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  • 1.
    Brandis, Gerrit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Biased Evolution: Causes and Consequences2016Doctoral 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.

    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, article id 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.

    Keywords
    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, p. 1004-1016Article 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.

    Keywords
    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: 2018-01-11Bibliographically 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)
    Keywords
    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)
    Keywords
    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)
    Keywords
    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, p. 142-151Article 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. 

    Keywords
    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, p. 2493-2497Article 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.

    Keywords
    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, p. 324-332Article 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.

    Keywords
    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, p. 680-685Article 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.

    Keywords
    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: 2018-01-11
    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, p. 1029-1039Article 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.

    Keywords
    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)000399373300001 ()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: 2018-09-04Bibliographically approved
  • 2.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bergman, Jessica M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Autoregulation of the tufB operon in Salmonella2016In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 100, no 6, p. 1004-1016Article in journal (Refereed)
    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.

  • 3.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Co-evolution with recombination affects the stability of mobile genetic element insertions within gene families of Salmonella2018In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 108, no 6, p. 697-710Article in journal (Refereed)
    Abstract [en]

    Bacteria can have multiple copies of a gene at separate locations on the same chromosome. Some of these gene families, including tuf (translation elongation factor EF-Tu) and rrl (ribosomal RNA), encode functions critically important for bacterial fitness. Genes within these families are known to evolve in concert using homologous recombination to transfer genetic information from one gene to another. This mechanism can counteract the detrimental effects of nucleotide sequence divergence over time. Whether such mechanisms can also protect against the potentially lethal effects of mobile genetic element insertion is not well understood. To address this we constructed two different length insertion cassettes to mimic mobile genetic elements and inserted these into various positions of the tuf and rrl genes. Wemeasured rates of recombinational repair that removed the inserted cassette and studied the underlying mechanism. Our results indicate that homologous recombination can protect the tuf and rrl genes from inactivation by mobile genetic elements, but forinsertions within shorter gene sequences the efficiency of repair is very low. Intriguingly, we found that physical distance separating genes on the chromosome directly affects the rate of recombinational repair suggesting that relative location will influence the ability of homologous recombination to maintain homogeneity.

  • 4.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Measuring Homologous Recombination Rates between Chromosomal Locations in Salmonella2019In: BIO-PROTOCOL, ISSN 2331-8325, Vol. 9, no 3, article id e3159Article in journal (Refereed)
    Abstract [en]

    Homologous recombination between two similar DNA molecules, plays an important role in the repair of double-stranded DNA breaks. Recombination can occur between two sister chromosomes, or between two locations of similar sequence identity within the same chromosome. The assay described here is designed to measure the rate of homologous recombination between two locations with sequence similarity within the same bacterial chromosome. For this purpose, a selectable/counter-selectable genetic cassette is inserted into one of the locations and homologous recombination repair rates are measured as a function of recombinational removal of the inserted cassette. This recombinational repair process is called gene conversion, non-reciprocal recombination. We used this method to measure the recombination rates between genes within gene families and to study the stability of mobile genetic elements inserted into members of gene families.

  • 5.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huseby, Douglas L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Having your cake and eating it - Staphylococcus aureus small colony variants can evolve faster growth rate without losing their antibiotic resistance2017In: MICROBIAL CELL, ISSN 2311-2638, Vol. 4, no 8, p. 275-277Article in journal (Refereed)
    Abstract [en]

    Staphylococcus aureus can produce small colony variants (SCVs) during infections. These cause significant clinical problems because they are difficult to detect in standard microbiological screening and are associated with persistent infections. The major causes of the SCV phenotype are mutations that inhibit respiration by inactivation of genes of the menadione or hemin biosynthesis pathways. This reduces the production of ATP required to support fast growth. Importantly, it also decreases cross-membrane potential in SCVs, resulting in decreased uptake of cationic compounds, with reduced susceptibility to aminoglycoside antibiotics as a consequence. Because SCVs are slow-growing (mutations in men genes are associated with growth rates in rich medium similar to 30% of the wild-type growth rate) bacterial cultures are very susceptible to rapid takeover by faster-growing mutants (revertants or suppressors). In the case of reversion, the resulting fast growth is obviously associated with the loss of antibiotic resistance. However, direct reversion is relatively rare due to the very small genetic target size for such mutations. We explored the phenotypic consequences of SCVs evolving faster growth by routes other than direct reversion, and in particular whether any of those routes allowed for the maintenance of antibiotic resistance. In a recent paper (mBio 8: e00358-17) we demonstrated the existence of several different routes of SCV evolution to faster growth, one of which maintained the antibiotic resistance phenotype. This discovery suggests that SCVs might be more adaptable and problematic that previously thought. They are capable of surviving as a slow-growing persistent form, before evolving into a significantly faster-growing form without sacrificing their antibiotic resistance phenotype.

  • 6.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    A large chromosomal inversion causes the formation of an unstable tufA/B hybrid operonManuscript (preprint) (Other academic)
  • 7.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Co-evolution protects the tuf genes from inactivation by mobile genetic elementsManuscript (preprint) (Other academic)
  • 8.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Genetic characterization of compensatory evolution in strains carrying rpoB Ser531Leu, the rifampicin resistance mutation most frequently found in clinical isolates2013In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 11, p. 2493-2497Article in journal (Refereed)
    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.

  • 9.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mechanisms of fitness cost reduction for rifampicin-resistant strains with deletion or duplication mutations in rpoB.2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 17488Article in journal (Refereed)
    Abstract [en]

    Rifampicin resistance (Rif(R)) is caused by mutations in rpoB, encoding the beta-subunit of RNA polymerase. Rif(R )mutations generally incur a fitness cost and in resistant isolates are frequently accompanied by compensatory mutations in rpoA, rpoB or rpoC. Previous studies of fitness compensation focused on Rif(R) caused by amino acid substitutions within rpoB. Rif(R) is also caused by deletion and duplication mutations in rpoB but it is not known whether or how such mutants can ameliorate their fitness costs. Using experimental evolution of Salmonella carrying Rif(R) deletion or duplication mutations we identified compensatory amino acid substitution mutations within rpoA, rpoB or rpoC in 16 of 21 evolved lineages. Additionally, we found one lineage where a large deletion was compensated by duplication of adjacent amino acids (possibly to fill the gap within the protein structure), two lineages where mutations occurred outside of rpoABC, and two lineages where a duplication mutant reverted to the wild-type sequence. All but the two revertant mutants maintained the Rif(R) phenotype. These data suggest that amino acid substitution mutations are the major compensatory mechanism regardless of the nature of the primary Rif(R) mutation.

  • 10.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The Selective Advantage of Synonymous Codon Usage Bias in Salmonella2016In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 12, no 3, article id e1005926Article in journal (Refereed)
    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.

  • 11.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pietsch, Franziska
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Alemayehu, Rahel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Comprehensive phenotypic characterization of rifampicin resistance mutations in Salmonella provides insight into the evolution of resistance in Mycobacterium tuberculosis2015In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 70, no 3, p. 680-685Article in journal (Refereed)
    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.

  • 12.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wrande, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Liljas, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Fitness-compensatory mutations in rifampicin-resistant RNA polymerase2012In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 85, no 1, p. 142-151Article in journal (Refereed)
    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. 

  • 13.
    Cao, Sha
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandis, Gerrit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Alternative Evolutionary Pathways for Drug-Resistant Small Colony Variant Mutants in Staphylococcus aureus2017In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 3, article id e00358-17Article in journal (Refereed)
    Abstract [en]

    Staphylococcus aureus is known to generate small colony variants (SCVs) that are resistant to aminoglycoside antibiotics and can cause persistent and recurrent infections. The SCV phenotype is unstable, and compensatory mutations lead to restored growth, usually with loss of resistance. However, the evolution of improved growth, by mechanisms that avoid loss of antibiotic resistance, is very poorly understood. By selection with serial passaging, we isolated and characterized different classes of extragenic suppressor mutations that compensate for the slow growth of small colony variants. Compensation occurs by two distinct bypass mechanisms: (i) translational suppression of the initial SCV mutation by mutant tRNAs, ribosomal protein S5, or release factor 2 and (ii) mutations that cause the constitutive activation of the SrrAB global transcriptional regulation system. Although compensation by translational suppression increases growth rate, it also reduces antibiotic susceptibility, thus restoring a pseudo-wild-type phenotype. In contrast, an evolutionary pathway that compensates for the SCV phenotype by activation of SrrAB increases growth rate without loss of antibiotic resistance. RNA sequence analysis revealed that mutations activating the SrrAB pathway cause upregulation of genes involved in peptide transport and in the fermentation pathways of pyruvate to generate ATP and NAD(+), thus explaining the increased growth. By increasing the growth rate of SCVs without the loss of aminoglycoside resistance, compensatory evolution via the SrrAB activation pathway represents a threat to effective antibiotic therapy of staphylococcal infections. IMPORTANCE Small colony variants (SCVs) of Staphylococcus aureus are a significant clinical problem, causing persistent and antibiotic-resistant infections. However, SCVs are unstable and can rapidly evolve growth-compensated mutants. Previous data suggested that growth compensation only occurred with the loss of antibiotic resistance. We have used selection with serial passaging to uncover four distinct pathways of growth compensation accessible to SCVs. Three of these paths (reversion, intragenic suppression, and translational suppression) increase growth at the expense of losing antibiotic resistance. The fourth path activates an alternative transcriptional program and allows the bacteria to produce the extra ATP required to support faster growth, without losing antibiotic resistance. The importance of this work is that it shows that drug-resistant SCVs can evolve faster growth without losing antibiotic resistance.

  • 14.
    Garmendia, Eva
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandis, Gerrit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Transcriptional Regulation Buffers Gene Dosage Effects on a Highly Expressed Operon in Salmonella2018In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, no 5, article id e01446-18Article in journal (Refereed)
    Abstract [en]

    Highly expressed genes are commonly located close to the origin of replication of bacterial chromosomes (OriC). This location skew is thought to reflect selective advantages associated with gene dosage effects during the replication cycle. The expression of constitutively expressed genes can vary up to fivefold based on chromosomal location, but it is not clear what level of variation would occur in naturally regulated operons. We tested the magnitude of the chromosome location effect using EF-Tu (tufA, tufB), an abundant protein whose cellular level correlates with, and limits, the maximum growth rate. We translocated the Salmonella tufB operon to four locations across the chromosome. The distance from OriC had only a small effect on growth rate, consistent with this operon having the natural ability to upregulate expression and compensate for reduced gene dosage. In contrast, when the total EF-Tu concentration was limiting for the growth rate (tufA deleted), we observed a strong gene dosage effect when tufB was located further from OriC. However, only a short period of experimental evolution was required before the bacteria adapted to this EF-Tu starvation situation by acquiring genetic changes that increased expression levels from the translocated tufB gene, restoring growth rates. Our findings demonstrate that, at least for the tufB operon, gene dosage is probably not the dominant force selecting for a chromosomal location close to OriC. We suggest that the colocation of highly expressed genes close to OriC might instead be selected because it enhances their coregulation during various growth states, with gene dosage being a secondary benefit. IMPORTANCE A feature of bacterial chromosomes is that highly expressed essential genes are usually located close to the origin of replication. Because bacteria have overlapping cycles of replication, genes located close to the origin will often be present in multiple copies, and this is thought to be of selective benefit where high levels of expression support high growth rate. However, the magnitude of this selective effect and whether other forces could be at play are poorly understood. To study this, we translocated a highly expressed essential operon, tufB, to different locations and measured growth fitness. We found that transcriptional regulation buffered the effects of translocation and that even under conditions where growth rate was reduced, genetic changes that increased the expression of tufB were easily and rapidly selected. We conclude, at least for tufB, that forces other than gene dosage may be significant in selecting for chromosomal location.

  • 15.
    Huseby, Douglas L.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pietsch, Franziska
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandis, Gerrit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Garoff, Linnéa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tegehall, Angelica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mutation supply and relative fitness shape the genotypes of ciprofloxacin-resistant Escherichia coli2017In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 34, no 5, p. 1029-1039Article in journal (Refereed)
    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.

  • 16.
    Huseby, Douglas L.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Praski-Alzrigat, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandis, Gerrit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    High-level intrinsic suppression of a frameshift mutation in an essential geneManuscript (preprint) (Other academic)
  • 17.
    Meftahi, Nedra
    et al.
    Univ Tunis El Manar, Inst Pasteur Tunis, Lab Mol Microbiol Vaccinol & Biotechnol Dev, Unit Typing & Genet Mycobacteria, Tunis, Tunisia.
    Namouchi, Amine
    Univ Tunis El Manar, Inst Pasteur Tunis, Lab Mol Microbiol Vaccinol & Biotechnol Dev, Unit Typing & Genet Mycobacteria, Tunis, Tunisia.
    Mhenni, Besma
    Univ Tunis El Manar, Inst Pasteur Tunis, Lab Mol Microbiol Vaccinol & Biotechnol Dev, Unit Typing & Genet Mycobacteria, Tunis, Tunisia.
    Brandis, Gerrit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mardassi, Helmi
    Univ Tunis El Manar, Inst Pasteur Tunis, Lab Mol Microbiol Vaccinol & Biotechnol Dev, Unit Typing & Genet Mycobacteria, Tunis, Tunisia.
    Evidence for the critical role of a secondary site rpoB mutation in the compensatory evolution and successful transmission of an MDR tuberculosis outbreak strain2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 2, p. 324-332Article in journal (Refereed)
    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.

  • 18. Pietsch, Franziska
    et al.
    Bergman, Jessica
    Brandis, Gerrit
    Marcusson, Linda
    Zorzet, Anna
    Huseby, Douglas L.
    Hughes, Diarmaid
    RNA polymerase mutations contribute to the evolution of ciprofloxacin resistance in Escherichia coliManuscript (preprint) (Other academic)
  • 19.
    Pietsch, Franziska
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bergman, Jessica M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandis, Gerrit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Marcusson, Linda L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Zorzet, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huseby, Douglas L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ciprofloxacin selects for RNA polymerase mutations with pleiotropic antibiotic resistance effects2017In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 72, no 1, p. 75-84Article in journal (Refereed)
    Abstract [en]

    Objectives: Resistance to the fluoroquinolone drug ciprofloxacin is commonly linked to mutations that alter the drug target or increase drug efflux via the major AcrAB-TolC transporter. Very little is known about other mutations that might also reduce susceptibility to ciprofloxacin. We discovered that an Escherichia coli strain experimentally evolved for resistance to ciprofloxacin had acquired a mutation in rpoB, the gene coding for the beta-subunit of RNA polymerase. The aim of this work was to determine whether this mutation, and other mutations in rpoB, contribute to ciprofloxacin resistance and, if so, by which mechanism. Methods: Independent lineages of E. coli were evolved in the presence of ciprofloxacin and clones from endpoint cultures were screened for mutations in rpoB. Ciprofloxacin-selected rpoB mutations were identified and characterized in terms of effects on susceptibility and mode of action. Results: Mutations in rpoB were selected at a high frequency in 3 out of 10 evolved lineages, in each case arising after the occurrence of mutations affecting topoisomerases and drug efflux. All ciprofloxacin-selected rpoB mutations had a high fitness cost in the absence of drug, but conferred a competitive advantage in the presence of ciprofloxacin. RNA sequencing and quantitative RT-PCR analysis showed that expression of mdtK, encoding a multidrug efflux transporter, was significantly increased by the ciprofloxacin-selected rpoB mutations. The susceptibility phenotype was shown to depend on the presence of an active mdtK and a mutant rpoB allele. Conclusions: These data identify mutations in RNA polymerase as novel contributors to the evolution of resistance to ciprofloxacin and show that the phenotype is mediated by increased MdtK-dependent drug efflux.

  • 20. Pietsch, Franziska
    et al.
    Garoff, Linnea
    Huseby, Douglas L.
    Lilja, Tua
    Brandis, Gerrit
    Hughes, Diarmaid
    Experimental evolution identifies a new class of genes selected during the development of ciprofloxacin resistance in Escherichia coliManuscript (preprint) (Other academic)
  • 21.
    Praski Alzrigat, Lisa
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandis, Gerrit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Fitness cost constrains the spectrum of marR mutations in ciprofloxacin-resistant Escherichia coli: Multiple Antibiotic-Resistance, Gram-Negative Bacteria, Multidrug Efflux Pump, Urinary-Tract-Infections, Fluoroquinolone Resistance, Quinolone Resistance, Mechanisms, Expression, Sequence, Soxs2017In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 2, no 11, p. 3016-3024Article in journal (Refereed)
    Abstract [en]

    Objectives: To determine whether the spectrum of mutations in marR in ciprofloxacin-resistant clinical isolates of Escherichia coli shows evidence of selection bias, either to reduce fitness costs, or to increase drug resistance. MarR is a repressor protein that regulates, via MarA, expression of the Mar regulon, including the multidrug efflux pump AcrAB-TolC. Methods: Isogenic strains carrying 36 different marR alleles identified in resistant clinical isolates, or selected for resistance in vitro, were constructed. Drug susceptibility and relative fitness in growth competition assays were measured for all strains. The expression level of marA, and of various efflux pump components, as a function of specific mutations in marR, was measured by qPCR. Results: The spectrum of genetic alterations in marR in clinical isolates is strongly biased against inactivating mutations. In general, the alleles found in clinical isolates conferred a lower level of resistance and imposed a lower growth fitness cost than mutations selected in vitro. The level of expression of MarA correlated well with the MIC of ciprofloxacin. This supports the functional connection between mutations in marR and reduced susceptibility to ciprofloxacin. Conclusions: Mutations in marR selected in ciprofloxacin-resistant clinical isolates are strongly biased against inactivating mutations. Selection favours mutant alleles that have the lowest fitness costs, even though these cause only modest reductions in drug susceptibility. This suggests that selection for high relative fitness is more important than selection for increased resistance in determining which alleles of marR will be selected in resistant clinical isolates.

1 - 21 of 21
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