A mechanism of transposon-mediated directed mutation

Abstract

Directed mutation is a proposed process that allows mutations to occur at higher frequencies when they are beneficial. Until now, the existence of such a process has been controversial. Here we describe a novel mechanism of directed mutation mediated by the transposon, IS5 in Escherichia coli. crp deletion mutants mutate specifically to glycerol utilization (Glp(+)) at rates that are enhanced by glycerol or the loss of the glycerol repressor (GlpR), depressed by glucose or glpR overexpression, and RecA-independent. Of the four tandem GlpR binding sites (O1-O4) upstream of the glpFK operon, O4 specifically controls glpFK expression while O1 primarily controls mutation rate in a process mediated by IS5 hopping to a specific site on the E. coli chromosome upstream of the glpFK promoter. IS5 insertion into other gene activation sites is unaffected by the presence of glycerol or the loss of GlpR. The results establish an example of transposon-mediated directed mutation, identify the protein responsible and define the mechanism involved.

Figure 1. The appearance of Glp⁺ mutations in a crp genetic background

(A) The glpFK promoter region. The transcriptional initiation site (+1), the −10 and −35 hexamers, the ribosome binding site (RBS) and the start codon for glpF translation are shaded. The GlpR binding sites (O1–O4; lines above the sequence) and Crp binding sites (CrpI and CrpII; lines under the sequence) are also shown. The location of the IS5 element upstream of the promoter in crp Glp⁺ cells is indicated by the vertical white arrow below the horizontal black arrow representing IS5. This arrow shows the orientation of IS5 with the 3’ end of its transposase gene linked to the downstream promoter. The 4-nucleotide IS5 target sequence (ctaa) at −126 to −122 is shaded. (B) Growth of E. coli wt (■), crp (◆), and crp Glp⁺ (●) cells in liquid glycerol M9 minimal medium. (C) Cumulative Glp⁺ mutations appearing per 10⁸ cells (determined on the day measured) as a function of time in various media: solid M9 minimal media + 1% glycerol (♦), 0.01% glucose (■) or 1% sorbitol (σ). Total Glp⁻ cell populations on these plates are denoted by the lines with ◊ (glycerol), □ (glucose) or △ (sorbitol). (D) The same minimal glycerol agar plates, where the crp mutant cells were plated together with various numbers of cells from a crp Glp⁺ strain: (□, 72; ◊, 38; ③, 19; σ, 10; ⑤, 5; and ♦, 0). The crp Glp⁺ cells were mixed with crp cells (10⁸) and then applied onto M9 glycerol agar plates. Six independently isolated Glp⁺ mutant strains were tested and they behaved similarly. The results from one such Glp⁺ strain were shown in Figure 1D.

Figure 2. Gel electrophoresis of the PCR products of the glpFK regulatory regions in crp Glp⁻ and crp Glp⁺ colonies (A) and effects of alteration of the IS5 insertion site (CTAA) on the appearance of Glp⁺ mutants (B)

In (A), the five crp Glp⁺ mutants shown were independently isolated. The glpFK regulatory regions were amplified using primers PglpFK-EcoR and PglpFK-Bam (Table S2). Size standards are shown on the far right. In (B) crp cells containing an 85-bp DNA fragment at −122.5, −124.5 and −126.5 relative to +1 of PglpFK, or lacking such an insert (none), as indicated on the X axis, were assayed for appearance of Glp⁺ colonies on M9 + glycerol agar plates. Numbers of colonies were scored after day 8.

Figure 3. Dependency of Glp⁺ mutation rate on GlpR

(A) Effect of the loss of GlpR on the appearance of Glp⁺ mutants in the absence of glycerol. crp or crp glpR cells (<100 cells/plate) were applied to LB agar plates without glycerol. After two days of incubation at 30°C, ~50 colonies of each strain from different plates were used for determination of the total number of cells and the total number of Glp⁺ cells. Glp⁺ mutation frequency refers to the ratio of Glp⁺ cell number to total cell number. (B) Effect of the loss of GlpR on Glp⁺ mutation in crp cells in the presence of glycerol. Glp⁺ mutations were assayed on M9 + glycerol agar plates. (C) Effects of the losses of GlpR and its operators, O1 and O4, on mutation rates (mutations/10⁸ cells/h) when grown in liquid LB medium at 30°C. Fresh overnight cultures were inoculated into 20 ml of fresh LB in 125 ml flasks (initial OD₆₀₀ = 0.1). The flasks were shaken at 30°C. Samples were taken at one hour intervals for five hours for determination of total populations and Glp⁺ cell populations. The frequencies of Glp⁺ mutations relative to the total cell populations were plotted versus time, and the mutation rates were determined from the slopes of the curves.

Figure 4. Effects of glpR overexpression on the appearance of Glp⁺ mutants in a crp glpR genetic background when cells were grown on LB agar plates without glycerol (A) or on minimal M9 plates plus glycerol (B)

glpR expression in pBAD24 was induced by adding L-arabinose and IAA (inducers) to the media at 1 mM and 0.5 mM final concentrations, respectively. For the LB agar plate assay, ~100 cells were applied onto each plate. After incubation at 30°C for two days, individual colonies were examined for total and Glp⁺ cell populations. For the M9 + glycerol plate assay, Glp⁺ mutations and total populations were determined as described in Methods.

Figure 5. Effects of glycerol and GlpR on IS5 insertions at other IS5 insertional hot spots

(A) Effect of glycerol and sorbitol on IS5 insertion upstream of the flhDC promoter. Swarming mutation assay was conducted on soft-agar plates of minimal M9 + sorbitol or glycerol and soft-agar plates of nutrient broth ± sorbitol or glycerol. The swarming mutations were normalized as outgrowing subpopulations (mutations) per 9-cm cell streak. ‘sorb’: sorbitol; and ‘gly’: glycerol. (B) Effects of the loss of GlpR on IS5 hopping into its insertion sites in the flhDC master switch operon controlling motility, the bglGFB β-glucoside utilization operon, and the fucAO fucose/propanediol operon. The swarming mutation assay was conducted on nutrient broth soft agar plates, and the mutations were normalized as outgrowing subpopulations (mutations) per streak. Bgl⁺ or propanediol⁺ mutation assays were conducted on minimal M9 arbutin plates or M9 minimal propanediol plates, and colonies (mutants) were counted daily and were normalized as mutations (colonies) per 10⁸ cells.

Figure 6. Effects of mutations in GlpR operators on promoter activity and the appearance of Glp⁺ mutants

The graphs show the effects of mutations in GlpR operators, O1 and O4 (see Figure 1A), on (A) PglpFK activity in crp cells grown in LB liquid medium, and (B) Glp⁺ mutation frequencies in crp cells grown on LB solid medium. The bars represent standard deviation values for three independently conducted experiments. wt: control with no mutation in either O1 or O4; O1: only operator 1 was mutated (see Methods); O4: only operator 4 was mutated; O1O4: both operators were mutated.

Figure 7. Effects of GlpR on the frequencies of IS5 insertion upstream of PglpFK when this promoter drives expression of a cat [chlorophenicol resistance (Cm)] gene

The graph in A reveals the effect of the loss of GlpR on the frequency of IS5 insertion upstream of PglpFK in crp (◆) and crp glpR (■) genetic backgrounds. The graph in B shows the effect of GlpR overproduction on IS5 insertion using crp glpR cells that carry the pBAD24 vector alone (−GlpR) or pBAD24 expressing glpR (+GlpR). glpR expression is induced by inducers (1mM L-arabinose + 0.5 mM IAA). In both A and B, PglpFK is fused to a cat gene. Cells were incubated on LB + Cm (50 µg/ml) agar plates at 30°C, and Cm resistant colonies were counted after the time intervals indicated.

Figure 8. Schematic diagram illustrating GlpR-mediated control of (right) glpFK transcription and (left) the rate of IS5 hopping (directed mutation) into the CTAA site upstream of the glpFK promoter

With GlpR bound to its operators (O1–O4), transcription and IS5 hopping both occur at low rates. When GlpR is not bound to its operators, both transcriptional initiation and IS5 hopping increase about 10x. Binding of GlpR to operator O1 blocks IS5 insertion, while binding of GlpR to operator O4 blocks transcription as indicated.