Letter to the Editor

Adaptive Mutation Requires No Mutagenesis—Only Growth Under Selection: A Response

Corresponding author: John R. Roth, 1 Shields Ave., University of California, Davis, CA 95616., jrroth{at}ucdavis.edu (E-mail)

IN a bacterial system devised by CAIRNS and FOSTER 1991 , a lac mutant population, starved in the presence of lactose, gives rise to 100 revertant colonies over 6 days. In this time, the plated population is not growing and is not experiencing general mutagenesis. Three models have been proposed to explain this. The directed mutation model (DMM) suggests that stress induces mutagenesis that is focused on the relevant target (lac, or the F' plasmid that carries it) to the exclusion of the chromosome at large (CAIRNS et al. 1988 ; CAIRNS and FOSTER 1991 ; FOSTER and CAIRNS 1992 ). The hypermutable state model (HSM) suggests that stress induces genome-wide mutagenesis in a subset of the population (10⁵ cells); this generates Lac⁺ revertants but kills the rest of the mutagenized population (HALL 1990 ; TORKELSON et al. 1997 ). According to the HSM, mutation appears directed because only Lac⁺ revertants survive mutagenesis. We have proposed (see below) the amplification mutagenesis model (AMM), in which growth under selection increases revertant number with no required change in the rate or target specificity of mutation (ANDERSSON et al. 1998 ; HENDRICKSON et al. 2002 ; SLECHTA et al. 2003 ).

The article under discussion (ROTH et al. 2003 ) examines some quantitative predictions of the HSM and finds that this model requires an implausibly high intensity of genome-wide mutagenesis—vastly higher than that estimated experimentally (ROSCHE and FOSTER 1999 ; SLECHTA et al. 2002B ). If realized, this mutation rate would add so many lethal mutations that one could not recover the number of lac mutations that are observed. We argued that the behavior of the Cairns-Foster system must be explainable by some means other than the HSM.

The letter from CAIRNS and FOSTER 2003 (this issue) suggests that our calculations are in error because we extrapolated from rates observed or inferred for genes on an F' plasmid to predict the number of mutations that would be caused in the chromosome. Their argument is based on three assumptions: (1) that mutation rates are 100-fold lower in the chromosome than on the F', (2) that stress induces mutagenesis regardless of where lac is located, and (3) that reversion is not detectably enhanced when lac is in the chromosome, simply because the basal reversion rate of a lac allele at that position is too low. We do not accept these assumptions. We have moved the lac allele used in the Cairns experiment to 34 different sites in the chromosome and compared unselected reversion rates to that of the same allele on the F' plasmid. At all chromosomal sites the unselected reversion rates clustered around the 10^-8 value found for lac on F'₁₂₈ (SLECHTA et al. 2002A ; S. SLECHTA, unpublished data). The chromosomal reversion rates were not affected by the presence of F'₁₂₈ (SLECHTA et al. 2003 ). The 100-fold lower mutation rate suggested for chromosomal lac in the Cairns-Foster letter would be reasonable for a typical chromosomal frameshift mutation (BULL et al. 2001 ), but the lac allele used in this system is not typical. That allele has a constellation of three mutations—an I^Q promoter, a +1 frameshift in lacI, and a deletion that fuses the lacI and lacZ genes (CAIRNS and FOSTER 1991 ). This combination provides a leaky Lac^- phenotype that can be corrected by any -1 mutation (or other mutation having the same effect on reading frame) in 100 bp (about one-tenth of the lacI gene sequence). This allele would therefore be expected to revert at 100 times the rate of a typical +1 frameshift, and our measurements suggest that it does. There seems to be no effect of gene position on reversion in the absence of selection.

When selection is imposed, a position effect is seen. When lac is on the F' plasmid, 100 revertants accumulate over 6 days and these revertants show an average 20- to 50-fold increase in associated mutations distributed genome-wide (TORKELSON et al. 1997 ; ROSCHE and FOSTER 1999 ; SLECHTA et al. 2002A ). In contrast, when selection is applied to strains with lac in the chromosome, very few revertants appear and we find that these few revertants show no increase in associated mutations (SLECHTA et al. 2002A ). We conclude that selection enhances reversion (and causes associated mutations) only when lac is on a conjugative plasmid. The HSM model does not address this position effect, but assumes that general mutagenesis is induced in a subset of the stressed population and is responsible for the lac revertants. We ignored this shortfall of HSM and simply tested the quantitative implications of that model as it has been enunciated (ROSENBERG 2001 ).

The assumptions underlying the above critique appear to be based on reversion tests of lac at a single chromosomal site (ROSCHE and FOSTER 2000 ). There the reversion rate (10^-10) was lower than that of lac on F'₁₂₈ (10^-8) and the very few recovered lac revertants did show evidence of general mutagenesis. We have not seen unselected mutations associated with reversion of a chromosomal lac allele either in an Escherichia coli strain like that used (ROSCHE and FOSTER 2000 ) or in Salmonella strains with lac at chromosomal sites. We do not know why the particular strain tested by Rosche and Foster showed a lower reversion rate or associated mutations, but we suggest that problems may have arisen in the course of genetically transferring the triply mutant lac allele from the F' plasmid to the chromosome; this procedure sometimes generates a duplication of the chromosomal lac-din region, because a duplication join point is inherent in the structure of F'₁₂₈ (KOFOID et al. 2003 ). If this occurred, the behavior of their strain may be explainable by the amplification model (see below).

We agree with Cairns and Foster that our cost estimate ignores any contribution to lac reversion by the nonhypermutable majority of plated cells. We ignored it because it is ignored by the HSM (which we were testing). However, we did consider this population as described by HSM in showing (ROTH et al. 2003 , Table 1, line 11) that 10⁸ cells with the normal mutation rate are expected to produce <1 Lac⁺ revertant under the HSM model. We also considered the predicted result if that population experienced the fourfold increase estimated by BULL et al. 2001 ; then the HSM predicts four revertants. Further increasing general mutagenesis of the whole population (ROTH et al. 2003 , Table 1, lines 9 and 10), can ultimately predict the observed 100 mutants, but eliminates the apparent directedness of mutation that is a hallmark of this system (i.e., the population at large would show as many associated mutations as do the Lac⁺ revertants).

We considered the demonstration by ROSCHE and FOSTER 1999 that the majority of lac revertants (90%) experience little or no general mutagenesis while 10% experience a 200-fold increase (an average 20-fold increase). We agreed with their conclusion and pointed out that the low average rate they estimated is in stark contrast to the 10⁵-fold increase required by HSM, leading to our conclusion that HSM is unlikely to explain the observed lac revertants. Further support for this conclusion is the observation that revertant yield decreases only slightly when general mutagenesis is eliminated by a dinB or lexA^Ind mutation (MCKENZIE et al. 2000 ; SLECHTA et al. 2002B , SLECHTA et al. 2003 ; TOMPKINS et al. 2003 ). We imagine that Cairns and Foster would agree with us—general mutagenesis is neither necessary nor sufficient to explain selection-enhanced revertant frequency in their system.

If general mutagenesis as posited by HSM is set aside as the cause of reversion, how then can one explain the lac⁺ revertants that arise under selection? The DMM (FOSTER 1993 ) posits that selection induces mutagenesis focused on lac (or on the F' plasmid that carries it). Such directed mutagenesis could certainly explain reversion without costly associated mutations. This model was initially supported by experiments in which starvation of the lac mutant (on F'₁₂₈) had very little effect on reversion of chromosomal tetA frameshifts in the nonrevertant parent population (BULL et al. 2001 ), but caused a striking increase in unselected revertants of a tetA frameshift inserted (within Tn10) on F' lac (FOSTER 1997 ). However, the Tn10 element used in this F' experiment is actually inserted very close to lac in the mhpC gene (KOFOID et al. 2003 ). A Tn10 insertion in this gene has been shown to co-amplify with lac during selection for improved growth on lactose (GODOY and FOX 2000 ); insertions far from lac on F'₁₂₈ do not appear to co-amplify (HENDRICKSON et al. 2002 ). Thus the high reversion rate seen for tetA on F'₁₂₈ during lactose selection (like that of the lac mutation) is likely to reflect selected amplification of the target site (tetA) with lac (see below) rather than mutagenesis directed to the plasmid.

We suggest that the Cairns-Foster phenomenology requires no induced mutagenesis, directed or general. The AMM proposes that rare preexisting cells with a lac duplication grow slowly when placed under selection and improve their growth by further lac amplification within each developing colony. Ultimately there are so many copies of lac (within colonies) that reversion can occur without any increase in the underlying (per base pair) mutation rate (ANDERSSON et al. 1998 ; HENDRICKSON et al. 2002 ). Selection appears to direct mutations to lac because this gene is amplified during growth under selection and because only the lac⁺ revertant allele is maintained during subsequent selected loss of the many (now deleterious) copies of the mutant lac allele. This process is enhanced for lac on F^'₁₂₈ because the transfer origin of this plasmid makes DNA ends that stimulate duplication, amplification, and segregation (GALITSKI and ROTH 1995 ; SLECHTA et al. 2002A ). We have recently presented evidence that the genome-wide general mutagenesis experienced by 10% of lac revertant clones (ROSCHE and FOSTER 1999 ) occurs because these few clones include within their amplified lac region the nearby dinB gene for an error-prone polymerase (KOFOID et al. 2003 ; SLECHTA et al. 2003 ); the majority (90%) of revertants arise without mutagenesis by amplifying lac alone. Thus the interesting predictions of Ninio do not seem to be required here (NINIO 1991 ). We submit that selection increases the number of lac revertants primarily by increasing the number of mutational targets (AMM), not by increasing the general mutation rate (as proposed by HSM) and not by directing mutation (as proposed by DMM).

² Present address: Microbiology Section (DBS), University of California, Davis, CA 95616.

LITERATURE CITED

ANDERSSON, D. I., E. S. SLECHTA, and J. R. ROTH, 1998  Evidence that gene amplification underlies adaptive mutability of the bacterial lac operon. Science 282:1133-1135.[Abstract/Free Full Text]

BULL, H., M.-J. LOMBARDO, and S. ROSENBERG, 2001  Stationary-phase mutation in the bacterial chromosome: recombination protein and DNA polymerase IV dependence. Proc. Natl. Acad. Sci. USA 98:8334-8341.[Abstract/Free Full Text]

CAIRNS, J. and P. L. FOSTER, 1991  Adaptive reversion of a frameshift mutation in Escherichia coli.. Genetics 128:695-701.[Abstract]

CAIRNS, J. and P. L. FOSTER, 2003  The risk of lethals for hypermutating bacteria in stationary phase. Genetics 165:2317-2318.[Free Full Text]

CAIRNS, J., J. OVERBAUGH, and S. MILLER, 1988  The origin of mutants. Nature 335:142-145.[Medline]

FOSTER, P. L., 1993  Adaptive mutation: the uses of adversity. Annu. Rev. Microbiol. 47:467-504.[Medline]

FOSTER, P. L., 1997  Nonadaptive mutations occur on the F' episome during adaptive mutation conditions in Escherichia coli.. J. Bacteriol. 179:1550-1554.[Abstract/Free Full Text]

FOSTER, P. L. and J. CAIRNS, 1992  Mechanisms of directed mutation. Genetics 131:783-789.[Abstract]

GALITSKI, T. and J. R. ROTH, 1995  Evidence that F plasmid transfer replication underlies apparent adaptive mutation. Science 268:421-423.[Abstract/Free Full Text]

GODOY, V. G. and M. S. FOX, 2000  Transposon stability and a role for conjugational transfer in adaptive mutability. Proc. Natl. Acad. Sci. USA 97:7393-7398.[Abstract/Free Full Text]

HALL, B. G., 1990  Spontaneous point mutations that occur more often when advantageous than when neutral. Genetics 126:5-16.[Abstract]

HENDRICKSON, H., E. S. SLECHTA, U. BERGTHORSSON, D. I. ANDERSSON, and J. R. ROTH, 2002  Amplification-mutagenesis: evidence that "directed" adaptive mutation and general hypermutability result from growth with a selected gene amplification. Proc. Natl. Acad. Sci. USA 99:2164-2169.[Abstract/Free Full Text]

KOFOID, E., U. BERGTHORSSON, E. S. SLECHTA, and J. R. ROTH, 2003  Formation of an F' plasmid by recombination between imperfectly repeated chromosomal Rep sequences: a closer look at an old friend (F'(128) pro lac). J. Bacteriol. 185:660-663.[Abstract/Free Full Text]

MCKENZIE, G. J., R. S. HARRIS, P. L. LEE, and S. M. ROSENBERG, 2000  The SOS response regulates adaptive mutation. Proc. Natl. Acad. Sci. USA 97:6646-6651.[Abstract/Free Full Text]

NINIO, J., 1991  Transient mutators: a semiquantitative analysis of the influence of translation and transcription errors on mutations rates. Genetics 129:957-962.[Abstract]

ROSCHE, W. A. and P. L. FOSTER, 1999  The role of transient hypermutators in adaptive mutation in Escherichia coli.. Proc. Natl. Acad. Sci. USA 96:6862-6867.[Abstract/Free Full Text]

ROSCHE, W. A. and P. L. FOSTER, 2000  Determining mutation rates in bacterial populations. Methods 20:4-17.[Medline]

ROSENBERG, S., 2001  Evolving responsively: adaptive mutation. Nat. Rev. Genet. 2:504-514.[Medline]

ROTH, J. R., E. KOFOID, F. P. ROTH, O. G. BERG, and J. SEGER et al., 2003  Regulating general mutation rates: examination of the hypermutable state model for Cairnsian adaptive mutation. Genetics 163:1483-1496.[Abstract/Free Full Text]

SLECHTA, E. S., J. HAROLD, D. I. ANDERSSON, and J. R. ROTH, 2002a  The effect of genomic position on reversion of a lac frameshift mutation (lacIZ33) during non-lethal selection (adaptive mutation). Mol. Microbiol. 44:1017-1032.[Medline]

SLECHTA, E. S., J. LIU, D. I. ANDERSSON, and J. R. ROTH, 2002b  Evidence that selected amplification of a bacterial lac frameshift allele stimulates Lac(+) reversion (adaptive mutation) with or wthout general hypermutability. Genetics 161:945-956.[Abstract/Free Full Text]

SLECHTA, E. S., K. L. BUNNY, E. KUGELBERG, E. KOFOID, and D. I. ANDERSSON et al., 2003  Adaptive mutation: General mutagenesis is not a programmed response to stress, but results from rare co-amplification of dinB with lac. Proc. Natl. Acad. Sci. USA 100:12847-12852.[Abstract/Free Full Text]

TOMPKINS, J. D., J. L. NELSON, J. C. HAZEL, S. L. LEUGERS, and J. D. STUMPF et al., 2003  Error-prone polymerase, DNA polymerase IV, is responsible for transient hypermutation during adaptive mutation in Escherichia coli. J. Bacteriol. 185:3469-3472.[Abstract/Free Full Text]

TORKELSON, J., R. S. HARRIS, M.-J. LOMBARDO, J. NAGENDRAN, and C. THULIN et al., 1997  Genome-wide hypermutation in a subpopulation of stationary-phase cells underlies recombination-dependent adaptive mutation. EMBO J. 16:3303-3311.[Medline]