Letter to the Editor

Response to John Cairns: The Contribution of Transiently Hypermutable Cells to Mutation in Stationary Phase

Corresponding author: Susan M. Rosenberg, Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Rm. S809A, Mail Stop 225, Houston, TX 77030-3498., smr{at}bcm.tmc.edu (E-mail)

IN his letter, John Cairns reiterates the model described in his Appendix (CAIRNS 1999 ) to ROSCHE and FOSTER 1999 and seems to be concerned that we have not adopted it as an explanation for our observations (BULL et al. 2000 ). His model is that most Lac⁺ adaptive mutants arise in one transiently mutating cell population while concurrently, secondary (unselected) mutations, and a smaller number of Lac⁺ adaptive mutants associated with them, arise in a different transiently hypermutating subpopulation. The two populations were proposed to produce mutations via different mechanisms, using different gene products (ROSCHE and FOSTER 1999 ).

In fact, we did not reject a multiple-population model (BULL et al. 2000 ). We favor the simpler model of a single transiently mutable subpopulation producing both Lac⁺ and secondary mutations (HALL 1990 ; TORKELSON et al. 1997 ) partly because the data that can be used to distinguish between the two models are very sparse such that other interpretations of those data remain possible. In the absence of more extensive data, a simpler model is more attractive.

We also favor the single subpopulation model because the data presented by BULL et al. 2000 imply a recombinational mechanism of chromosomal secondary mutation, as is found for all Lac⁺ adaptive mutation (HARRIS et al. 1994 ). This contradicts the proposal that one population produces most Lac⁺ recombinationally, but that chromosomal secondary mutations (and the Lac⁺ associated with them) arise in a separate subpopulation that is mutating via a different mechanism.

Cairns' principal criticism is the use of a Poisson distribution when individual events have widely different mutation rates. We did not calculate a Poisson distribution based on mutation frequencies that covered an 50-fold range. When TORKELSON et al. 1997 made this calculation, the very low frequency for mutation to fructose nonutilization (Fru^-) was omitted. We know now that the aberrantly low mutation frequency observed was caused by an inability of most Fru^- cells to utilize lactose (see FRAENKEL 1996 ), such that Fru^- cells Lac⁺ are counterselected. The remaining frequencies show a range of only fourfold. The appropriate numbers from TORKELSON et al. 1997 are 286 double mutants (Lac⁺ plus one secondary mutation) and 5 triple mutants (Lac⁺ plus two secondary mutations) among 42,000 Lac⁺ mutants. These numbers omit Experiment 1, Table 2 of TORKELSON et al. 1997 , because only 5-fluorocytosine (5-FC) resistance was scored in that experiment. Also omitted is the quadruple mutant (Lac⁺ plus three other mutations) and four of the double mutants, because these isolates have a stable mutator phenotype. Both the size of the subpopulation and the mutation rates within that population are unknown initially. We assume that an average mutation rate can be applied to the remaining targets. The mutation rate to Lac⁺ (4 Lac⁺ per 850 Tet^R or 4.7 x 10^-3; FOSTER 1997 ) can be seen to be comparable to that of the remaining targets (3 x 10^-3–0.7 x 10^-3 secondary mutants per Lac⁺; TORKELSON et al. 1997 ). A reasonable fit to a Poisson distribution can be obtained by using an aggregate mutation rate for the five targets other than lac of 0.007 mutations/cell/4 days (the length of time over which these mutants formed under starvation). At this mutation rate, we expect 292 Lac⁺ isolates to have one other mutation, and 1 to have two other mutations among 42,000 Lac⁺ isolates. (Compare this with 286 and 5 observed in the same two classes.) Given that the individual mutation rate to Lac⁺ would be about one-fifth of the aggregate mutation rate, the hypermutating subpopulation from which they arose would be 3 x 10⁷ cells (42,000 mutations per 4 days ÷ 0.0014 mutations per hypermutating cell per 4 days). With the 42,000 Lac⁺ being 10^-6 of all cells (4.2 x 10¹⁰), the frequency of hypermutating cells would be 7.1 x 10^-4 of the whole population. These numbers are revised from those estimated by TORKELSON et al. 1997 , in which we mistakenly applied the aggregate mutation rate (rather than one-fifth of the aggregate) to Lac⁺, and thereby arrived at a 10-fold smaller subpopulation and 2-fold higher mutation rate. The net conclusion does not differ.

We agree with Cairns that an excess of observed triple mutants relative to expected would argue in favor of multiple populations. However, on the basis of the current scant data, we cannot take the observed numbers as showing a significant deviation from the expectation, though further data might perhaps do so. Specifically, we are not persuaded that the 1 triple expected (per 292 doubles) deviates significantly from the 5 observed (per 286 doubles). This means that, on the basis of the data of Torkelson et al., we do not reject the simpler hypothesis of a single transiently hypermutating subpopulation giving rise to all Lac⁺ and the secondary mutations.

The data of ROSCHE and FOSTER 1999 cited by Cairns are very interesting. Although the relevant numbers in that work are larger than those discussed here, we feel that more are needed to discriminate between single- and multiple-subpopulation models. Because the simpler model of one subpopulation is also more harmonious with our observation of recombination-promoted mechanisms for both secondary (BULL et al. 2000 ) and all Lac⁺ mutations (HARRIS et al. 1994 ), it seems the more economical model at present. Investigating the mechanism of the unselected hypermutation (e.g., BULL et al. 2000 ) should be an effective tactic for addressing the key issue here: Is Lac⁺ adaptive mutation a process that generates only adaptive mutations, or is the observed, concurrent chromosomal hypermutation part of the same process?

¹ Present address: Department of Microbiology and Immunology, University of Saskatchewan, A231 Health Sciences Bldg., 107 Wiggins Rd., Saskatoon, Saskatchewan S7N 5E5, Canada.

ACKNOWLEDGMENTS

We are indebted to Russ Maurer for advice on the mathematics. This work is supported by National Institutes of Health grants R01-GM53158 and R01-AI43917.

Manuscript received June 8, 2000; Accepted for publication June 14, 2000.

LITERATURE CITED

BULL, H. J., G. J. MCKENZIE, P. J. HASTINGS, and S. M. ROSENBERG, 2000  Evidence that stationary-phase hypermutation in the Escherichia coli chromosome is promoted by recombination. Genetics 154:1427-1437[Abstract/Full Text].

CAIRNS, J., 1999  Appendix. Proc. Natl. Acad. Sci. USA 96:6866-6867.

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

FRAENKEL, D. G., 1996 Glycolysis, pp. 189–198 in Escherichia coli and Salmonella Cellular and Molecular Biology, Ed. 2, edited by F. C. NEIDHARDT, R. CURTISS III, J. L. INGRAHAM, E. C. C. LINN, K. B. LOW et al. ASM Press, Washington, DC.

HALL, B. G., 1990 Spontaneous point mutations that occur more often when advantageous than when neutral. 126: 5–16.

HARRIS, R. S., S. LONGERICH, and S. M. ROSENBERG, 1994  Recombination in adaptive mutation. Science 264:258-260[Medline].

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/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[Abstract/Full Text].

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