| 1. |
- Bergman, Jessica M.
(author)
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Genetics and Growth Regulation in Salmonella enterica
- 2014
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Doctoral thesis (other academic/artistic)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
(author)
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Multi-omics investigation into bacterial evolution
- 2022
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Doctoral thesis (other academic/artistic)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-
(author)
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Antibiotic Resistance: A Multimethod Investigation of Individual Responsibility and Behaviour
- 2021
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Doctoral thesis (other academic/artistic)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. |
- Jagdmann, Jennifer, 1993-
(author)
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Antibiotic resistance in the pan-genome of E. coli
- 2022
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Doctoral thesis (other academic/artistic)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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| 5. |
- Knöppel, Anna, 1984-
(author)
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Experimental Evolution : and Fitness Effects of Mutations
- 2016
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Doctoral thesis (other academic/artistic)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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| 6. |
- Lind, Peter A, 1980-
(author)
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Evolutionary Dynamics of Mutation and Gene Transfer in Bacteria
- 2010
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Doctoral thesis (other academic/artistic)abstract
- The study of bacterial evolution is fundamental for addressing current problems of antibiotic resistance and emerging infectious diseases and lays a solid foundation for successful and rational design in biotechnology and synthetic biology. The main aim of this thesis is to test evolutionary hypotheses, largely based on theoretical considerations and sequence analysis, by designing scenarios in a laboratory setting to obtain experimental data. Paper I examines how genomic GC-content can be reduced following a change in mutation rate and spectrum. Transcription-related biases in mutation location were found, but no replicative bias was detected. Paper II explores the distribution of fitness effects of random substitutions in two ribosomal protein genes using a highly sensitive fitness assay. The substitutions had a weakly deleterious effect, with low frequencies of both neutral and inactivating mutations. The surprising finding that synonymous and non-synonymous substitutions have very similar distribution of fitness effects suggests that, at least for these genes, fitness constraints are present mainly on the level of mRNA instead of protein. Paper III examines selective barriers to inter-species gene transfer by constructing mutants with a native gene replaced by an orthologue from another species. Results suggest that the fitness costs of these gene replacements are large enough to provide a barrier to this kind of horizontal gene transfer in nature. The paper also examines possible compensatory mechanisms that can reduce the cost of the poorly functioning alien genes and found that gene amplification acts as a first step to improve the selective contribution after transfer. Paper IV investigates the fitness constraints on horizontal gene transfer by inserting DNA from other species into the Salmonella chromosome. Results suggest that insertion of foreign DNA often is neutral and the manuscript provides new experimental data for theoretical analysis of interspecies genome variation and horizontal gene transfer between species.
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| 7. |
- Linkevičius, Marius, 1985-
(author)
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Evolution and Mechanisms of Tigecycline Resistance in Escherichia coli
- 2015
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Doctoral thesis (other academic/artistic)abstract
- Antibiotic resistance is an ongoing global medical crisis and we are in great need of new antibacterial agents to combat rapidly emerging resistant pathogens. Tigecycline is one of few drugs that have been introduced into medicine during the last two decades. It is a broad-spectrum third generation tetracycline that is active against multidrug-resistant bacteria that cause complicated infections.In this thesis I examined the development of tigecycline resistance in Escherichia coli and associated in vitro and in vivo fitness effects. Selections of spontaneous E. coli mutants revealed relatively high accumulation rates of changes in the multidrug efflux system AcrAB-TolC regulation network and in heptose biosynthesis and transport pathways important for lipopolysaccharide (LPS) synthesis. Both groups of mutations led to reduced susceptibility to tigecycline and slower growth compared to the wild-type bacteria. Additional in vitro fitness assays and in vivo competitions showed that LPS mutants were less fit than efflux mutants, providing a possible explanation for why up-regulation of multidrug efflux pumps is the main tigecycline resistance mechanism reported in clinical isolates.Tigecycline was designed to evade the two most common tetracycline resistance mechanisms conferred by Tet proteins, efflux and ribosomal protection. However, tigecycline is a substrate for the tetracycline modifying enzyme Tet(X). Screening of Tet protein mutant libraries showed that it is possible to select Tet mutants with minimal inhibitory concentrations of tigecycline that reach clinically relevant levels. Mutations in Tet proteins that permitted a better protection from tigecycline frequently exhibited reduced activity against earlier generations of tetracyclines, except for the Tet(X) enzyme mutants, which were better at inactivating all tested tetracyclines. This is particularly worrisome because different variants of Tet(X) have recently spread to multidrug-resistant pathogens through horizontal gene transfer. Therefore, Tet(X) mutants with improved activity threaten the medical future of tetracyclines.Multidrug resistance is easily disseminated through horizontally spreading conjugative plasmids. pUUH239.2 is an example of a successful conjugative plasmid that caused the first clonal outbreak of extended spectrum β-lactamase-producing Klebsiella pneumoniae in Scandinavia. This plasmid was formed after rearrangements between two different plasmid backbones and it carries resistance genes to multiple antibiotic classes, heavy metals, and detergents.
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| 8. |
- Lofton Tomenius, Hava, 1976-
(author)
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Mechanisms and Biological Costs of Bacterial Resistance to Antimicrobial Peptides
- 2016
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Doctoral thesis (other academic/artistic)abstract
- The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development.In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4.The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways.Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host.In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.
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| 9. |
- Thulin, Elisabeth, 1984-
(author)
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Mechanisms and Dynamics of Mecillinam Resistance in Escherichia coli
- 2017
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Doctoral thesis (other academic/artistic)abstract
- The introduction of antibiotics in healthcare is one of the most important medical achievements with regard to reducing human morbidity and mortality. However, bacterial pathogens have acquired antibiotic resistance at an increasing rate, and due to a high prevalence of resistance to some antibiotics they can no longer be used therapeutically. The antibiotic mecillinam, which inhibits the penicillin-binding protein PBP2, however, is an exception since mecillinam resistance (MecR) prevalence has remained low. This is particularly interesting since laboratory experiments have shown that bacteria can rapidly acquire MecR mutations by a multitude of different types of mutations.In this thesis, I examined mechanisms and dynamics of mecillinam resistance in clinical and laboratory isolates of Escherichia coli. Only one type of MecR mutations (cysB) was found in the clinical strains, even though laboratory experiments demonstrate that more than 100 genes can confer resistance Fitness assays showed that cysB mutants have higher fitness than most other MecR mutants, which is likely to contribute to their dominance in clinical settings.To determine if the mecillinam resistant strains could compensate for their fitness cost, six different MecR mutants (cysB, mrdA, spoT, ppa, aspS and ubiE) were evolved for 200-400 generations. All evolved mutants showed increased fitness, but the compensation was associated with loss of resistance in the majority of cases. This will also contribute to the rarity of clinical MecR isolates with chromosomal resistance mutations.How MecR is mediated by cysB mutations was previously unclear, but in this thesis I propose and test a model for the mechanism of resistance. Thus, inactivation of CysB results in cellular depletion of cysteine that triggers an oxidative stress response. The response alters the intracellular levels of 450 proteins, and MecR is achieved by the increase of two of these, the LpoB and PBP1B proteins, which rescue the cells with a mecillinam-inhibited PBP2.Mecillinam is used for UTI treatments and to investigate mecillinam resistance in a more host-like milieu, MecR strains were grown in urine and resistance was examined. Interestingly, this study showed that neither laboratory, nor clinical cysB mutants are resistant in urine, most likely because the cysteine present in the urine phenotypically reverts the bacteria to susceptibility. These findings suggest that mecillinam can be used to treat also those clinical strains that are identified as MecR in standard laboratory tests, and that testing of mecillinam susceptibility in the laboratory ought to be performed in media that mimics urine to obtain clinically relevant results.In summary, the work described in this thesis has increased ourgeneral knowledge of mecillinam resistance and its evolution. Hopefully this knowledge can be put to good use in clinical settings to reduce the negative impact of antibiotic resistance.
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| 10. |
- Tobin, Christina, 1980-
(author)
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Removal and Replacement of Ribosomal Proteins : Effects on Bacterial Fitness and Ribosome Function
- 2011
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Doctoral thesis (other academic/artistic)abstract
- Protein synthesis is a complex process performed by sophisticated cellular particles known as ribosomes. Although RNA constitutes the major structural and functional component, ribosomes from all kingdoms contain an extensive array of proteins with largely undefined functional roles. The work presented in this thesis addresses ribosomal complexity using mutants of Salmonella typhimurium to examine the physiological effects of ribosomal protein (r-protein) removal and orthologous replacement on bacterial fitness and ribosome function. The results of paper I demonstrate that removal of small subunit protein S20 conferred two independent translation initiation defects: (i) a significant reduction in the rate and extent of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in subunit association. The topographical location of S20 in mature 30S subunits suggests that these perturbations are the result of improper orientation of helix 44 of the 16S rRNA when S20 is absent. In paper II we show that the major functional impairment associated with loss of large subunit protein L1 manifested as an increase in free ribosomal subunits at the expense of translationally active 70S particles. Furthermore, the formation of free ribosomal subunits was imbalanced suggesting that L1 is required to suppress degradation or promote formation of 30S subunits. Compensatory evolution revealed that mutations in other large subunit proteins mitigate the cost of L1 removal, in one case seemingly via an increase in 70S complex formation. As shown in paper III, the large fitness costs associated with complete removal of r-proteins is in contrast to the generally mild costs of orthologous protein replacement, even in the absence of a high degree of homology to the native protein. This clearly demonstrates the robustness and plasticity of the ribosome and protein synthesis in general and it also implies that functional constraints are highly conserved between these proteins. The findings of paper III also allowed us to examine the barriers that constrain horizontal gene transfer and we find that increased gene dosage of the sub-optimal heterologous protein may be an initial response to stabilize deleterious transfer events. Overall the results highlight the requirement of r-proteins for the maintenance of ribosomal structural integrity.
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