10 research outputs found
Experimental Evolution : and Fitness Effects of Mutations
Bacteria have small, streamlined genomes and evolve rapidly. Their large population sizes allow selection to be the main driver of evolution. With advances in sequencing technologies and precise methods for genetic engineering, many bacteria are excellent models for studying elementary questions in evolutionary biology. The work in this thesis has broadly been devoted to adaptive evolution and fitness effects of different types of mutations. In Paper I we experimentally tested the fitness constrains of horizontal gene transfer (HGT), which could be used to predict how the fixation of HGT events are affected by selection and fitness effects. We found that the majority of the examined HGT inserts were indistinguishable from neutral, implying that extra DNA transferred by HGT, even though it does not confer an immediate selective advantage, could be maintained at transfer-selection balance and serve as a reservoir for the evolution of novel beneficial functions. Paper II examined why four synonymous mutations in rpsT (encoding ribosomal protein S20) reduced fitness, and how this cost could be genetically compensated. We found that the cause for the fitness reduction was low S20 levels and that this lead to a defective subpopulation of 30S subunits lacking S20. In an adaptive evolution experiment, these impairments were compensated by up-regulation of S20 though various types of mutations. In Paper III we continued the studies of how the deleterious rpsT mutations could be compensated. The mutations either down-regulated the global regulator Fis or altered a subunit of the RNA polymerase (rpoA). We found that the decreased S20 levels in the cells causes an assembly defect of the 30S particles and that the fis and rpoA mutations restored the skewed S20:ribosome ratio by both increasing S20 levels and decreasing other ribosomal components. Paper IV examined adaptation of two bacterial species to different growth media. A total of 142 different adaptive mutations were identified and 112 mutants were characterized in terms of fitness. We found that the experimental variation in fitness measurements could be reduced 10-fold by introducing some adaptive mutations prior to the experiment, allowing measurements of fitness differences as small as 0.04%
Compensating the fitness costs of synonymous mutations
Synonymous mutations do not change the sequence of the polypeptide but they may still influence fitness. We investigated in Salmonella enterica how four synonymous mutations in the rpsT gene (encoding ribosomal protein S20) reduce fitness (i.e. growth rate) and the mechanisms by which this cost can be genetically compensated. The reduced growth rates of the synonymous mutants were correlated with reduced levels of the rpsT transcript and S20 protein. In an adaptive evolution experiment these fitness impairments could be compensated by mutations that either caused up-regulation of S20 through increased gene dosage (due to duplications), increased transcription of the rpsT gene (due to an rpoD mutation or mutations in rpsT), or increased translation from the rpsT transcript (due to rpsT mutations). We suggest that the reduced levels of S20 in the synonymous mutants result in production of a defective subpopulation of 30S subunits lacking S20 that reduce protein synthesis and bacterial growth and that the compensatory mutations restore S20 levels and the number of functional ribosomes. Our results demonstrate how specific synonymous mutations can cause substantial fitness reductions and that many different types of intra- and extragenic compensatory mutations can efficiently restore fitness. Furthermore, our study highlights that also synonymous sites can be under strong selection, which may have implications for the use of dN/dS ratios as signature for selection
Duplication-Insertion Recombineering : a fast and scar-free method for efficient transfer of multiple mutations in bacteria
We have developed a new λ Red recombineering methodology for generating transient selection markers that can be used to transfer mutations between bacterial strains of both Escherichia coli and Salmonella enterica. The method is fast, simple and allows for the construction of strains with several mutations without any unwanted sequence changes (scar-free). The method uses λ Red recombineering to generate a marker-held tandem duplication, termed Duplication-Insertion (Dup-In). The Dup-Ins can easily be transferred between strains by generalized transduction and are subsequently rapidly lost by homologous recombination between the two copies of the duplicated sequence, leaving no scar sequence or antibiotic resistance cassette behind. We demonstrate the utility of the method by generating several Dup-Ins in E. coli and S. enterica to transfer genetically linked mutations in both essential and non-essential genes. We have successfully used this methodology to re-construct mutants found after various types of selections, and to introduce foreign genes into the two species. Furthermore, recombineering with two overlapping fragments was as efficient as recombineering with the corresponding single large fragment, allowing more complicated constructions without the need for overlap extension PCR
Evolution of antibiotic resistance without antibiotic exposure
Antibiotic use is the main driver in the emergence of antibiotic resistance. Another unexplored possibility is that resistance evolves coincidentally in response to other selective pressures. We show that selection in the absence of antibiotics can co-select for decreased susceptibility to several antibiotics. Thus, genetic adaptation of bacteria to natural environments may drive resistance evolution by generating a pool of resistance mutations that selection could act on to enrich resistant mutants when antibiotic exposure occurs
Synonymous mutations in rpsT lead to ribosomal assembly defects that can be compensated by mutations in fis and rpoA
We previously described how four deleterious synonymous mutations in the Salmonella enterica rpsT gene (encoding ribosomal protein S20) result in low S20 levels that can be compensated by mutations that restore [S20]. Here, we have further studied the cause for the deleterious effects of S20 deficiency and found that the S20 mutants were also deficient in four other 30S proteins (S1, S2, S12, and S21), which is likely due to an assembly defect of the S20 deficient 30S subunits. We examined the compensatory effect by six additional mutations affecting the global regulator Fis and the C-terminal domain of the α subunit of RNA polymerase (encoded by rpoA). The fis and rpoA mutations restored the S20 levels, concomitantly restoring the assembly defect and the levels of S1, S2, S12, and S21. These results illustrate the complexity of compensatory evolution and how the negative effects of deleterious mutations can be suppressed by a multitude of mechanisms. Additionally, we found that the mutations in fis and rpoA caused reduced expression of other ribosomal components. Notably, some of the fis mutations and the rpoA mutation restored the fitness of the rpsT mutants to wild-type levels, although expression of other ribosomal components was reduced compared to wild-type. This finding raises new questions regarding the relation between ribosome concentration and growth rate
Regulatory elements coordinating initiation of chromosome replication to the Escherichia coli cell cycle
Escherichia coli coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thou-sands of division cycles in wild-type and mutant strains, we were able to compare the relative importance of previously described control systems. We found that accurate triggering of initiation does not require synthesis of new DnaA. The initiation size increased only marginally as DnaA was diluted by growth after dnaA expression had been turned off. This suggests that the conversion of DnaA between its active ATP -and inactive ADP-bound states is more important for initiation size control than the total free concentration of DnaA. In addition, we found that the known ATP/ADP converters DARS and datA compensate for each other, although the removal of them makes the initiation size more sensitive to the concentration of DnaA. Only disruption of the regulatory inactivation of DnaA mechanism had a radical impact on replication initiation. This result was corroborated by the finding that termination of one round of replication correlates with the next initiation at intermediate growth rates, as would be the case if RIDA-mediated conversion from DnaA-ATP to DnaA-ADP abruptly stops at termination and DnaA-ATP starts accumulating
Genetic adaptation to growth under laboratory conditions in Escherichia coli and Salmonella enterica
Experimental evolution under controlled laboratory conditions is becoming increasingly important to address various evolutionary questions, including, for example, the dynamics and mechanisms of genetic adaptation to different growth and stress conditions. In such experiments, mutations typically appear that increase the fitness under the conditions tested (medium adaptation), but that are not necessarily of interest for the specific research question. Here, we have identified mutations that appeared during serial passage of E. coli and S. enterica in four different and commonly used laboratory media and measured the relative competitive fitness and maximum growth rate of 111 genetically re-constituted strains, carrying different single and multiple mutations. Little overlap was found between the mutations that were selected in the two species and the different media, implying that adaptation occurs via different genetic pathways. Furthermore, we show that commonly occurring adaptive mutations can generate undesired genetic variation in a population and reduce the accuracy of competition experiments. However, by introducing media adaptation mutations with large effects into the parental strain that was used for the evolution experiment, the variation (standard deviation) was decreased 10-fold, and it was possible to measure fitness differences between two competitors as small as |s| < 0.001.Michael Knopp, Lisa M. Albrecht, Erik Lundin and Ulrika Lustig contributed equally to this work.</p
Duplication-Insertion Recombineering: a fast and scar-free method for efficient transfer of multiple mutations in bacteria
Table_1_Genetic Adaptation to Growth Under Laboratory Conditions in Escherichia coli and Salmonella enterica.xlsx
<p>Experimental evolution under controlled laboratory conditions is becoming increasingly important to address various evolutionary questions, including, for example, the dynamics and mechanisms of genetic adaptation to different growth and stress conditions. In such experiments, mutations typically appear that increase the fitness under the conditions tested (medium adaptation), but that are not necessarily of interest for the specific research question. Here, we have identified mutations that appeared during serial passage of E. coli and S. enterica in four different and commonly used laboratory media and measured the relative competitive fitness and maximum growth rate of 111 genetically re-constituted strains, carrying different single and multiple mutations. Little overlap was found between the mutations that were selected in the two species and the different media, implying that adaptation occurs via different genetic pathways. Furthermore, we show that commonly occurring adaptive mutations can generate undesired genetic variation in a population and reduce the accuracy of competition experiments. However, by introducing media adaptation mutations with large effects into the parental strain that was used for the evolution experiment, the variation (standard deviation) was decreased 10-fold, and it was possible to measure fitness differences between two competitors as small as |s| < 0.001.</p