Plasmid copy number underlies adaptive mutability in bacteria

Abstract

The origin of mutations under selection has been intensively studied using the Cairns-Foster system, in which cells of an Escherichia coli lac mutant are plated on lactose and give rise to 100 Lac+ revertants over several days. These revertants have been attributed variously to stress-induced mutagenesis of nongrowing cells or to selective improvement of preexisting weakly Lac+ cells with no mutagenesis. Most revertant colonies (90%) contain stably Lac+ cells, while others (10%) contain cells with an unstable amplification of the leaky mutant lac allele. Evidence is presented that both stable and unstable Lac+ revertant colonies are initiated by preexisting cells with multiple copies of the F'lac plasmid, which carries the mutant lac allele. The tetracycline analog anhydrotetracycline (AnTc) inhibits growth of cells with multiple copies of the tetA gene. Populations with tetA on their F'lac plasmid include rare cells with an elevated plasmid copy number and multiple copies of both the tetA and lac genes. Pregrowth of such populations with AnTc reduces the number of cells with multiple F'lac copies and consequently the number of Lac+ colonies appearing under selection. Revertant yield is restored rapidly by a few generations of growth without AnTc. We suggest that preexisting cells with multiple F'lac copies divide very little under selection but have enough energy to replicate their F'lac plasmids repeatedly until reversion initiates a stable Lac+ colony. Preexisting cells whose high-copy plasmid includes an internal lac duplication grow under selection and produce an unstable Lac+ colony. In this model, all revertant colonies are initiated by preexisting cells and cannot be stress induced.

Keywords: adaptive mutation; gene amplification; mutation under selection; selection; stress-induced mutagenesis.

Figure 1

The process of gene amplification by tandem duplication. The sequence of events is essentially a random walk with two boundary conditions—amplification initiation is limited by a low initial duplication rate and expansion is limited by increasing fitness cost and copy loss rate in higher amplifications. Only single-copy changes are depicted, but exchanges between more distant copies can cause larger copy-number shifts. Each amplification and loss step (red arrows) depends on recombination between a pair of long repeats. These recombination-dependent events are expected to occur at the same rate, which is higher than that of RecA-independent initial duplication formation (black arrow). The whole system (frequency of all copy-number variants) comes to a steady state as a unit due to the high loss rate and increasing fitness cost (S. Maisnier-Patin, M. Savageau, and J. R. Roth, unpublished results; Reams et al. 2010). Fitness cost can be offset (and steady-state frequencies can rise) when growth is stimulated by the increased copy number of a gene within the amplified region.

Figure 2

Inhibition by AnTc of growth of a strain with high tetA copy number. The strain used (TT26900) is a derivative of the standard reversion tester FC40 (TR7178) carrying a nonconjugative R1 plasmid having a tetracycline resistance cassette cloned from Tn10. The cassette includes the tetR tetA genes and their regulatory sequences. Cells were grown in NCE glycerol (0.1%) (open circles), with anhydrotetracycline (AnTc) added at 216 nM where indicated (solid circles). Samples were withdrawn, diluted, and plated on LB medium.

Figure 3

Effect of AnTc on growth of strains with Tn10dTc on the F′ plasmid. The strain without Tn10 (left) is the standard tester strain (TR7178). Strains with Tn10dTc (right) have an insertion in lac (TT26933), in the yebB gene, far from lac (TT26932), or at both positions (TT26935). Growth was measured in minimal NCE medium with glycerol using an automated plate reader.

Figure 4

Competition between strains with and without Tn10dTc. To better observe effects of AnTc on growth, pairs of strains with and without a Tn10dTc insertion in F′lac were mixed and grown together in minimal glycerol medium with or without AnTc. The cultures were grown for two passages and the ratio of cells with and without Tn10dTc was scored by plating diluted cultures on rich medium with and without tetracycline. Strains: parent with no Tn10 (TR7178); one insertion, yebB::Tn10dTc (TT26932); one insertion of Tn10dTc in lacA (TT26933); and two Tn10dTc insertions, one in lacA and the other in yebB (TT26935).

Figure 5

Concentrations of AnTc in pregrowth medium that reduce revertant yield. Reversion was tested as described in Materials and Methods, using strain TT26327 with a Tn10dTc insertion in the lacA gene of plasmid F′₁₂₈lac. The revertant colony number is presented per viable cell of the tester strain plated.

Figure 6

Effect of AnTc on revertant yield requires Tn10dTc and exposure during pregrowth. The strain without Tn10dTc (TR7178) is the original tester strain of Cairns and Foster (1991) and the strain with Tn10dTc in lac (TT26327) carried an insertion at the distal end of the lac operon of the F′₁₂₈ plasmid. Cells were pregrown for 20 generations in minimal glycerol with and without AnTc (216 nM) and plated on lactose medium with or without AnTc.

Figure 7

Effect of Tn10dTc position on sensitivity of reversion to AnTc. Cells were pregrown to stationary phase in glycerol with or without AnTc (216 nM) and then washed and plated on selective lactose plates with or without AnTc (216 nM). The strain at the top carries Tn10dTc in the chromosomal btuR gene (TT26328). The strain at the bottom carries Tn10dTc in the yebB gene (TT26323). Revertant number is expressed relative to viable cells plated. This normalization understates the effect of pregrowth with AnTc, by including only cells that have survived the effects of AnTc and eliminating those multicopy cells lost during pregrowth.

Figure 8

Insertions of Tn10dTc at points far from lac on the F′ plasmid allowed AnTc to inhibit revertant yield. A series of tester strains with Tn10dTc inserted at various points in the F′lac plasmid were compared for their yield of Lac⁺ revertants following pregrowth with and without AnTc. These strains are described in Table 1. Revertant yields following pregrowth without and with AnTc are described in the top left and top right, respectively. The map positions of the Tn10dTc insertions are described in the bottom left.

Figure 9

Effect of Tn10dTc on lac reversion under selection. Strains with tetA in plasmid R1 are compared to strains with one and two copies of Tn10dTc in F′lac. The standard tester strain (TR7178) with F′lac but no Tn10 is at top left. The strain at top right (TT26900) carries plasmid R1 with a cloned tetR tetA cassette derived from Tn10. The strain at bottom left has Tn10dTc inserted in lacA and is the same strain described in Figure 6 and Figure 8. The strain at bottom right (TT26905) has two tetA genes, one within insertion yebB::Tn10dTc far from lac and the other within a derivative of zzf-1881::Tn10dTc inserted in inverse order ∼5 kb away from lac—this derivative has a Cm resistance marker and a GFP gene (Sun et al. 2009).

Figure 10

The effect of AnTc on revertant yield is reversed by growth without AnTc. Preexisting cells responsible for initiating revertants appearing under selection are thought to have multiple copies of the F′lac plasmid (including the tetA gene). The stability of such cells was tested using a strain with Tn10dTc in lacA (TT26327), a strain with Tn10dTc inserted into the chromosomal btuR gene (TT26328), and a strain with no Tn10 (TR7178). All strains were pregrown for 7 generations without (top left) or with (top right) AnTc and then were used in a standard reversion experiment. Immediately following growth with AnTc all three mutants were diluted 10-fold into fresh medium lacking AnTc, grown for 3 generations, and retested for reversion (bottom left). The same strain was passaged six times by 10-fold dilution into glycerol medium containing AnTc (20 additional generations with AnTc) (bottom right).