Positive selection at high temperature reduces gene transcription in the bacteriophage ϕX174

Abstract

Background: Gene regulation plays a central role in the adaptation of organisms to their environments. There are many molecular components to gene regulation, and it is often difficult to determine both the genetic basis of adaptation and the evolutionary forces that influence regulation. In multiple evolution experiments with the bacteriophage ϕX174, adaptive substitutions in cis-acting regulatory sequences sweep through the phage population as the result of strong positive selection at high temperatures that are non-permissive for laboratory-adapted phage. For one cis-regulatory region, we investigate the individual effects of four adaptive substitutions on transcript levels and fitness for phage growing on three hosts at two temperatures.

Results: The effect of the four individual substitutions on transcript levels is to down-regulate gene expression, regardless of temperature or host. To ascertain the conditions under which these substitutions are adaptive, fitness was measured by a variety of methods for several bacterial hosts growing at two temperatures, the control temperature of 37°C and the selective temperature of 42°C. Time to lysis and doublings per hour indicate that the four substitutions individually improve fitness over the ancestral strain at high temperature independent of the bacterial host in which the fitness was measured. Competition assays between the ancestral strain and either of two mutant strains indicate that both mutants out-compete the ancestor at high temperature, but the relative frequencies of each phage remain the same at the control temperature.

Conclusions: Our results strongly suggest that gene transcription plays an important role in influencing fitness in the bacteriophage ϕX174, and different point mutations in a single cis-regulatory region provided the genetic basis for this role in adaptation to high temperature. We speculate that the adaptive nature of these substitutions is due to the physiology of the host at high temperature or the need to maintain particular ratios of phage proteins during capsid assembly. Our investigation of regulatory evolution contributes to interpreting genome-level assessments of regulatory variation, as well as to understanding the molecular basis of adaptation.

Figure 1

Regulation of ϕX174 gene transcription. Gene transcription in ϕX174 is regulated by four promoters (P_A, P_B, P_D, *) and four transcription terminators (T_J, T_F, T_G, T_H). Bars indicate transcript start and stop and thickness of bars indicates relative amount of transcript. Ovals show approximate positions of sequences detected in qPCR experiments. Gene locations are shown below, and genes B, K and E are in different reading frames from the genes with which they overlap. The ϕX174 genome is a circular molecule; the figure was linearized for clarity. From [21,22].

Figure 2

Transcript levels differ between ancestor and promoter mutant strains at two temperatures in three bacterial hosts. The amount of transcript starting from the D promoter was measured relative to the amount of transcript starting from the B promoter for each phage strain using quantitative PCR. Injection of DNA into host was synchronized by attachment at 15°C and then addition to LB at 37°C. Samples were taken at 4 and 8 min after injection, which is prior to the start of cell lysis. The y-axis indicates the ratio of the D transcript to the B transcript. Bars are the mean and error bars the standard error of two (S. sonnei) or three replicate assays. Solid bars are mutant strains with non-synonymous substitutions in C, hatched bars are synonymous in C. Relative transcript level of the ancestor is significantly greater than the four mutants for all host/temperature/time combinations (Table 2). Note the difference in scale for the S. sonnei graph.

Figure 3

Fitness differences between ancestor and promoter mutant strains at two temperatures for three bacterial hosts. Doublings per hour were measured for each phage strain in three bacterial hosts and at two temperatures. Bars are the mean and error bars the standard error of three replicate assays. Solid bars are mutant strains with non-synonymous substitutions in C, hatched bars are synonymous in C. Doublings per hour of the ancestor is significantly lower than at least one of the four mutant strains for all host/temperature combinations except E. coli and S. sonnei at 37°C, where the ancestor's fitness is significantly greater than at least one of the four mutant strains (Table 3).

Figure 4

Competition assays confirm the greater fitness of mutant strains at 42°C in E. coli. The ancestor and one mutant strain were grown together in a two-stage chemostat on E. coli C at 37°C or 42°C. Concentration of each strain was determined by allele specific PCR at the beginning and end of each chemostat. The y-axis shows the average increase in frequency of the mutant strain after two hours. Three replicate chemostats were run for each mutant at each temperature. Mut323 is a non-synonymous substitution in C, and mut 324 is synonymous. The increase in mutant strain frequency was significantly different between the two temperatures for both mutants (Wilcoxon rank sum test, p < 0.005).