| 1. |
- Bergman, Jessica M.
(författare)
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Genetics and Growth Regulation in Salmonella enterica
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Most free-living bacteria will encounter different environments and it is therefore critical to be able to rapidly adjust to new growth conditions in order to be competitively successful. Responding to changes requires efficient gene regulation in terms of transcription, RNA stability, translation and post-translational modifications.Studies of an extremely slow-growing mutant of Salmonella enterica, with a Glu125Arg mutant version of EF-Tu, revealed it to be trapped in a stringent response. The perceived starvation was demonstrated to be the result of increased mRNA cleavage of aminoacyl-tRNA synthetase genes leading to lower prolyl-tRNA levels. The mutant EF-Tu caused an uncoupling of transcription and translation, leading to increased turnover of mRNA, which trapped the mutant in a futile stringent response.To examine the essentiality of RNase E, we selected and mapped three classes of extragenic suppressors of a ts RNase E phenotype. The ts RNase E mutants were defective in the degradation of mRNA and in the processing of tRNA and rRNA. Only the degradation of mRNA was suppressed by the compensatory mutations. We therefore suggest that degradation of at least a subset of cellular mRNAs is an essential function of RNase E.Bioinformatically, we discovered that the mRNA of tufB, one of the two genes encoding EF-Tu, could form a stable structure masking the ribosomal binding site. This, together with previous studies that suggested that the level of EF-Tu protein could affect the expression of tufB, led us to propose three models for how this could occur. The stability of the tufB RNA structure could be affected by the elongation rate of tufB-translating ribosomes, possibly influenced by the presence of rare codons early in the in tufB mRNA.Using proteomic and genetic assays we concluded that two previously isolated RNAP mutants, each with a growth advantage when present as subpopulations on aging wild-type colonies, were dependent on the utilization of acetate for this phenotype. Increased growth of a subpopulation of wild-type cells on a colony unable to re-assimilate acetate demonstrated that in aging colonies, acetate is available in levels sufficient to sustain the growth of at least a small subpopulation of bacteria.
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| 2. |
- Dyrhage, Karl
(författare)
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Multi-omics investigation into bacterial evolution
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The focus of this thesis is the investigation of the evolution and cellular processes of Tuwongella immobilis and Apilactobacillus kunkeei, two bacterial species with different levels of genomic and cellular complexity, using a multi-omics approach.In the first study we examined the proteome of T. immobilis with LC-MS/MS after fractionation by differential solubilisation, yielding fractions corresponding to the cytoplasm, inner membrane, and outer membrane. The experiment was repeated with Escherichia coli and the results were compared. T. immobilis had five times as many predicted cytoplasmic proteins in the most hydrophobic fraction as E. coli. Among these are innovations in the Planctomycetota lineage and protein families that have undergone recent paralogisation followed by domain shuffling, including many enzymes related to information processing.The remaining three studies dealt with honeybee symbiont A. kunkeei. In the first of these, we sequenced and compared the chromosomal and extrachromosomal content of 102 novel A. kunkeei strains. We found that A. kunkeei has an open pangenome and an active set of transposable elements. Within the population we discovered three plasmids between 19.5 and 32.9 kb, one of which codes for enzymes involved in the synthesis of the antimicrobial compound kunkecin A which inhibits growth of the bee pathogen Melisococcus plutonius.In the next study we collected transcriptomic, proteomic, and metabolomic data from two growth phases from A. kunkeei strain A1401 and mapped the results to a metabolic pathway model. Enzymes involved in fermentation of fructose were highly expressed during the exponential growth phase. Enzymes involved in UMP biosynthesis were upregulated during stationary phase, as were protein involved in stress response and detoxification.The last study concerned the secretome of A. kunkeei. We characterised two types of extracellular particles from A. kunkeei strains A1401 and A0901. One type of particle was found to be proteinaceous, while the other type constituted membrane vesicles containing RNA. Comparison of transcriptomic data from the membrane vesicles and whole cells showed that the packing of the RNA was largely untargeted, but with a bias towards highly expressed mRNAs. We suggest that the cell uses membrane vesicles as a mechanism to get rid of superfluous mRNAs after rapid-response overexpression.Together these studies provide insights into the processes driving evolution in T. immobilis and A. kunkeei, and generate several testable hypotheses for future studies.
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| 3. |
- Ancillotti, Mirko, 1981-
(författare)
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Antibiotic Resistance: A Multimethod Investigation of Individual Responsibility and Behaviour
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The rapid development of antibiotic resistance is directly related to how antibiotics are used in society. The international effort to decrease and optimise the use of antibiotics should be sustained by the development of policies that are sensitive to social and cultural contexts.The overarching aim of the thesis was to explore and discuss the Swedish public’s beliefs, values and preferences influencing engagement in judicious antibiotic behaviour.Study I explored through focus group discussions lay people’s perceptions and beliefs about antibiotics and antibiotic resistance. The Health Belief Model was used to identify factors that could promote or hinder engagement in judicious antibiotic behaviour. Participants found antibiotic resistance to be a serious problem but were not equally worried about being affected by it. There was a tension between individual and collective reasons for engaging in judicious behaviour.Study II explored lay people’s views on the moral challenges posed by antibiotic resistance through focus group discussions. Participants identified in the decreasing availability of effective antibiotics a problem of justice, which involves individual as well as collective moral responsibility. Different levels of policy demandingness were discussed in light of these results.Study III investigated, through an online Discrete Choice Experiment, public preferences regarding antibiotic treatment and the relative weight of antibiotic resistance in decision-making. Public behaviour may be influenced by concerns over the rise of antibiotic resistance. Therefore, stressing individual responsibility for antibiotic resistance in clinical and societal communication may affect personal decision-making.Study IV clarified the notions of collective and individual moral responsibility for antibiotic resistance and suggested a virtue-based account thereof. While everyone is morally responsible for minimising his/her own contribution to antibiotic resistance, individuals do or do not engage in judicious antibiotic behaviour with different degrees of voluntariness.The findings suggest that people could change their behaviour due to concerns over their own contribution to antibiotic resistance. Effective health communication should be developed from an appraisal of people’s attitudes, beliefs and social norms that influence antibiotic resistance related behaviours. Policy demandingness should take into account socioeconomic factors characterising local realities.
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| 4. |
- Malik, Sohaib Zafar
(författare)
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Interaction of cyclotides and bacteria : A study of the cyclotide action and the bacterial reaction
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The growing problem of antibiotic resistance and the lack of promising prospective antibiotics have forced us to search for new classes of antibiotics. Among the candidates to develop into future antibacterials are antimicrobial peptides (AMPs). Thesepotent, broad spectrum compounds are important components of innate immunity of organism from all kingdoms of life. One such family of mini-proteins from plants is called cyclotides, whose members are defines by cyclic backbone and a cystine knot (CCK), which confers to them extreme stability in the face of biological, chemical and physical insults. Some cyclotides possess Gram-negative specific antibacterial activity; the purpose of this thesis was to characterize how these molecules kill bacteria, and how bacteria would respond to treatment with cyclotides. For this purpose, Salmonella enterica and Escherichia coli mutants resistant to the cyclotides cycloviolacin O2 and cycloviolacin O19, respectively, were selected. These mutants were characterized by whole genome sequencing, genetic reconstitution, fitness measurements, and cross-resistance studies. These studies identified a number of genetic pathways for resistance development to cyclotides. These mutants displayed variable fitness profiles in laboratory growth media and in mice competition experiments, with some mutants possessing a fitness advantage in mice. Cross-resistance studies resulted in the identification of several cases of cross-resistance and collateral sensitivity between cyclotides and other AMPs/antibiotics. Antimicrobial effects of cyclotides were assayed in different conditions and in bacterial organisms with different surface characteristics. In addition, immunolocalization experiments were performed to explore the biological distribution of cyclotides in plants and to determine the mechanism of action of cyclotides in bacteria, respectively. Antibodies raised against cyO2 were used for this purpose. Immunohistochemical techniques applied to plant cells, tissues and organs provided the information that cyclotides were distributed in all plant organs, and were found in tissues vulnerable to pathogen attack, and that cyclotides were stored in the vacuoles of plant cells. Immunogold staining of cyclotide treated cells of S. typhimurium, showed effects of cyclotide treatment on the cell envelope components as well as cytoplasm. A higher number of cyclotide molecules was associated with the cell envelope, but a considerable fraction of them penetrated into the cytoplasm.
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| 5. |
- Mellenius, Harriet, 1983-
(författare)
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Exploring and predicting DNA template dependent variation in transcription
- 2012
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Reliable transmission of information from DNA to proteins is a pre-requisite for all life, where substitution errors in the polypeptide chain may arise from transcription, aminoacylation of tRNAs or translation. The fidelity control mechanisms in transcription have nevertheless received little attention, based on the assumption that the transcriptional error is masked by the translational error. This thesis shows how accuracy theory can be applied to transcription to elucidate the principles of transcriptional accuracy. The DNA template dependent transcriptional accuracy variation is studied through modelling based on transition state theory, using thermodynamic properties of the nucleic acids in the transcription bubble. The models show that the error frequency variation in transcription causes it to surpass the translational error in some sequence contexts, making transcription a significant source of amino acids substitution errors.
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| 6. |
- Sun, Song, 1982-
(författare)
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Dynamics and Mechanisms of Adaptive Evolution in Bacteria
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Determining the properties of mutations is fundamental to understanding the mechanisms of adaptive evolution. The major goal of this thesis is to investigate the mechanisms of bacterial adaptation to new environments using experimental evolution. Different types of mutations were under investigations with a particular focus on genome rearrangements. Adaptive evolution experiments were focused on the development of bacterial resistance to antibiotics.In paper I, we performed stochastic simulations to examine the role of gene amplification in promoting the establishment of new gene functions. The results show that gene amplification can contribute to creation of new gene functions in nature. In paper II, the evolution of β-lactam resistance was studied by evolving S. typhimurium carrying a β-lactamase gene towards increased resistance against cephalosporins. Our results suggest that gene amplification is likely to provide an immediate solution at the early stage of adaptive evolution and subsequently facilitate further stable adaptation. In paper III, we isolated spontaneous deletion mutants with increased competitive fitness, which indicated that genome reduction could be driven by selection. To test this hypothesis, independent lineages of wild type S. typhimurium were serially passaged for 1000 generations and we observed fixation of deletions that significantly increased bacterial fitness when reconstructed in wild type genetic background. In paper IV, we developed a new strategy combining 454 pyrosequencing technology and a ‘split mapping’ computational method to identify unique junction sequences formed by spontaneous genome rearrangements. A high steady-state frequency of rearrangements in unselected bacterial populations was suggested from our results. In paper V, the rates, mechanisms and fitness effects of colistin resistance in S. typhimurium were determined. The high mutation rate and low fitness costs suggest that colistin resistance could develop in clinical settings. In paper VI, a novel Metallo-β-lactamase (MBL) with low resistance against β-lactam antibiotics was employed as the ancestral protein in a directed evolution experiment to examine how an enzyme evolves towards increased resistance. For most isolated mutants, in spite of their significantly increased resistance, both mRNA and protein levels were decreased as compared with the parental protein, suggesting that the catalytic activity had increased.
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| 7. |
- Elfström, Johan, 1991-
(författare)
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Reconceiving Public Reason : Neutrality, Civility, and the Self-Defeat Objection
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- How should we live together? The question is at the heart of social ethics and it is an as urgent political question as ever. In this thesis, one particularly attractive reply to this central issue is analysed—John Rawls’s theory of public reason, and three different objections that have been put against it. Rawls’s theory is an approach to democratic decision-making. According to it, the exercise of political power should be neutral. Secondly, the exercise of political power should restrict the reasons that have justificatory force in political decision-making procedures to reasons that do not rely on any particular worldview. Finally, the exercise of political power is legitimate only if it is in accordance with terms of cooperation that all reasonable and rational persons can accept.The objections each target one of these components. Cécile Laborde has challenged the conception of neutrality espoused by egalitarian liberals generally. Egalitarian liberal understandings of neutrality do not take sufficient account of all relevant dimensions of our worldviews and often confuse neutral policies with what conforms to the status quo. Jeffrey Stout, in turn, targets the constraints on public discourse and argues that imposing such constraints is unfair to religious citizens because it distributes the burdens of cooperation to their disadvantage. Finally, Steven Wall argues that the requirement that the legitimate exercise of political power be acceptable to citizens ends up defeating itself.These arguments are tested and I consider the alternative approaches that are presented by each of the three critics. I propose that neutrality should be rejected, as equality better captures the end pursued by demanding neutral treatment of different worldviews. I then go on to revise the constraints that Rawls impose. Although many of Stout’s arguments are persuasive, Rawls’s constraints on political discourse are introduced for very good reasons. Finally, I argue that Wall’s self-defeat argument fails and that Rawls’s principle of legitimacy need not be revised, but is defendable in its current form.
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| 8. |
- Jagdmann, Jennifer, 1993-
(författare)
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Antibiotic resistance in the pan-genome of E. coli
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The pan-genome of a species is made up of all gene families that can be included in any individual isolate of the species. Escherichia coli (E. coli) has an open pan-genome including at least 128000 gene families, while only about half of the genes found in each individual isolate are common to all isolates. This indicates a great intraspecies genetic diversity that is not often considered when studying antibiotic resistance. This thesis uses a comparatively large collection of isolates to include more intraspecies genetic diversity and assess its impact on resistance.One angle of this approach was to study the impact of the pan-genome on spontaneous resistance development. For this, we compared the development of resistance to several antibiotics in a 35-strain collection of E. coli isolates. We found that frequencies of resistant mutants varied greatly between strains, that this variation was largely independent from the initial resistance level of the isolates, and that an isolate’s frequency of mutants for one antibiotic was a poor predictor of the mutant frequencies for other antibiotics. In conclusion, there was a clear impact of genetic diversity on spontaneous antibiotic resistance development. Using this approach, we observed a previously undescribed pattern of resistance development for tigecycline, a last-line antibiotic, via amplifications of a known efflux pump. In addition, we found a mutated allele of the pump with a reduced level of induction that did not allow for resistance development through amplifications. We showed that a fitness advantage at low antibiotics concentrations and clonal spread were likely contributing to the high occurrence of the mutated pump among E. coli isolates. While this efflux pump is common and well-studied, the lack of pre-existing knowledge of the mutated allele highlights the value of including many isolates in studies of antibiotic resistance. Another angle of this thesis was to determine whether intraspecies genetic diversity also impacts plasmid-borne resistance. For this, we transferred several multiresistance plasmids into a collection of E. coli hosts and characterized the plasmid-host combinations. We observed strain- and plasmid-dependent variations in resistance as well as inconsistencies in the clinical resistance categorization of different hosts with the same plasmid.In conclusion, this work reveals the impact of intraspecies genetic diversity on the development of antibiotic resistance, both through spontaneous mutations and the acquisition of resistance plasmids, highlighting the need to include intraspecies genetic diversity in studies of antibiotic resistance.
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| 9. |
- Knopp, Michael, 1984-
(författare)
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Mechanisms of Antibiotic Resistance Evolution
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The continuing emergence and spread of antibiotic resistant bacteria are a threat to various applications in modern medicine and impose a strong economic burden on health systems. The development of new antibiotics is slow and cannot counterbalance the dissemination of resistant bacteria. Thus, we need to find ways to reduce the rate of antibiotic resistance development. For this, we need to acquire a deeper understanding of the mechanisms underlying the evolution of antibiotic resistance.Here, we investigate the factors that govern how antibiotic resistance mechanisms affect bacterial fitness and the overall level of resistance. Using porin-deficient mutants of Escherichia coli, we show that upregulation of alternative porins provides compensatory mechanisms that can ameliorate the fitness costs associated with resistance. Furthermore, we demonstrate that the phenotypic effects of antibiotic resistance mutations are largely predictable, both in combination with each other as well as in different bacterial strains. However, outliers from this trend exemplify the limitations of solely relying on laboratory strains for the characterization of antibiotic resistance mechanisms. In contrast, strong epistatic interactions were observed in mutants evolved at sub-lethal concentrations of streptomycin. Despite these low concentrations and weak selective pressure, strains of Salmonella Typhimurium evolved high-level resistance, which followed completely different mutational pathways compared to high-level selection. Finally, we show that aminoglycoside resistance genes can be selected de novo from the expression of completely randomized nucleotide sequences. This demonstrates that new genes can arise from pools of non-coding sequences and that this process is relatively common.The studies presented in this thesis provide insights into the mechanistic basis of resistance evolution, including the mutational spectrum causing antibiotic resistance, compensatory pathways for growth-restoration and the influence of epistatic interactions on the phenotypic expression of resistance mutations. Understanding these factors in detail will enable us to better predict and prevent the emergence of antibiotic resistance development, through improvements in surveillance, treatment regimens and drug development.
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| 10. |
- Knöppel, Anna, 1984-
(författare)
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Experimental Evolution : and Fitness Effects of Mutations
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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%.
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