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Biased Evolution: Causes and Consequences
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In evolution alternative genetic trajectories can potentially lead to similar phenotypic outcomes. However, certain trajectories are preferred over others. These preferences bias the genomes of living organisms and the underlying processes can be observed in ongoing evolution.

We have studied a variety of biases that can be found in bacterial chromosomes and determined the selective causes and functional consequences for the cell. We have quantified codon usage bias in highly expressed genes and shown that it is selected to optimise translational speed. We further demonstrated that the resulting differences in decoding speed can be used to regulate gene expression, and that the use of ‘non-optimal’ codons can be detrimental to reading frame maintenance. Biased gene location on the chromosome favours recombination between genes within gene families and leads to co-evolution. We have shown that such recombinational events can protect these gene families from inactivation by mobile genetic elements, and that chromosome organization can be selectively maintained because inversions can lead to the formation of unstable hybrid operons.

We have used the development of antibiotic resistance to study how different bacterial lifestyles influence evolutionary trajectories. For this we used two distinct pairs of antibiotics and disease-causing bacteria, namely (i) Mycobacterium tuberculosis that is treated with rifampicin and (ii) Escherichia coli that is treated with ciprofloxacin. We have shown that in the slow-growing Mycobacterium tuberculosis, resistance mutations are selected for high-level resistance. Fitness is initially less important, and over time fitness costs can be ameliorated by compensatory mutations. The need for rapid growth causes the selection of ciprofloxacin resistance in Escherichia coli not only to be selected on the basis of high-level resistance but also on high fitness. Compensatory evolution is therefore not required and is not observed.

Taken together, our results show that the evolution of a phenotype is the product of multiple steps and that many factors influence which trajectory is the most likely to occur and be most beneficial. Over time, selection will favour this particular trajectory and lead to biased evolution, affecting genome sequence and organization.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1198
Keyword [en]
Evolution, Codon usage bias, Post-transcriptional regulation, Recombination, Inversion, EF-Tu, Frameshift suppression, Antibiotic resistance, Rifampicin, Ciprofloxacin, Compensatory evolution, Drug efflux, RNA polymerase, DNA gyrase
National Category
Microbiology
Research subject
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-276456ISBN: 978-91-554-9518-3 (print)OAI: oai:DiVA.org:uu-276456DiVA: diva2:912652
Public defence
2016-05-09, A1:107a, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2016-04-13 Created: 2016-02-13 Last updated: 2016-04-21
List of papers
1. The Selective Advantage of Synonymous Codon Usage Bias in Salmonella
Open this publication in new window or tab >>The Selective Advantage of Synonymous Codon Usage Bias in Salmonella
2016 (English)In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 12, no 3, e1005926Article in journal (Refereed) Published
Abstract [en]

The genetic code in mRNA is redundant, with 61 sense codons translated into 20 different amino acids. Individual amino acids are encoded by up to six different codons but within codon families some are used more frequently than others. This phenomenon is referred to as synonymous codon usage bias. The genomes of free-living unicellular organisms such as bacteria have an extreme codon usage bias and the degree of bias differs between genes within the same genome. The strong positive correlation between codon usage bias and gene expression levels in many microorganisms is attributed to selection for translational efficiency. However, this putative selective advantage has never been measured in bacteria and theoretical estimates vary widely. By systematically exchanging optimal codons for synonymous codons in the tuf genes we quantified the selective advantage of biased codon usage in highly expressed genes to be in the range 0.2–4.2 x 10−4 per codon per generation. These data quantify for the first time the potential for selection on synonymous codon choice to drive genome-wide sequence evolution in bacteria, and in particular to optimize the sequences of highly expressed genes. This quantification may have predictive applications in the design of synthetic genes and for heterologous gene expression in biotechnology.

Keyword
EF-Tu, Synonymous codon usage bias
National Category
Evolutionary Biology Microbiology Genetics
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-276274 (URN)10.1371/journal.pgen.1005926 (DOI)000373268900033 ()
Funder
Swedish Research Council, 521-2013-2904Swedish Research Council, 621-2012-2188Swedish Foundation for Strategic Research , RBa08-0063Knut and Alice Wallenberg Foundation
Available from: 2016-02-10 Created: 2016-02-10 Last updated: 2017-11-30Bibliographically approved
2. Autoregulation of the tufB operon in Salmonella
Open this publication in new window or tab >>Autoregulation of the tufB operon in Salmonella
2016 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 100, no 6, 1004-1016 p.Article in journal (Refereed) Published
Abstract [en]

In Salmonella enterica and related species, translation elongation factor EF-Tu is encoded by two widely separated but near-identical genes, tufA and tufB. Two thirds of EF-Tu is expressed from tufA with the remaining one third coming from tufB. Inactivation of tufA is partly compensated by a doubling in the amount of EF-TuB but the mechanism of this up-regulation is unknown. By experimental evolution selecting for improved growth rate in a strain with an inactive tufA we selected six different noncoding or synonymous point mutations close to the tufB start codon. Based on these results we constructed a total of 161 different point mutations around the tufB start codon, as well as tufB 3'-truncations, and measured tufB expression using tufB-yfp transcriptional and translational fusions. The expression data support the presence of two competing stem-loop structures that can form in the 5'-end of the tufB mRNA. Formation of the 'closed' structure leads to Rho-dependent transcriptional termination of the tufB mRNA. We propose a model in which translational speed is used as a sensor for EF-Tu concentration and where the expression of tufB is post-transcriptionally regulated. This model describes for the first time how expression of the most abundant Salmonella protein is autoregulated.

Keyword
Salmonella enterica, tufA, tufB, EF-Tu, Rho, post-transcriptional regulation
National Category
Microbiology in the medical area
Research subject
Biology with specialization in Microbiology; Microbiology; Molecular Genetics
Identifiers
urn:nbn:se:uu:diva-235218 (URN)10.1111/mmi.13364 (DOI)000379687100008 ()26934594 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, KAW 2009.0251Swedish Research Council, 621-2012-2188; 521-2013-2904
Available from: 2014-10-29 Created: 2014-10-29 Last updated: 2017-12-05Bibliographically approved
3. High-level intrinsic suppression of a frameshift mutation in an essential gene
Open this publication in new window or tab >>High-level intrinsic suppression of a frameshift mutation in an essential gene
(English)Manuscript (preprint) (Other academic)
Keyword
rpoB, frameshift supression
National Category
Microbiology
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-276454 (URN)
Available from: 2016-02-13 Created: 2016-02-13 Last updated: 2016-04-21
4. Co-evolution protects the tuf genes from inactivation by mobile genetic elements
Open this publication in new window or tab >>Co-evolution protects the tuf genes from inactivation by mobile genetic elements
(English)Manuscript (preprint) (Other academic)
Keyword
tufA, tufB, recombination, evolution
National Category
Microbiology Evolutionary Biology
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-276280 (URN)
Available from: 2016-02-10 Created: 2016-02-10 Last updated: 2016-04-21
5. A large chromosomal inversion causes the formation of an unstable tufA/B hybrid operon
Open this publication in new window or tab >>A large chromosomal inversion causes the formation of an unstable tufA/B hybrid operon
(English)Manuscript (preprint) (Other academic)
Keyword
tufA, tufB, EF-G, IF2, chromosomal inversion
National Category
Microbiology Evolutionary Biology
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-276281 (URN)
Available from: 2016-02-10 Created: 2016-02-10 Last updated: 2016-04-21
6. Fitness-compensatory mutations in rifampicin-resistant RNA polymerase
Open this publication in new window or tab >>Fitness-compensatory mutations in rifampicin-resistant RNA polymerase
2012 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 85, no 1, 142-151 p.Article in journal (Refereed) Published
Abstract [en]

Mutations in rpoB (RNA polymerase beta-subunit) can cause high-level resistance to rifampicin, an important first-line drug against tuberculosis. Most rifampicin-resistant (RifR) mutants selected in vitro have reduced fitness, and resistant clinical isolates of M. tuberculosis frequently carry multiple mutations in RNA polymerase genes. This supports a role for compensatory evolution in global epidemics of drug-resistant tuberculosis but the significance of secondary mutations outside rpoB has not been demonstrated or quantified. Using Salmonella as a model organism, and a previously characterized RifR mutation (rpoB R529C) as a starting point, independent lineages were evolved with selection for improved growth in the presence and absence of rifampicin. Compensatory mutations were identified in every lineage and were distributed between rpoA, rpoB and rpoC. Resistance was maintained in all strains showing that increased fitness by compensatory mutation was more likely than reversion. Genetic reconstructions demonstrated that the secondary mutations were responsible for increasing growth rate. Many of the compensatory mutations in rpoA and rpoC individually caused small but significant reductions in susceptibility to rifampicin, and some compensatory mutations in rpoB individually caused high-level resistance. These findings show that mutations in different components of RNA polymerase are responsible for fitness compensation of a RifR mutant. 

Keyword
Salmonella enterica, rpoA, rpoC, structure analysis
National Category
Natural Sciences
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-172391 (URN)10.1111/j.1365-2958.2012.08099.x (DOI)000305582700012 ()
Available from: 2012-04-10 Created: 2012-04-10 Last updated: 2017-12-07Bibliographically approved
7. Genetic characterization of compensatory evolution in strains carrying rpoB Ser531Leu, the rifampicin resistance mutation most frequently found in clinical isolates
Open this publication in new window or tab >>Genetic characterization of compensatory evolution in strains carrying rpoB Ser531Leu, the rifampicin resistance mutation most frequently found in clinical isolates
2013 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 11, 2493-2497 p.Article in journal (Refereed) Published
Abstract [en]

Objectives: The evolution of rifampicin resistance in Mycobacterium tuberculosis is a major threat to effective tuberculosis therapy. Much is known about the initial emergence of rifampicin resistance, but the further evolution of these resistant strains has only lately been subject to investigation. Although resistance can be caused by many different mutations in rpoB, among clinical M. tuberculosis isolates the mutation rpoB S531L is overwhelmingly the most frequently found. Clinical isolates with rpoB S531L frequently carry additional mutations in genes for RNA polymerase subunits, and it has been speculated that these are fitness-compensatory mutations, ameliorating the fitness cost of the primary resistance mutation. We tested this hypothesis using Salmonella as a model organism. Methods: We created the rpoB S531L mutation in Salmonella and then evolved independent lineages with selection for mutants with increased relative fitness. Relative fitness associated with putative compensatory mutations was measured after genetic reconstruction in isogenic strains. Results: Compensatory mutations were identified in genes coding for different subunits of RNA polymerase: rpoA, rpoB and rpoC. Genetic reconstructions demonstrated that each of these secondary mutations reduced the fitness cost of the rpoB S531L resistance mutation. Conclusions: The compensatory mutations identified in Salmonella cluster in similar locations to the additional mutations found in M. tuberculosis isolates. These new data strongly support the idea that many of the previously identified rpoA, rpoB and rpoC mutations in rifampicin-resistant M. tuberculosis (rpoB S531L) are indeed fitness-compensatory mutations.

Keyword
antibiotic resistance, S531L, Mycobacterium tuberculosis
National Category
Medical and Health Sciences Natural Sciences
Identifiers
urn:nbn:se:uu:diva-213822 (URN)10.1093/jac/dkt224 (DOI)000326978600014 ()
Available from: 2014-01-05 Created: 2014-01-04 Last updated: 2017-12-06
8. Evidence for the critical role of a secondary site rpoB mutation in the compensatory evolution and successful transmission of an MDR tuberculosis outbreak strain
Open this publication in new window or tab >>Evidence for the critical role of a secondary site rpoB mutation in the compensatory evolution and successful transmission of an MDR tuberculosis outbreak strain
Show others...
2016 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 2, 324-332 p.Article in journal (Refereed) Published
Abstract [en]

Background MDR Mycobacterium tuberculosis clinical strains that cause large outbreaks, particularly among HIV-negative patients, are likely to have undergone the most successful compensatory evolution. Hence, mutations secondary to the acquisition of drug resistance are worthy of consideration in these highly transmissible strains. Here, we assessed the role of a mutation within rpoBrpoB V615M, secondary to the rifampicin resistance-conferring mutation rpoB S531L, which is associated with a major MDR tuberculosis outbreak strain that evolved in an HIV-negative context in northern Tunisia.

Methods Using BCG as a model organism, we engineered strains harbouring either the rpoB S531L mutation alone or the double mutation rpoB S531L, V615M. Individual and competitive in vitro growth assays were performed in order to assess the relative fitness of each BCG mutant.

Results The rpoB V615M mutation was found to be invariably associated with rpoB S531L. Structural analysis mapped rpoB V615M to the same bridge helix region as rpoB compensatory mutations previously described in Salmonella. Compared with the rpoB single-mutant BCG, the double mutant displayed improved growth characteristics and fitness rates equivalent to WT BCG. Strikingly, the rpoB double mutation conferred high-level resistance to rifampicin.

Conclusions Here, we demonstrated the fitness compensatory role of a mutation within rpoB, secondary to the rifampicin resistance mutation rpoB S531L, which is characteristic of an MDR M. tuberculosis major outbreak strain. The finding that this secondary mutation concomitantly increased the resistance level to rifampicin argues for its significant contribution to the successful transmission of the MDR-TB strain.

Keyword
M. tuberculosis, rpoB, compensatory evolution
National Category
Microbiology
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-276455 (URN)10.1093/jac/dkv345 (DOI)000372427600007 ()26538504 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 245 872
Available from: 2016-02-13 Created: 2016-02-13 Last updated: 2017-11-30Bibliographically approved
9. Comprehensive phenotypic characterization of rifampicin resistance mutations in Salmonella provides insight into the evolution of resistance in Mycobacterium tuberculosis
Open this publication in new window or tab >>Comprehensive phenotypic characterization of rifampicin resistance mutations in Salmonella provides insight into the evolution of resistance in Mycobacterium tuberculosis
2015 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 70, no 3, 680-685 p.Article in journal (Refereed) Published
Abstract [en]

Objectives: Mutations in the beta-subunit of RNA polymerase (RNAP), encoded by rpoB, are responsible for rifampicin resistance (Rif(R)). Although many mutations in rpoB can reduce susceptibility, only a few are frequent amongst Rif(R) clinical Mycobacterium tuberculosis (MTB) isolates. It has been suggested that there is a negative correlation between the fitness costs of Rif(R) mutations and their respective clinical frequency, but so far comparable fitness cost measurements have only been conducted for a very limited number of Rif(R) mutations. We tested this hypothesis using Salmonella and Mycobacterium smegmatis as model organisms. Methods: We constructed 122 different Rif(R) mutations in Salmonella. MICs and relative fitness costs in the presence and absence of rifampicin were determined for each mutant, including for a smaller number of Rif(R) M. smegmatis strains. Results were compared with available mutation frequency data from clinical MTB isolates. Results: (i) Rif(R) mutations frequently found in MTB isolates have a fitness cost in Salmonella Typhimurium and M. smegmatis. (ii) Clinically frequent Rif(R) mutations have a high rifampicin MIC. (iii) There is a strong correlation between the magnitude of the fitness cost of a Rif(R) mutation in Salmonella Typhimurium or M. smegmatis and the frequency with which that mutation is associated with secondary (putative compensatory) mutations in RNAP of clinical MTB isolates. Conclusions: This suggests that the success of Rif(R) mutations in clinical MTB isolates may be dependent not only on a low initial fitness cost, but rather the results of three factors: (i) a high rifampicin MIC; (ii) a relatively low initial fitness cost; and (iii) the ability to additionally acquire compensatory mutations selected to further reduce fitness cost.

Keyword
Rif(R), resistance genetics, M. tuberculosis
National Category
Infectious Medicine Microbiology in the medical area Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-251501 (URN)10.1093/jac/dku434 (DOI)000350214700008 ()25362573 (PubMedID)
Available from: 2015-04-22 Created: 2015-04-20 Last updated: 2017-12-04
10. Experimental evolution identifies a new class of genes selected during the development of ciprofloxacin resistance in Escherichia coli
Open this publication in new window or tab >>Experimental evolution identifies a new class of genes selected during the development of ciprofloxacin resistance in Escherichia coli
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-264883 (URN)
Available from: 2015-10-20 Created: 2015-10-19 Last updated: 2016-04-21
11. RNA polymerase mutations contribute to the evolution of ciprofloxacin resistance in Escherichia coli
Open this publication in new window or tab >>RNA polymerase mutations contribute to the evolution of ciprofloxacin resistance in Escherichia coli
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-264876 (URN)
Available from: 2015-10-20 Created: 2015-10-19 Last updated: 2016-04-21
12. Mutation supply and relative fitness shape the genotypes of ciprofloxacin-resistant Escherichia coli
Open this publication in new window or tab >>Mutation supply and relative fitness shape the genotypes of ciprofloxacin-resistant Escherichia coli
Show others...
2017 (English)In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 34, no 5, 1029-1039 p.Article in journal (Refereed) Published
Abstract [en]

Ciprofloxacin is an important antibacterial drug targeting Type II topoisomerases, highly active against Gram-negatives including Escherichia coli. The evolution of resistance to ciprofloxacin in E. coli always requires multiple genetic changes, usually including mutations affecting two different drug target genes, gyrA and parC. Resistant mutants selected in vitro or in vivo can have many different mutations in target genes and efflux regulator genes that contribute to resistance. Among resistant clinical isolates the genotype, gyrA S83L D87N, parC S80I is significantly overrepresented suggesting that it has a selective advantage. However, the evolutionary or functional significance of this high frequency resistance genotype is not fully understood. By combining experimental data and mathematical modeling, we addressed the reasons for the predominance of this specific genotype. The experimental data were used to model trajectories of mutational resistance evolution under different conditions of drug exposure and population bottlenecks. We identified the order in which specific mutations are selected in the clinical genotype, showed that the high frequency genotype could be selected over a range of drug selective pressures, and was strongly influenced by the relative fitness of alternative mutations and factors affecting mutation supply. Our data map for the first time the fitness landscape that constrains the evolutionary trajectories taken during the development of clinical resistance to ciprofloxacin and explain the predominance of the most frequently selected genotype. This study provides strong support for the use of in vitro competition assays as a tool to trace evolutionary trajectories, not only in the antibiotic resistance field.

Keyword
ciprofloxacin, multistep evolution, population bottleneck, modeling evolution, clinical isolates
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-264874 (URN)10.1093/molbev/msx052 (DOI)28087782 (PubMedID)
Funder
Swedish Research CouncilSwedish Foundation for Strategic Research Knut and Alice Wallenberg Foundation
Available from: 2015-10-20 Created: 2015-10-19 Last updated: 2017-05-11Bibliographically approved

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