The effect of mobile element IS10 on experimental regulatory evolution in Escherichia coli

Abstract

Mobile genetic elements are widespread in bacteria, where they cause several kinds of mutations. Although their effects are on the whole negative, rare beneficial mutations caused by insertion sequence elements are frequently selected in some experimental evolution systems. For example, in earlier work, we found that strains of Escherichia coli that lack the sigma factor RpoS adapt to a high-osmolarity environment by the insertion of element IS10 into the promoter of the otsBA operon, rewiring expression from RpoS dependent to RpoS independent. We wished to determine how the presence of IS10 in the genome of this strain shaped the evolutionary outcome. IS10 could influence the outcome by causing mutations that confer adaptive phenotypes that cannot be achieved by strains without the element. Alternatively, IS10 could influence evolution by increasing the rate of appearance of certain classes of beneficial mutations even if they are no better than those that could be achieved by a strain without the element. We found that populations evolved from an IS10-free strain did not upregulate otsBA. An otsBA-lacZY fusion facilitated the recovery of a number of mutations that upregulate otsB without involving IS10 and found that two caused greater fitness increases than IS10 insertion, implying that evolution could have upregulated otsBA in the IS10-free strain. Finally, we demonstrate that there is epistasis between the IS10 insertion into the otsBA promoter and the other adaptive mutations, implying that introduction of IS10 into the otsBA promoter may alter the trajectory of adaptive evolution. We conclude that IS10 exerts its effect not by creating adaptive phenotypes that could not otherwise occur but by increasing the rate of appearance of certain adaptive mutations.

FIG. 1.

Transcription of otsB from evolved lines that lacked IS10 as measured by QPCR. otsB levels are not significantly higher in any of the evolved lines than in their ancestor (P > 0.2, Tukey’s HSD on log-transformed data). As a control, note that otsB message levels are much higher in both the wt (rpoS⁺) and P_otsBA::IS10 lines (P < 0.001, Tukey’s HSD). otsB mRNA levels are normalized to rho mRNA and expressed as a fold difference relative to the ΔrpoS ancestor. mRNA levels were measured on three biological replicates.

FIG. 2.

Mutations recovered by selection with an otsBA::lacZY fusion. Mutants above the sequence were isolated in strain DMS2098, a background that contains IS10, and were previously reported (Stoebel et al. 2009). Mutants below the sequence were isolated in DMS2191, a background that does not contain IS10. All mutants were recovered once, except for the C to T transition at +11 relative to the transcriptional start site, isolated five times, and the IS10 insertion between bases +12 and +13, which was isolated 19 times. The IS10 insertion between +11 and +12 is the insertion found in the experimentally evolved lines. The start of translation is at +56. The transcriptional start site is from Becker and Hengge-Aronis (2001), and the −10 and −35 sites are inferred from the data in Shultzaberger et al. (2007).

FIG. 3.

Activity of the isolated promoter mutations as assayed by β-galactosidase activity. Strains are labeled as seen in figure 2. Wt refers to an rpoS⁺ line (DMS2096) and IS10 to a strain with IS10 inserted at the same location as recovered in our experimental evolution. Data are from four independent replicates. Error bars represent the standard error of the mean.

FIG. 4.

(A) Fitness effects of mutations in P_otsBA. Promoter mutations isolated from the otsB::lacZY fusion were recombined into the wild-type chromosome and competed against a ΔrpoS strain with wild-type P_otsBA. Two mutations (the −35 deletion and duplication II) have higher fitness than an IS10 insertion. (B) Relationship between transcription of otsBA and fitness increase. For small increases in transcription, the relationship between otsBA transcription and fitness increase is approximately linear. A very large increase in transcription (the −35 deletion) does not increase fitness much beyond that achievable with less transcription (by duplication II).

FIG. 5.

Epistasis between P_otsBA::IS10 and other unknown adaptive mutations. Fitness of the ten evolved lines that lack IS10 was measured relative to their ancestor. In addition, P_otsBA::IS10 was added to each of these lines, and they were competed against a strain with only P_otsBA::IS10. The unknown adaptive mutations were more beneficial on the ancestral background than on the background P_otsBA::IS10, significantly so in nine of ten cases (P < 0.05, t-test with P values modified by the sequential adjustment method of Holm, 1979). The diagonal line shows equal fitness on the two backgrounds.