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Dynamics and Mechanisms of Adaptive Evolution in Bacteria
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 770
Keyword [en]
adaptive evolution, mutation, genome rearrangements, antibiotic resistance, gene amplification, genome reduction, directed evolution
National Category
Microbiology
Research subject
Biology with specialization in Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-172786ISBN: 978-91-554-8354-8 (print)OAI: oai:DiVA.org:uu-172786DiVA: diva2:515938
Public defence
2012-06-05, C10:305, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2012-05-14 Created: 2012-04-15 Last updated: 2012-08-01Bibliographically approved
List of papers
1. Evolution of new gene functions: simulation and analysis of the amplification model.
Open this publication in new window or tab >>Evolution of new gene functions: simulation and analysis of the amplification model.
2009 (English)In: Genetica, ISSN 0016-6707, E-ISSN 1573-6857, Vol. 135, no 3, 309-324 p.Article in journal (Refereed) Published
Abstract [en]

Creation of new genes and functions is a central feature of evolution. Duplication of existing genes has long been assumed to be the source of new genes, but the precise mechanism has remained unclear. One suggestion is that new genes are created via temporary amplifications, which simultaneously increase both the selective advantage of weak, pre-existing secondary functions and the target for optimizing mutations. This paper examines the amplification model by formalizing it into a mathematical framework. This framework is used to perform stochastic (Monte Carlo) simulations. In addition, experimental data from Salmonella typhimurium LT2 are used to support the modelling, by providing estimates for parameter values. The results show that amplification of tandem repeats is likely to contribute to creation of new genes in nature.

Keyword
Gene amplification, gene innovation, population dynamics, stochastic modelling
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-98894 (URN)10.1007/s10709-008-9289-z (DOI)000263833900007 ()18568430 (PubMedID)
Available from: 2009-03-04 Created: 2009-03-04 Last updated: 2017-12-13Bibliographically approved
2. Contribution of gene amplification to evolution of increased antibiotic resistance in Salmonella typhimurium
Open this publication in new window or tab >>Contribution of gene amplification to evolution of increased antibiotic resistance in Salmonella typhimurium
2009 (English)In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 182, no 4, 1183-1195 p.Article in journal (Refereed) Published
Abstract [en]

The use of beta-lactam antibiotics has led to the evolution and global spread of a variety of resistance mechanisms, including beta-lactamases, a group of enzymes that degrade the beta-lactam ring. The evolution of increased beta-lactam resistance was studied by exposing independent lineages of Salmonella typhimurium to progressive increases in cephalosporin concentration. Each lineage carried a beta-lactamase gene (bla(TEM-1)) that provided very low resistance. In most lineages, the initial response to selection was an amplification of the bla(TEM-1) gene copy number. Amplification was followed in some lineages by mutations (envZ, cpxA, or nmpC) that reduced expression of the uptake functions, the OmpC, OmpD, and OmpF porins. The initial resistance provided by bla(TEM-1) amplification allowed the population to expand sufficiently to realize rare secondary point mutations. Mathematical modeling showed that amplification often is likely to be the initial response because events that duplicate or further amplify a gene are much more frequent than point mutations. These models show the importance of the population size to appearance of later point mutations. Transient gene amplification is likely to be a common initial mechanism and an intermediate in stable adaptive improvement. If later point mutations (allowed by amplification) provide sufficient adaptive improvement, the amplification may be lost.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-119727 (URN)10.1534/genetics.109.103028 (DOI)000270214000021 ()19474201 (PubMedID)
Available from: 2010-03-01 Created: 2010-03-01 Last updated: 2017-12-12Bibliographically approved
3. Selection-driven genome reduction in bacteria
Open this publication in new window or tab >>Selection-driven genome reduction in bacteria
2012 (English)In: PLOS genetics, ISSN 1553-7404, Vol. 8, no 6, e1002787- p.Article in journal (Refereed) Published
Abstract [en]

Gene loss by deletion is a common evolutionary process in bacteria, as exemplified by bacteria with small genomes that have evolved from bacteria with larger genomes by reductive processes. The driving force(s) for genome reduction remains unclear, and here we examined the hypothesis that gene loss is selected because carriage of superfluous genes confers a fitness cost to the bacterium. In the bacterium Salmonella enterica, we measured deletion rates at 11 chromosomal positions and the fitness effects of several spontaneous deletions. Deletion rates varied over 200-fold between different regions with the replication terminus region showing the highest rates. Approximately 25% of the examined deletions caused an increase in fitness under one or several growth conditions, and after serial passage of wild-type bacteria in rich medium for 1,000 generations we observed fixation of deletions that substantially increased bacterial fitness when reconstructed in a non-evolved bacterium. These results suggest that selection could be a significant driver of gene loss and reductive genome evolution.

Keyword
selection, genome reduction, bacteria
National Category
Genetics
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-172783 (URN)10.1371/journal.pgen.1002787 (DOI)000305961000046 ()
Available from: 2012-04-15 Created: 2012-04-15 Last updated: 2012-08-01Bibliographically approved
4. High frequencies of genome rearrangements in bacterial chromosomes
Open this publication in new window or tab >>High frequencies of genome rearrangements in bacterial chromosomes
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Genome rearrangements have important effects on bacterial phenotypes and influence the evolution of bacterial genomes. Conventional strategies for characterizing rearrangements in bacterial genomes rely on comparisons of sequenced genomes from related species. However, the spectra of spontaneous rearrangements in supposedly homogenous and clonal bacterial populations are still poorly characterized. Here we used 454 pyrosequencing technology and a ‘split mapping’ computational method to identify unique junction sequences caused by spontaneous genome rearrangements in chemostat cultures of Salmonella enterica Var. Typhimurium LT2. We were able to confirm 22 unique junction sequences with a junction homology more than 10bp and this led to an estimation of 51 true junction sequences, of which 28, 12 and 11 were likely to be formed by deletion, duplication and inversion events, respectively.  All experimentally confirmed rearrangements had short inverted (inversions) or direct (deletions and duplications) homologous repeat sequences at the endpoints. This study demonstrates the feasibility of genome wide characterization of spontaneous genome rearrangements in bacteria and the high steady-state frequency of rearrangements in bacterial populations.

Keyword
genome rearrangements, bacteria
National Category
Genetics
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-172784 (URN)
Available from: 2012-04-15 Created: 2012-04-15 Last updated: 2012-08-01
5. Genetic analysis of colistin resistance in Salmonella enterica serovar Typhimurium
Open this publication in new window or tab >>Genetic analysis of colistin resistance in Salmonella enterica serovar Typhimurium
2009 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 53, no 6, 2298-2305 p.Article in journal (Refereed) Published
Abstract [en]

Colistin is a cyclic cationic peptide that kills gram-negative bacteria by interacting with and disrupting the outer membrane. We isolated 44 independent mutants in Salmonella enterica serovar Typhimurium with reduced susceptibility to colistin and identified 27 different missense mutations located in the pmrA and pmrB genes (encoding the regulator and sensor of a two-component regulatory system) that conferred increased resistance. By comparison of the two homologous sensor kinases, PmrB and EnvZ, the 22 missense mutations identified in pmrB were shown to be located in four different structural domains of the protein. All five pmrA mutations were located in the phosphate receiver domain of the regulator protein. The mutants appeared at a mutation rate of 0.6 x 10(-6) per cell per generation. The MICs of colistin for the mutants increased 2- to 35-fold, and the extent of killing was reduced several orders of magnitude compared to the susceptible strain. The growth rates of the mutants were slightly reduced in both rich medium and M9-glycerol minimal medium, whereas growth in mice appeared unaffected by the pmrA and pmrB mutations. The low fitness costs and the high mutation rate suggest that mutants with reduced susceptibility to colistin could emerge in clinical settings.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-119728 (URN)10.1128/AAC.01016-08 (DOI)000266244500010 ()19332669 (PubMedID)
Available from: 2010-03-01 Created: 2010-03-01 Last updated: 2017-12-12Bibliographically approved
6. Evolution of increased ß-lactam resistance in an engineered Metallo-ß-lactamase
Open this publication in new window or tab >>Evolution of increased ß-lactam resistance in an engineered Metallo-ß-lactamase
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The extensive use and misuse of antibiotics during the last 60 years has led to the evolution and global spread of a variety of resistance mechanisms. Of high medical importance are ß-lactamases, a group of enzymes that can hydrolyze the ß-lactam ring present in all ß-lactam antibiotics. Metallo-ß-lactamases (MBLs) are particularly problematic due to their ability to hydrolyze virtually all classes of ß-lactam antibiotics. A novel MBL (evMBL9) with low-level resistance against ß-lactam antibiotics was designed and employed as the ancestral MBL during an experiment to examine how an enzyme evolved towards increased resistance. We designed and synthesized a mutant library in which the substrate binding profile was varied by randomizing six amino acid residues. Mutants with increased resistance against seven different ß-lactam antibiotics (penicillin G, ampicillin, cefalotin, cefaclor, cefuroxime, cefoperazone and cefotaxime) were isolated and characterized. For the majority of mutants, in spite of their significantly increased resistance, both mRNA and protein levels were reduced (up to >20 fold) relative to those of parental evMBL9, indicating that the catalytic activities of these mutant MBLs were highly increased. Multivariate analysis showed that the majority of mutant enzymes became generalists, conferring increased resistance against most of the examined ß-lactams. The increased resistance and decreased protein level suggest that the improved hydrolysis in these novel MBLs is associated with decreased protein stability.

Keyword
Metallo-beta-lactamase, directed protein evolution
National Category
Other Biological Topics
Identifiers
urn:nbn:se:uu:diva-172785 (URN)
Available from: 2012-04-15 Created: 2012-04-15 Last updated: 2012-08-01

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