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Amplification–Mutagenesis: Evidence That ‘‘Directed’’ Adaptive Mutation and General Hypermutability Result from Growth with a Selected Gene Amplification

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Amplification–Mutagenesis: Evidence That ‘‘Directed’’ Adaptive Mutation and General Hypermutability Result from Growth with a Selected Gene Amplification Amplification–mutagenesis: Evidence that ‘‘directed’’ adaptive mutation and general hypermutability result from growth with a selected gene amplification Heather Hendrickson*†, E. Susan Slechta*, Ulfar Bergthorsson*, Dan I. Andersson‡, and John R. Roth*§ *Department of Biology, University of Utah, Salt Lake City, UT 84112; and ‡Swedish Institute for Infectious Disease Control, S 17182 Solna, Sweden Communicated by Nancy Kleckner, Harvard University, Cambridge, MA, December 18, 2001 (received for review September 9, 2001) When a particular lac mutant of Escherichia coli starves in the presence We have suggested (9) that apparently directed mutation of lactose, nongrowing cells appear to direct mutations preferentially could be explained if the leaky lac allele used in this experiment to sites that allow growth (adaptive mutation). This observation were amplified so as to allow slow growth of a clone on lactose. suggested that growth limitation stimulates mutability. Evidence is Reversion would then occur when the growing clone has accu- provided here that this behavior is actually caused by a standard mulated enough mutant lac copies. What appears to be a Darwinian process in which natural selection acts in three sequential directed single step mutation in a nongrowing cell can result steps. First, growth limitation favors growth of a subpopulation with from selection acting on growing cells within a colony. The ؉ an amplification of the mutant lac gene; next, it favors cells with a lac required growth will not be evident in the lawn between colonies, revertant allele within the amplified array. Finally, it favors loss but can be revealed by appropriate analysis of developing ؉ of mutant copies until a stable haploid lac revertant arises and colonies (9). overgrows the colony. By increasing the lac copy number, selection Evidence is presented that amplification is essential for Lacϩ enhances the likelihood of reversion within each developing clone. reversion in the Cairns system and that associated general This sequence of events appears to direct mutations to useful sites. mutability is an unavoidable side effect of growth with a selected General mutagenesis is a side-effect of growth with an amplification gene amplification. These results suggest that several previously ؅ (SOS induction). The F plasmid, which carries lac, contributes by proposed attributes of the Cairns system are incorrect; specifi- stimulating gene duplication and amplification. Selective stress has cally, (i) stress does not direct mutation to valuable sites (1, 6), no direct effect on mutation rate or target specificity, but acts to favor (ii) stress has no direct effect on the general mutation rate (7), a succession of cell types with progressively improved growth on (iii) reversion does not occur in stationary-phase cells (2, 5, 10), lactose. The sequence of events—amplification, mutation, segrega- (iv) there is no reason to suppose that mutation reflects error- tion—may help to explain both the origins of some cancers and the prone recombination (5, 11), and (v) reversion is actually initi- evolution of new genes under selection. ated before selection by formation of duplication-bearing cells during pregrowth; these cells, when placed on selection medium, selection system developed by John Cairns (1, 2) provided grow and evolve by a conventional Darwinian process. Aevidence that bacteria might sense growth-limiting stress and The amplification–mutagenesis model suggests a general direct mutations to sites that enhance growth (adaptive mutation). mechanism by which selection may enhance the rate of genetic The behavior of Cairns’ system does not contradict classical dem- change during growth under strong selection without altering the onstrations that some mutations arise independent of selection (3, rate or specificity of mutation. The model may be applicable to 4). However, the behavior raises the possibility that another fraction origin of some cancers and to the evolution of new genes. of mutations might be induced or even directed by growth limita- tion. Stress-induced mutations (general or directed) would contra- Materials and Methods dict the neo-Darwinian view that agents of selection play no role in Strains. All strains are derived from E. coli K-12; the tester strain causing mutations, but affect only the relative reproductive success FC40 (ϭTR7178) and scavenger FC29 (ϭTR7177) were pro- of organisms with different genotypes. vided by Patricia Foster (2). A Tn10 insertion near his was Experiments supporting directed mutation used an Esche- obtained from Sidney Kushner (SK2651) and transduced into richia coli strain with a lac deletion on the chromosome and a ϩ Ј strain FC40 (producing TT23242). Insertion nadB210::Tn10 was revertible lacZ ( 1) frameshift mutation on an F128 plasmid (2). Ϫ8 introduced into a lac deletion mutant to make the tetracycline- During growth, the lac point mutation reverts at a rate of 10 R 8 Ϫ resistant (Tc ) scavenger TT23241. When this scavenger was per cell per division. When 10 of these Lac cells are starved used, nicotinic acid was added to the selection plate. By P22- in the presence of lactose, about 100 revertant colonies accu- mediated crosses in Salmonella enterica,Tn10 insertions were mulate over a period of 6 days, during which the plated popu- Ј added to plasmid F128 (from strain FC40) and their positions lation does not grow and does not accumulate unselected were mapped; plasmids were returned to a plasmid-free E. coli mutations (5). This behavior suggested that selection directs strain isogenic with FC40 (TT23250). mutations to growth-promoting sites (2, 6). Subsequent studies showed that lacϩ revertants have a 10- to Reversion Experiments. Testers (FC40) and scavenger cells (FC29 100-fold higher probability of carrying an unselected mutation or TT23241) were pregrown in NCE medium (34) containing than do unselected cells or starved nonrevertant cells (7). Thus, glycerol (0.2%), thiamin (50 ␮M), and (for TT23241) nicotinic the revertants (but not the starved population as a whole) were generally mutagenized in the process of reversion. Once isolated, the revertants show a normal mutation rate. This finding sug- Abbreviations: Tc, tetracycline; CTc, chlortetracycline; TetA, tetracycline-inducible gested that stress induces a temporary general hypermutable exporter. state. This general mutagenesis requires induction of the SOS †Present address: Department of Biology, University of Pittsburgh, Pittsburgh, PA 15260. system of DNA repair with its error-prone polymerase DinB (8). §To whom reprint requests should be addressed. E-mail [email protected]. Induction of SOS mutagenesis was attributed to a beneficial The publication costs of this article were defrayed in part by page charge payment. This regulatory mechanism that evolved under selection because it article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. facilitates genetic adaptation. §1734 solely to indicate this fact. 2164–2169 ͉ PNAS ͉ February 19, 2002 ͉ vol. 99 ͉ no. 4 www.pnas.org͞cgi͞doi͞10.1073͞pnas.032680899 Downloaded by guest on September 27, 2021 acid (100 ␮M); cells were pelleted and resuspended in sterile saline. Each reversion plate [NCE, 0.2% lactose, 25 mg 5-bromo- 4-chloro-3-indolyl-␤-D-galactoside (X-Gal)] was seeded with about 2 ϫ 109 scavenger cells and incubated for 24 h to remove contaminating carbon sources before 108 tester cells were plated. For Fig. 4A, cells were pregrown with tetracycline (Tc; 10 ␮g/ml) to induce the tetA (resistance) gene before plating on minimal lactose medium containing Tc. This preinduction proved unnec- essary and was not included in the experiment in Fig. 4B. Testing Stability of Lac؉ Phenotypes. Revertants in Fig. 2 were identified (some with a dissecting microscope) as new colonies not present on the previous day. Each colony was removed from the selection plate with a plug of agar. Cells were suspended and stored at 4°C. Samples were diluted and plated on nutrient broth (Difco) plates containing rifampicin (50 mg/liter) to eliminate scavengers and X-Gal (40 mg/liter) to visualize lac phenotypes; this medium and X-Gal concentration facilitate visualization of the sectored colonies that indicate an unstable Lacϩ phenotype. Testing the Clonal Relatedness of Stable and Unstable Lac؉ Cells. The S R Fig. 1. Amplification–mutagenesis model for reversion during growth un- two testers FC40 (TR7178) (Tc ) and TT23242 (Tc ) were der selection and associated hypermutability. (Left) The basic amplification 8 plated at a 30:1 ratio (total 10 ) on plates with the scavenger model without general mutagenesis. (Right) Induction of SOS mutagenesis is R strain TT23241 (Tc ) as described above. Of 500 Day 6 rever- a side effect of growth with an unstable amplification. tants, 7 contained a substantial number of TcR,Lacϩ cells. These 7 suspensions were diluted and plated to determine the Lacϩ stability and genetic background of individual cells. overgrow the clone, and thus ultimately predominate in the mature revertant colony. Testing Amplifications by Pulsed-Field Gel Electrophoresis. Unstable In this process, the probability of a reversion event increases Lacϩ cells were mated with a recA recipient (TT23249) and Proϩ with the number of replicating lac targets added to the clone. exconjugants were selected. DNA was prepared (12) and cut The process is accelerated because the lac gene is on an FЈ with either BlnIorSfiI before pulse-field gel electrophoresis. plasmid, which stimulates
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