Environmental stresses can alleviate the average deleterious effect of mutations
© Kishony and Leibler, licensee BioMed Central Ltd. 2003
Received: 13 December 2002
Accepted: 2 May 2003
Published: 29 May 2003
Fundamental questions in evolutionary genetics, including the possible advantage of sexual reproduction, depend critically on the effects of deleterious mutations on fitness. Limited existing experimental evidence suggests that, on average, such effects tend to be aggravated under environmental stresses, consistent with the perception that stress diminishes the organism's ability to tolerate deleterious mutations. Here, we ask whether there are also stresses with the opposite influence, under which the organism becomes more tolerant to mutations.
We developed a technique, based on bioluminescence, which allows accurate automated measurements of bacterial growth rates at very low cell densities. Using this system, we measured growth rates of Escherichia coli mutants under a diverse set of environmental stresses. In contrast to the perception that stress always reduces the organism's ability to tolerate mutations, our measurements identified stresses that do the opposite – that is, despite decreasing wild-type growth, they alleviate, on average, the effect of deleterious mutations.
Our results show a qualitative difference between various environmental stresses ranging from alleviation to aggravation of the average effect of mutations. We further show how the existence of stresses that are biased towards alleviation of the effects of mutations may imply the existence of average epistatic interactions between mutations. The results thus offer a connection between the two main factors controlling the effects of deleterious mutations: environmental conditions and epistatic interactions.
We first built a library containing 65 random mutations generated by chemical mutagenesis, along with 12 copies of the parental strain as controls. Importantly, we avoided as far as possible any selection against slow-growing mutants during the library construction procedures. The library was screened for growth under various environmental conditions and the growth rate of each mutant culture was defined as the reciprocal of the doubling time of the population during exponential growth.
It should be noted that our assay is designed to measure absolute growth rates of the mutants in isolation, rather than their relative fitness in competition. Such an absolute measurement is important for some of the analyses presented (in particular the analysis relevant to Figure 4, below). In general, since actual fitness depends on many factors – such as the particular environment, the specific competitors or the population densities – it is always being defined only in an operational way. In our case, the growth-rate measurements should be considered simply as direct measurements of a fitness-related trait.
The stresses tested, and their influence on average relative mutation effects
30% old supernatant
600 mM NaCl
In total, several thousand growth curves were measured. Typically, at least two replicates of each mutant were grown in each of the environmental conditions. An example of the growth curve of one mutant from the library compared to the parental strain, in the favorable environment and under chloramphenicol stress, is shown in Figure 2b.
The results of the acidic stress, on the other hand, are qualitatively different, showing a small but significant (p < 0.01) aggravation of the effects of mutations. As shown in Figure 3d, the distribution of distances from the equal-effect line is now more centered and shifted slightly towards the negative region. Note also that a relatively large number of mutations become lethal under acidic stress. For the high osmolarity stress and the unsupplemented stress, mutations occur equally on both sides of the equal-effect line (Figure S3), indicating a neutral or non-significant influence of these stresses on the average mutation effect.
Explaining the observed qualitative diversity of the average impacts of stress on mutations, ranging continuously from alleviation to aggravation of average mutation phenotypic effects, is beyond the scope of this paper. We briefly discuss, however, some possible mechanisms that could be evoked to explain the existence of stresses that alleviate the average mutation effect. First, certain stresses – in particular the bacteriostatic antibiotics chloramphenicol and trimethoprim – may target a specific functional module in the bacterium, thus generating a rate-limiting step for growth. The data on the effects of these stresses may, to some extent, be interpreted in terms of an extremely idealized picture in which cell growth results from the combined functionalities of many parallel modules . Assuming that proliferation rate is determined by the 'slowest module' and that the mutation and the stress target different modules, the mutant growth rate under the stress should be μS = min[μF, νS], where μF is the growth rate of the mutant in favorable conditions and νS is the parental strain growth rate under the stress (Figure 1). This necessarily implies that the effect of the mutation on the relative growth rate is decreased under the stress (αS < αF). A similar argument stating that the "genetic potential of organisms is not reached under poor nutrition" was also made as a possible explanation for evidence of reduced heritability of natural populations seen under certain stressful conditions . Second, it is known that certain bactericidal antibiotics, such as penicillin, confer an advantage on non-growing mutants [21, 22]. In sub-lethal concentrations, which allow slow growth of the parental strain, these reagents could potentially reduce the deleterious effect of mutations on relative growth rates. This does not seem to be the mechanism behind the results described here, however. One reason is that there would have to have been a positive correlation between the reduction in relative growth rate and the level of buffering by the stress, while the results indicated in Figure 3b do not show such a correlation. Third, chemicals such as chloramphenicol and dithiothreitol may cause increased error rates of translation and protein folding, respectively. The effects of mutations could then be obscured by the already high error rates imposed by the stress.
Our results show that organisms may actually become more tolerant to genetic perturbations when put under certain environmental stresses. This intriguing result implies a connection between the two main factors controlling the deleterious effects of mutations: environmental conditions and epistatic interactions (for additional support see ). Such a connection may allow a unification of environmental and mutational theories for the advantage of sexual reproduction [2, 24, 35]. While the current study was aimed at the statistical characteristics of random mutations, the same approach and experimental techniques can also be applied to libraries of known and marked mutants, which should give further insight into the modular structure of the organism [29, 36, 37]. Finally, double and triple mutants constructed from such libraries may make it possible to test our prediction for the existence of epistasis and its dependence on environmental conditions.
Materials and methods
Strains and media
E. coli K12 strain DL41 (λ-, metA28) was obtained from the E. coli Genetic Stock Center, CGSC# 7177. Plasmid pCS16 (SC101 ori, a luxCDABE operon and a KanR marker) was obtained from M. Surette. The luciferase promoter in pCS16 was BamHI-excised and a synthetic lambda promoter  was ligated instead to form pCS-λ. The parental strain of the current study is the constitutively bright DL41 strain bearing pCS-λ.
The standard favorable medium (FM) is a M63 minimal medium , supplemented with 0.2% glucose, 0.01% casamino acids, 0.5 μg/ml thiamine, 33 μg/ml methionine and 40 μg/ml kanamycin. Growth temperature was 30°C unless otherwise indicated. Stressful environments were formed by supplementing FM as indicated in Table 1.
Mutant library construction
The parental strain culture was mutagenized by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) according to standard methods . The mutagen dose used (7.5 μg/ml NTG for 10 minutes) corresponds to a relatively low number of mutations per genome (rifampicin resistance frequency of 3 × 10-5). It should be noted that the exact number of mutations per genome may vary between the mutants, but none of the arguments made in the current study assume, in any way, a specific constant number of mutations per mutant (see in particular the legend to Figure 4). After mutagenesis, cells were allowed to recover in LB for only 2 hours to avoid considerable selection against slow-growing mutants. Cells were then plated for single colonies on FM agar plates and incubated at 30°C. At five time points (21, 24, 34, 50 and 73 hours after plating), newly arising colonies were counted (there were 1,268, 58, 29, 18 and 6, respectively) and colonies (7, 35, 20, 13 and 3, respectively) were randomly picked and re-streaked on FE agar plates. Each re-streaked plate was placed at 4°C when small visible colonies first appeared. Once all re-streaked mutants formed visible colonies, they were picked into separate wells on a 96-well microtiter plate containing 100 μl FM per well. Twelve parental strain controls, which went through the same procedure with no mutagen, were also included in the library. The library microtiter plate was then used as a master plate from which the library was replicated to initiate the growth rate assays. Frozen -80°C copies of the library were also made by replicating the master plate into M63 + 3.5% v/v DMSO.
The growth rates measured for the seven clones picked in the first time point were equal to the parental strain growth rate under all tested environments, and were therefore excluded from the statistical analysis. Mutants picked at the four later time points were assigned a statistical weight equal to the ratio of the total number of new colonies that appeared at a given time point divided by the number of colonies picked at that time point. This statistical weight was used to properly weight the growth-rate measurements for the statistical analysis shown in Figures 3d and S2 and Table 1.
Growth curve assay
The 96-well plates (Costar 3792 black, round bottom) were filled with 100 μl per well of the tested media, inoculated with the library cells using a 96-pin replicator and tightly sealed with a clear adhesive tape (Perkin-Elmer 1450–461). For a given medium, at least two replications of several cell inoculations (typically three different inoculations aimed around 0.15, 3 and 25 cells per well) were made. Photon counting was done in Packard's TopCount NXT Microplate Scintillation and Luminescence Counter. The instrument was placed in a 30°C (or 17°C for the cold-temperature experiment) environmental room and the same temperature was also set in the instrument's reading chamber. Acquisition time was 2 seconds per well. A total of 10–20 microtiter plates were typically assayed in parallel using the instrument stacker. No shaking for aeration was performed. A calibration of counts per second (cps) in the detector to number of cells per well is 30 cells per cps during exponential growth of the parental strain in favorable conditions (see Figure S1).
Special thanks to M.G. Surette for kindly providing plasmid pCS16, to A.W. Murray for important comments and to M. Elowitz and R. Chait for proofreading the manuscript. We thank the following for helpful discussions: B.L. Bassler, D. Fisher, D. Kahne, P. Model, M. Russel, T.J. Silhavy, M.G. Surette and all the members of our lab. This work was partially supported by the National Institutes of Health and the Human Frontiers Science Program.
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