strains, we assayed for rpsL and rpoB mutations that confer resistance to 500 µg/ml streptomycin sulfate and 100 µg/ml rifampicin, respectively. Estimates of the rates of the streptomycin-resistant mutations were derived from two or more batches of 1770 cultures per strain grown and plated as described. Estimates of the rates of mutations to rifampicin resistance for each strain were derived from four batches of 18 cultures grown and plated as follows: tubes containing 0.3 ml LB media were inoculated with approximately 106 cells and incubated at 37°C for 6.5 h with moderate shaking. Fifteen cultures per batch were plated directly onto LB-rifampicin plates, and the remaining three were plated to enumerate the numbers of total cells.
Calculation of Mutation Rates
When calculating mutation rates, it is necessary to distinguish between the frequencies of mutants and those of mutations because each early exponential-phase mutation gives rise to several mutants. Furthermore, determination of mutation rates from information about the frequencies of mutants requires mathematical models of all the relevant mutational processes. Standard models for mutations occurring in the exponential phase predict that mutants follow a Luria-Delbrück (LD) distribution (Luria and Delbrück 1943
), whereas models for stationary-phase and postplating mutations predict a Poisson distribution. As our procedures yielded cultures that were predominantly, but not exclusively, in the exponential phase, we expected that the mutants followed some particular combined Poisson-LD distribution. The distribution parameters, m, the overall mutation rate and
, the proportion of the mutations that occurred in the stationary phase and after plating, were estimated for the given batches from the data (to estimate the latter with sufficient accuracy, batches of 4570 cultures were required).
Data were analyzed using a program made available by P. Gerrish (ftp-t10.lanl.gov). This program calculates the likelihood of distributions based on different values of . Because few postexponential-phase mutations were expected, we assumed that mutations followed a pure LD distribution (
= 0), unless the best-fitting combined Poisson-LD distribution described the observed distribution much better, as indicated by a likelihood 10-fold greater than that of the pure LD distribution. When the pure LD distribution was rejected, we employed the value of
that yielded the maximum likelihood. Because the estimates of
for strains containing the same lacZ mutation were similar, we grouped the strains by mutation type and produced one estimate for each group by averaging those derived from two different strains.
Estimates of overall (LD + Poisson) mutations per culture for each batch were generated by a maximum-likelihood method. Batch mutation rates were used in the following formula to estimate the mutation rate of each strain:
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Estimates of exponential-phase (LD) mutation rates and their confidence intervals were derived using the same procedures. Estimates of LD mutations per culture for each batch were generated by determining the value of this parameter that maximized likelihood, using the value of chosen for that strain.
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Results |
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Effects of Growth Phase and Mutation Type
The majority of A·T G·C mutations, but few if any of T·A
G·C mutations, occur after the exponential phase:
, the frequency of mutations arising postexponentially, is 0.52 for A·T
G·C mutations and 0.00 for T·A
G·C mutations. For strains reverting by A·T
G·C, the likelihood is maximized at
= 0.51 for a batch of 70 cultures of strain TT22912 and at
= 0.53 for a batch of 69 cultures of strain TT22911 (fig. 2
);
was 0 for strains reverting by all other mutations tested. The combined Poisson-LD distributions based on these
values fit the data much better than the pure LD distributions, with likelihood ratios exceeding 22. Conversely, the pure LD distributions fit the data well for strains reverting by T·A
G·C, yielding an estimate for
of 0 for both a batch of 70 cultures of strain TT22907 and a batch of 48 cultures of strain TT22910. In the former case, the pure LD distribution had the highest likelihood, but in the latter,
was set to 0 because the likelihood of the optimum combined distribution exceeded that of the pure distribution by only a very small amount. To investigate the reliability of our procedures for estimating
, we analyzed the distribution of revertant colonies arising on the fourth through the eighth day of plate incubation, which most likely reflect postplating mutations; as expected, the pure Poisson distribution (
= 1.00) best fit these data.
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Discussion |
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The higher reversion rates of alleles inserted at the envZ locus could reflect either a genome-wide increase in mutation rates caused by the inactivation of envZ or a locally elevated mutation rate at envZ. These alternatives were distinguished by determining the rates of streptomycin- and rifampicin-resistance mutations, which arise via mutations within the rpsL and the rpoB genes, respectively (Jin and Gross 1988
; Lisitsyn, Monastyrskaya, and Sverdlov 1988
; Timms et al. 1992
; Ito et al. 1994
), in each of the lacZ strains. Neither of the mutations conferring antibiotic resistance occurred at a significantly higher rate in strains harboring inserts at the envZ locus (table 4
), although power calculations indicate differences equal to or greater than 50% and 30%, respectively, which would have been significant at the 5% level. Thus, the increase in reversion rates in these strains was specific to the inserts at envZ.
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Whereas mutation rates are influenced by the orientation of a gene (Fijalkowska et al. 1998
), this does not contribute to the variation in mutation rates observed in this study. At each locus, the orientation of the inserted constructs, the disrupted gene, and the leading-strand replication all coincide. Although it is also possible that the increased mutation rates observed in the inserts at the envZ locus were caused by elevated transcription from the disrupted gene's promoter, this hypothesis is inconsistent with other data. Transcription increases the rate of this A·T
G·C reversion over twofold more than that of the T·A
G·C reversion (unpublished data). However, the increased rates observed for the envZ inserts follow a different pattern, with the former being augmented only about one-third as much as the latter.
Although we detected no systematic increase in mutation rates with distance from the replication origin, the frequency of synonymous substitutions among enterobacterial genes, as determined from sequence comparisons, increases with distance (Sharp et al. 1989
). This disparity could be the result of the differences in mutation rates under certain conditions, such as prolonged starvation or anaerobiosis, which might be evolutionarily relevant but not tested in our experiments. Alternatively, the differences in the inherent properties of the genes included in the substitution-rate comparisons might underlie the variation in these rates. The analysis of Sharp et al. (1989)
controlled for the effects of codon usage bias, thereby eliminating this factor as the cause of the variation in substitution rates with chromosomal position; however, genes closer to the origin may more often possess other properties associated with more stringent selective constraints or lower mutation rates.
Based on our estimates for , T·A
G·C transversions are more frequent than A·T
G·C transitions during the exponential phase, but less frequent in other phases. In the exponential phase, chromosomal T·A
G·C transversions occur approximately twofold more frequently than A·T
G·C transitions, in agreement with the mutation rates determined using plasmids (MacKay, Han, and Samson 1994
) and with the analyses of spontaneous mutational spectra (Hutchinson 1996
). Because in mismatch-repairdeficient cells, T·A
G·C transversions are generated at a lower frequency than are A·T
G·C transitions (Fujii et al. 1999
), these results suggest either that mismatch repair is more efficient at correcting the mismatches that lead to this transition or that DNA repair creates more T·A
G·C than A·T
G·C mutations.
Although half of the A·T G·C transitions detected in this study were attributed to postplating mutations, none of the T·A
G·C transversions were. The strains reverting by A·T
G·C transitions have higher postplating reversion rates because they are leakier, that is, they produce more wild-type lacZ proteins via mis-transcription or mis-translation (Cupples and Miller 1988
; Andersson, Slechta, and Roth 1998
). In contrast to these results, postplating Tcoding strand·A
Gcoding strand·C mutations were detected using a hisG428 reversion assay; however, these arose on plates lacking scavengers after prolonged incubation (Prival and Cebula 1992
).
The spontaneous mutation rates that we estimated for the lacZ reversions on the S. enterica chromosome are lower than those previously reported for the same alleles. Reversion rates on an E. coli episome are approximately 10-fold higher than those observed in this study (MacKay, Han, and Samson 1994
), and the reversion rate of the A·T
G·C transversion on the E. coli chromosome is 100-fold higher (Fijalkowska et al. 1998
). The differences are consistent with data reporting higher mutation rates in nutrient-rich media (Smith 1992
) and lower mutation rates in media that enable cells to skip the mutagenic steps of glycolysis (Lee and Cerami 1990
). The low mutation rates that we observed are not the result of plating high numbers of Lac- cells because the average number of colonies formed on minimal lactose plates by lacZ+ revertants plated in the presence or absence of 1010 Lac- cells was similar (36.2 vs. 33.6 per plate, respectively; difference was not significant by Student's t test: t = 1.30, P = 0.23, df = 8).
The volume of cultures that we employed in these studies permitted the detection of frequencies of mutants well below the threshold of other protocols. In addition, our methods yielded more accurate mutation rates by providing the following advantages: (1) statistical power was enhanced by using a maximum-likelihoodestimation procedure (Stewart 1994
); (2) the confounding effects of postexponential-phase mutations on the estimates of exponential-phase mutation rates were removed by partitioning the mutations into LD and Poisson fractions; and (3) spurious significant differences, which can arise, for example, when different strains have different carrying capacities, were avoided by the use of multiple batches to estimate the mutation rate of each strain and by enumerating the total cells.
Applying an experimental approach, we have shown that both the chromosomal location and the type of mutation affect the rates of spontaneous point mutations. However, a general increase in mutation rate with distance from the origin of replication, as suggested by sequence divergence of homologous genes in E. coli and S. enterica, is not supported by our results. Variation in mutation rates among sites can also be influenced by other factors, such as frequency of transcription and direction of DNA replication (Beletskii and Bhagwat 1996, 1998
; Francino and Ochman 1997
); therefore, future work will exploit this system to establish the effects of additional factors on the rates of different point mutations.
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Acknowledgements |
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Footnotes |
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Keywords: chromosomal location
molecular evolution
Salmonella enterica
spontaneous mutations
base substitutions
Address for correspondence and reprints: Howard Ochman, Department of Ecology and Evolutionary Biology, 310 Biosciences West, University of Arizona, Tucson, Arizona 85721. hochman{at}email.arizona.edu
.
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