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Growth in Aging Colonies: The Importance of Being Different
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
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: urn:nbn:se:uu:diva-172393ISBN: 978-91-554-8342-5 (print)OAI: oai:DiVA.org:uu-172393DiVA: diva2:514535
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
List of papers
1. Accumulation of mutants in "aging" bacterial colonies is due to growth under selection, not stress-induced mutagenesis
Open this publication in new window or tab >>Accumulation of mutants in "aging" bacterial colonies is due to growth under selection, not stress-induced mutagenesis
2008 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 33, 11863-11868 p.Article in journal (Refereed) Published
Abstract [en]

Several bacterial systems show behavior interpreted as evidence for stress-induced mutagenesis (adaptive mutation), a postulated process by which nongrowing cells temporarily increase their general mutation rate. Theoretical considerations suggest that periodic stress-induced general mutagenesis would not be advantageous in the long term, due to the high cost of deleterious mutations. Alternative explanations have been tested for very few of the systems used as evidence for stress-induced mutation. In one prominent system, mutants resistant to rifampicin (RifR; rpoB; RNA polymerase) accumulate in cell populations that “age” on solid medium with little net growth. Mutant accumulation was initially attributed to stress-induced general mutagenesis in nongrowing cells. Evidence is presented that these RifR mutants accumulate because they grow faster than parent cells during the aging period. Direct tests revealed no increase in the frequency of other mutant types during the aging period.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-109091 (URN)10.1073/pnas.0804739105 (DOI)000258723800051 ()
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13Bibliographically approved
2. Importance of acetate utilization for the growth of mutant sub-populations in aging bacterial colonies
Open this publication in new window or tab >>Importance of acetate utilization for the growth of mutant sub-populations in aging bacterial colonies
Show others...
(English)Article in journal (Refereed) Submitted
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-172392 (URN)
Available from: 2012-04-10 Created: 2012-04-10 Last updated: 2012-08-01Bibliographically approved
3. 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

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