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Fitness-compensatory mutations in rifampicin-resistant RNA polymerase
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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. 

Place, publisher, year, edition, pages
2012. Vol. 85, no 1, 142-151 p.
Keyword [en]
Salmonella enterica, rpoA, rpoC, structure analysis
National Category
Natural Sciences
Research subject
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-172391DOI: 10.1111/j.1365-2958.2012.08099.xISI: 000305582700012OAI: oai:DiVA.org:uu-172391DiVA: diva2:514530
Available from: 2012-04-10 Created: 2012-04-10 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Growth in Aging Colonies: The Importance of Being Different
Open this publication in new window or tab >>Growth in Aging Colonies: The Importance of Being Different
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The accumulation of rifampicin resistant (RifR) mutants in aging bacterial colonies has previously been attributed to stress-induced mutagenesis. Mutations giving rise to RifR are located in the rpoB gene, coding for the β subunit of RNA polymerase, RpoB. We showed that these mutants accumulate because they grow faster than the wild-type in the aging colonies. We found no evidence of increased mutagenesis in the RifR cells and their distribution, as localized clones in the aging colonies, indicated that they accumulated by selection and growth rather than by an increased rate of mutagenesis. Colony competition experiments with reconstructed strains showed that the RifR mutations were responsible for the growth advantage. We also found that deletion of rpoS, coding for the stationary phase sigma factor (RpoS), also gives a growth advantage on bacterial cells in aging colonies.

We suggest that mutants lacking RpoS, having a different transcription pattern than the wild-type, may override the signals to enter stationary phase together with the rest of the population and instead keep growing for as long as possible. We found that the rpoB mutants mimicked the transcription pattern of the rpoS deletion mutant, thereby displaying a similar phenotype in the aging colonies. The pathways used in acetate metabolism (consisting of the enzymes Acs, AckA-Pta, PoxB and AceBAK) were shown to be important for the growth advantage mutants suggesting that acetate is one of the main carbon sources used to support their prolonged growth in the aging colonies.

Rifampicin is a first-line drug used to treat M. tuberculosis infections. We used S. enterica as a model system for experimental evolution to ask whether compensatory mutations might be important in RifR mutants. In every lineage evolved compensatory mutations arose without any significant reduction in resistance. These mutations altered genes for the α, β, and β’ subunits of RNA polymerase.  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 925
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-172393 (URN)978-91-554-8342-5 (ISBN)
Public defence
2012-05-25, B42, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2012-05-04 Created: 2012-04-10 Last updated: 2012-08-01Bibliographically approved
2. Biased Evolution: Causes and Consequences
Open this publication in new window or tab >>Biased Evolution: Causes and Consequences
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
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:nbn:se:uu:diva-276456 (URN)978-91-554-9518-3 (ISBN)
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

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Brandis, GerritWrande, MarieLiljas, LarsHughes, Diarmaid

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