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  • 1.
    Amlinger, Lina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hoekzema, Mirthe
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 10392Article in journal (Refereed)
    Abstract [en]

    CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

  • 2.
    Ghosh, Anirban
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Baltekin, Özden
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Wäneskog, Marcus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Elkhalifa, Dina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Larsson, Disa
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Contact-dependent growth inhibition induces high levels of antibiotic-tolerant persister cells in clonal bacterial populations2018In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 37, no 9, article id UNSP e98026Article in journal (Refereed)
    Abstract [en]

    Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. One example is non-growing cells in clonal bacterial populations that are able to persist antibiotic treatment. Previous studies suggest that persisters arise in bacterial populations either stochastically through variation in levels of global signalling molecules between individual cells, or in response to various stresses. Here, we show that toxins used in contact-dependent growth inhibition (CDI) create persisters upon direct contact with cells lacking sufficient levels of CdiI immunity protein, which would otherwise bind to and neutralize toxin activity. CDI-mediated persisters form through a feedforward cycle where the toxic activity of the CdiA toxin increases cellular (p)ppGpp levels, which results in Lon-mediated degradation of the immunity protein and more free toxin. Thus, CDI systems mediate a population density-dependent bet-hedging strategy, where the fraction of non-growing cells is increased only when there are many cells of the same genotype. This may be one of the mechanisms of how CDI systems increase the fitness of their hosts.

  • 3.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Dynamics of the Bacterial Genome: Rates and Mechanisms of Mutation2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Bacterial chromosomes are highly dynamic, continuously changing with respect to gene content and size via a number of processes, including deletions that result in gene loss. How deletions form and at what rates has been the focus of this thesis.

    In paper II we investigated how chromosomal location affects chromosomal deletion rates in S. typhimurium. Deletion rates varied more than 100-fold between different chromosomal locations and some large deletions significantly increased the exponential growth rate of the cells. Our results suggest that the chromosome is heterogeneous with respect to deletion rates and that deletions may be genetically fixed as a consequence of natural selection rather than by drift or mutational biases.

    In paper I we examined in a laboratory setting how rapidly reductive evolution, i.e. gene loss, could occur. Using a serial passage approach, we showed that extensive genome reduction potentially could occur on a very short evolutionary time scale. For most deletions we observed little or no homology at the deletion endpoints, indicating that spontaneous deletions often form through a RecA independent process.

    In paper III we examined further how large spontaneous deletions form and, unexpectedly, showed that 90% of all spontaneous chromosomal deletions required error-prone translesion DNA polymerases for their formation. We propose that the translesion polymerases stimulate deletion formation by allowing extension of misaligned single-strand DNA ends.

    In paper IV we investigated how the translesion DNA polymerase Pol IV, RpoS and different types of stresses affect mutation rates in bacteria. Derepression of the LexA regulon caused a small to moderate increase in mutation rates that was fully dependent on functional endonucleases but only partly dependent on translesion DNA polymerases. RpoS levels and growth stresses had only minor effects on mutation rates. Thus, mutation rates appear very robust and are only weakly affected by growth conditions and induction of translesion polymerases and RpoS.

    List of papers
    1. Translesion DNA polymerases are required for spontaneous deletion formation in Salmonella typhimurium
    Open this publication in new window or tab >>Translesion DNA polymerases are required for spontaneous deletion formation in Salmonella typhimurium
    2009 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 25, p. 10248-10253Article in journal (Refereed) Published
    Abstract [en]

    How spontaneous deletions form in bacteria is still a partly unresolved problem. Here we show that deletion formation in S. typhimurium requries the presence of functional translesion polymerases. First, in wild type bacteria, removal of the known translesion DNA polymerases: PolII (polB), PolIV (dinB), PolV (umuDC) and the PolV homologue SamAB (samAB) resulted in a 10-fold decrease in the deletion rate, indicating that 90% of all spontaneous deletions require these polymerases for their formation. Second, overexpression of these polymerases by de-repression of the DNA damage-inducible LexA regulon caused a 25-fold increase in deletion rate that depended on the presence of functional translesion polymerases. Third, overexpression of the polymerases PolII and PolIV from a plasmid increased the deletion rate 12- to 30-fold respectively. Last, in a recBC- mutant where dsDNA ends are stabilized due to the lack of the end-processing nuclease RecBC, the deletion rate was increased 20-fold. This increase depended on the translesion polymerases. In lexA(def) mutant cells with constitutive SOS-expression, a 10-fold increase in DNA breaks was observed. Inactivation of all 4 translesion polymerases in the lexA(def) mutant reduced the deletion rate 250-fold without any concomitant reduction in the amount of DNA breaks. Mutational inactivation of 3 endonucleases under LexA control, reduced the number of DNA breaks to the wild-type level in a lexA(def) mutant with a concomitant 50-fold reduction in deletion rate. These findings suggest that the translesion polymerases are not involved in forming the DNA breaks, but that they require them to stimulate deletion formation.

    Keywords
    bacteria, DNA homology, gene loss, RecA protein
    National Category
    Medical and Health Sciences
    Research subject
    Evolutionary Genetics
    Identifiers
    urn:nbn:se:uu:diva-111424 (URN)10.1073/pnas.0904389106 (DOI)000267292200034 ()
    Available from: 2009-12-14 Created: 2009-12-14 Last updated: 2017-12-12Bibliographically approved
    2. Variation in spontaneous deletion rates at different locations of the Salmonella typhimurium chromosome
    Open this publication in new window or tab >>Variation in spontaneous deletion rates at different locations of the Salmonella typhimurium chromosome
    (English)Manuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    How and at what rates spontaneous deletions form is still a partly unresolved question. Here we have constructed a genetic tool that can be used to determine spontaneous chromosomal deletion rates at any chromosomal location. We measured deletion rates at 12 chromosomal locations and identified the deletable region as the largest deletion found at each location. Our data shows that spontaneous deletion rates can at least vary 100-fold between the different chromosomal locations when normalized to the size of the deletable region. The isolated deletions ranged in size from 1-200 kbp and the highest deletion rates were found around 2 Mbp of the S. typhiumurium chromosome, suggesting a potential hotspot for deletion formation. No long repeat sequences were found in this region that could explain the high deletion rate. Furthermore, no obvious correlation between fitness (measured as exponential growth rate) and deletion size could be seen. Surprisingly, since deletions are commonly considered deleterious certain deletions (ranging from 18- to 38 kbp in size) increased the growth rate of the cells with ~5% in both rich and poor growth media. These results suggest that the bacterial chromosome is heterogeneous with respect to the rate of deletion formation and that some deletions could become fixed as a consequence of natural selection rather than by drift and/or mutational biases.

    Keywords
    bacteria, gene loss, chromosome
    National Category
    Microbiology in the medical area Microbiology in the medical area
    Research subject
    Evolutionary Genetics; Microbiology
    Identifiers
    urn:nbn:se:uu:diva-111427 (URN)
    Available from: 2009-12-14 Created: 2009-12-14 Last updated: 2018-01-12
    3. Effect of the translesion DNA polymerases, endonucleases and RpoS on mutation rates in Salmonella typhimurium
    Open this publication in new window or tab >>Effect of the translesion DNA polymerases, endonucleases and RpoS on mutation rates in Salmonella typhimurium
    2010 (English)In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 185, no 3, p. 783-795Article in journal (Refereed) Published
    Abstract [en]

    It has been suggested that bacteria have evolved mechanisms to increase their mutation rate in response to various stresses and that the translesion DNA polymerase Pol IV under control of the LexA regulon and the alternative sigma factor RpoS are involved in regulating this mutagenesis. Here we examined in Salmonella enterica serovar Typhimurium LT2 the rates for four different types of mutations (rifampicin-, nalidixic acid- and chlorate-resistance and Lac+ reversion) during various growth conditions and with different levels of four translesion DNA polymerases (Pol II, Pol IV, Pol V and SamAB) and RpoS. Constitutive de-repression of the LexA regulon by a lexA(def) mutation increased mutation rates 1.5- to 12-fold and the contribution of the translesion DNA polymerases to this mutagenesis varied with the type of mutation examined. In contrast, for all four types of mutations examined the increase in mutation rate in the lexA(def) mutant required the presence of the LexA-controlled endonucleases UvrB, UvrC and Cho. With regard to the potential involvement of RpoS in mutagenesis, neither an increase in RpoS levels conferred by artificial over-expression from a plasmid nor long-term stationary phase incubation or slow growth caused an increase in any of the four mutation rates measured, alone or in combination with over-expression of the translesion DNA polymerases. In conclusion, mutation rates are remarkably robust and no combination of growth conditions, induction of translesion polymerases by inactivation of LexA or increased RpoS expression could confer an increase in mutation rates higher than the moderate increase caused by de-repression of the LexA regulon alone.

    Keywords
    bacteria, stress, mutation
    National Category
    Medical and Health Sciences
    Research subject
    Evolutionary Genetics; Microbiology
    Identifiers
    urn:nbn:se:uu:diva-111426 (URN)10.1534/genetics.110.116376 (DOI)000281906800007 ()20421601 (PubMedID)
    Available from: 2009-12-14 Created: 2009-12-14 Last updated: 2017-12-12Bibliographically approved
    4. Bacterial genome size reduction by experimental evolution
    Open this publication in new window or tab >>Bacterial genome size reduction by experimental evolution
    Show others...
    2005 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, Vol. 102, no 34, p. 12112-12116Article in journal (Refereed) Published
    Abstract [en]

    Bacterial evolution toward endosymbiosis with eukaryotic cells is associated with extensive bacterial genome reduction and loss of metabolic and regulatory capabilities. Here we examined the rate and process of genome reduction in the bacterium Salmonella enterica by a serial passage experimental evolution procedure. The initial rate of DNA loss was estimated to be 0.05 bp per chromosome per generation for a WT bacterium and approximately 50-fold higher for a mutS mutant defective in methyl-directed DNA mismatch repair. The endpoints were identified for seven chromosomal deletions isolated during serial passage and in two separate genetic selections. Deletions ranged in size from 1 to 202 kb, and most of them were not associated with DNA repeats, indicating that they were formed via RecA-independent recombination events. These results suggest that extensive genome reduction can occur on a short evolutionary time scale and that RecA-dependent homologous recombination only plays a limited role in this process of jettisoning superfluous DNA.

    Keywords
    bacterial evolution, genome reduction, serial passage
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-80317 (URN)10.1073/pnas.0503654102 (DOI)16099836 (PubMedID)
    Available from: 2006-05-05 Created: 2006-05-05 Last updated: 2011-06-30Bibliographically approved
  • 4.
    Koskiniemi, Sanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Translesion DNA polymerases are required for spontaneous deletion formation in Salmonella typhimurium2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 25, p. 10248-10253Article in journal (Refereed)
    Abstract [en]

    How spontaneous deletions form in bacteria is still a partly unresolved problem. Here we show that deletion formation in S. typhimurium requries the presence of functional translesion polymerases. First, in wild type bacteria, removal of the known translesion DNA polymerases: PolII (polB), PolIV (dinB), PolV (umuDC) and the PolV homologue SamAB (samAB) resulted in a 10-fold decrease in the deletion rate, indicating that 90% of all spontaneous deletions require these polymerases for their formation. Second, overexpression of these polymerases by de-repression of the DNA damage-inducible LexA regulon caused a 25-fold increase in deletion rate that depended on the presence of functional translesion polymerases. Third, overexpression of the polymerases PolII and PolIV from a plasmid increased the deletion rate 12- to 30-fold respectively. Last, in a recBC- mutant where dsDNA ends are stabilized due to the lack of the end-processing nuclease RecBC, the deletion rate was increased 20-fold. This increase depended on the translesion polymerases. In lexA(def) mutant cells with constitutive SOS-expression, a 10-fold increase in DNA breaks was observed. Inactivation of all 4 translesion polymerases in the lexA(def) mutant reduced the deletion rate 250-fold without any concomitant reduction in the amount of DNA breaks. Mutational inactivation of 3 endonucleases under LexA control, reduced the number of DNA breaks to the wild-type level in a lexA(def) mutant with a concomitant 50-fold reduction in deletion rate. These findings suggest that the translesion polymerases are not involved in forming the DNA breaks, but that they require them to stimulate deletion formation.

  • 5.
    Koskiniemi, Sanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Variation in spontaneous deletion rates at different locations of the Salmonella typhimurium chromosomeManuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    How and at what rates spontaneous deletions form is still a partly unresolved question. Here we have constructed a genetic tool that can be used to determine spontaneous chromosomal deletion rates at any chromosomal location. We measured deletion rates at 12 chromosomal locations and identified the deletable region as the largest deletion found at each location. Our data shows that spontaneous deletion rates can at least vary 100-fold between the different chromosomal locations when normalized to the size of the deletable region. The isolated deletions ranged in size from 1-200 kbp and the highest deletion rates were found around 2 Mbp of the S. typhiumurium chromosome, suggesting a potential hotspot for deletion formation. No long repeat sequences were found in this region that could explain the high deletion rate. Furthermore, no obvious correlation between fitness (measured as exponential growth rate) and deletion size could be seen. Surprisingly, since deletions are commonly considered deleterious certain deletions (ranging from 18- to 38 kbp in size) increased the growth rate of the cells with ~5% in both rich and poor growth media. These results suggest that the bacterial chromosome is heterogeneous with respect to the rate of deletion formation and that some deletions could become fixed as a consequence of natural selection rather than by drift and/or mutational biases.

  • 6.
    Koskiniemi, Sanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Arming the Neighborhood2016In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 39, no 1, p. 5-6Article in journal (Other academic)
    Abstract [en]

    Bacteria use type 6 secretion systems in antagonistic behavior to compete for resources with other bacteria. In a recent issue of Cell, Vettiger and Basler (2016) show that bacteria can also use these systems to arm neighboring cells and force them to pass on a signal in the bacterial population.

  • 7.
    Koskiniemi, Sanna
    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 Science and Technology, Biology, Department of Cell and Molecular Biology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Effect of the translesion DNA polymerases, endonucleases and RpoS on mutation rates in Salmonella typhimurium2010In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 185, no 3, p. 783-795Article in journal (Refereed)
    Abstract [en]

    It has been suggested that bacteria have evolved mechanisms to increase their mutation rate in response to various stresses and that the translesion DNA polymerase Pol IV under control of the LexA regulon and the alternative sigma factor RpoS are involved in regulating this mutagenesis. Here we examined in Salmonella enterica serovar Typhimurium LT2 the rates for four different types of mutations (rifampicin-, nalidixic acid- and chlorate-resistance and Lac+ reversion) during various growth conditions and with different levels of four translesion DNA polymerases (Pol II, Pol IV, Pol V and SamAB) and RpoS. Constitutive de-repression of the LexA regulon by a lexA(def) mutation increased mutation rates 1.5- to 12-fold and the contribution of the translesion DNA polymerases to this mutagenesis varied with the type of mutation examined. In contrast, for all four types of mutations examined the increase in mutation rate in the lexA(def) mutant required the presence of the LexA-controlled endonucleases UvrB, UvrC and Cho. With regard to the potential involvement of RpoS in mutagenesis, neither an increase in RpoS levels conferred by artificial over-expression from a plasmid nor long-term stationary phase incubation or slow growth caused an increase in any of the four mutation rates measured, alone or in combination with over-expression of the translesion DNA polymerases. In conclusion, mutation rates are remarkably robust and no combination of growth conditions, induction of translesion polymerases by inactivation of LexA or increased RpoS expression could confer an increase in mutation rates higher than the moderate increase caused by de-repression of the LexA regulon alone.

  • 8.
    Michalska, Karolina
    et al.
    Argonne Natl Lab, Midwest Ctr Struct Genom, Argonne, IL USA; Argonne Natl Lab, Struct Biol Ctr, Biosci Div, Argonne, IL USA.
    Nhan, Dinh Quan
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA USA.
    Willett, Julia L. E.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA USA; Univ Minnesota, Dept Microbiol & Immunol, Minneapolis, MN USA.
    Stols, Lucy M.
    Argonne Natl Lab, Midwest Ctr Struct Genom, Argonne, IL USA.
    Eschenfeldt, William H.
    Argonne Natl Lab, Midwest Ctr Struct Genom, Argonne, IL USA.
    Jones, Allison M.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA USA.
    Nguyen, Josephine Y.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA USA.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala Univ, Dept Cell & Mol Biol, Uppsala, Sweden.
    Low, David A.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA USA; Univ Calif Santa Barbara, Biomol Sci & Engn Program, Santa Barbara, CA USA.
    Goulding, Celia W.
    Univ Calif Irvine, Dept Mol Biol & Biochem, Irvine, CA USA; Univ Calif Irvine, Pharmaceut Sci, Irvine, CA USA.
    Joachimiak, Andrzej
    Argonne Natl Lab, Midwest Ctr Struct Genom, Argonne, IL USA; Argonne Natl Lab, Struct Biol Ctr, Biosci Div, Argonne, IL USA; Univ Chicago, Dept Biochem & Mol Biol, Chicago, IL USA.
    Hayes, Christopher S.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA USA; Univ Calif Santa Barbara, Biomol Sci & Engn Program, Santa Barbara, CA USA.
    Functional plasticity of antibacterial EndoU toxins2018In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 109, no 4, p. 509-527Article in journal (Refereed)
    Abstract [en]

    Bacteria use several different secretion systems to deliver toxic EndoU ribonucleases into neighboring cells. Here, we present the first structure of a prokaryotic EndoU toxin in complex with its cognate immunity protein. The contact‐dependent growth inhibition toxin CdiA‐CTSTECO31 from Escherichia coli STEC_O31 adopts the eukaryotic EndoU fold and shares greatest structural homology with the nuclease domain of coronavirus Nsp15. The toxin contains a canonical His‐His‐Lys catalytic triad in the same arrangement as eukaryotic EndoU domains, but lacks the uridylate‐specific ribonuclease activity that characterizes the superfamily. Comparative sequence analysis indicates that bacterial EndoU domains segregate into at least three major clades based on structural variations in the N‐terminal subdomain. Representative EndoU nucleases from clades I and II degrade tRNA molecules with little specificity. In contrast, CdiA‐CTSTECO31 and other clade III toxins are specific anticodon nucleases that cleave tRNAGlu between nucleotides C37 and m2A38. These findings suggest that the EndoU fold is a versatile scaffold for the evolution of novel substrate specificities. Such functional plasticity may account for the widespread use of EndoU effectors by diverse inter‐bacterial toxin delivery systems.

  • 9. Nilsson, A I
    et al.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eriksson, S
    Kugelberg, E
    Hinton, J C D
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bacterial genome size reduction by experimental evolution2005In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, Vol. 102, no 34, p. 12112-12116Article in journal (Refereed)
    Abstract [en]

    Bacterial evolution toward endosymbiosis with eukaryotic cells is associated with extensive bacterial genome reduction and loss of metabolic and regulatory capabilities. Here we examined the rate and process of genome reduction in the bacterium Salmonella enterica by a serial passage experimental evolution procedure. The initial rate of DNA loss was estimated to be 0.05 bp per chromosome per generation for a WT bacterium and approximately 50-fold higher for a mutS mutant defective in methyl-directed DNA mismatch repair. The endpoints were identified for seven chromosomal deletions isolated during serial passage and in two separate genetic selections. Deletions ranged in size from 1 to 202 kb, and most of them were not associated with DNA repeats, indicating that they were formed via RecA-independent recombination events. These results suggest that extensive genome reduction can occur on a short evolutionary time scale and that RecA-dependent homologous recombination only plays a limited role in this process of jettisoning superfluous DNA.

  • 10.
    Ruhe, Zachary C.
    et al.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA..
    Nguyen, Josephine Y.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA..
    Xiong, Jing
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA..
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA..
    Beck, Christina M.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA.;Icahn Sch Med Mt Sinai, New York, NY 10029 USA..
    Perkins, Basil R.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA.;Barnard Coll, Dept Biol, New York, NY USA..
    Low, David A.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA.;Univ Calif Santa Barbara, Biomol Sci & Engn Program, Santa Barbara, CA 93106 USA..
    Hayes, Christopher S.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA.;Univ Calif Santa Barbara, Biomol Sci & Engn Program, Santa Barbara, CA 93106 USA..
    CdiA Effectors Use Modular Receptor-Binding Domains To Recognize Target Bacteria2017In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 2, article id e00290-17Article in journal (Refereed)
    Abstract [en]

    Contact-dependent growth inhibition (CDI) systems encode CdiA effectors, which bind to specific receptors on neighboring bacteria and deliver C-terminal toxin domains to suppress target cell growth. Two classes of CdiA effectors that bind distinct cell surface receptors have been identified, but the molecular basis of receptor specificity is not understood. Alignment of BamA-specific CdiAEC93 from Escherichia coli EC93 and OmpC-specific CdiA(EC536) from E. coli 536 suggests that the receptor-binding domain resides within a central region that varies between the two effectors. In support of this hypothesis, we find that CdiA(EC93) fragments containing residues Arg1358 to Phe1646 bind specifically to purified BamA. Moreover, chimeric CdiA(EC93) that carries the corresponding sequence from CdiA(EC536) is endowed with OmpC-binding activity, demonstrating that this region dictates receptor specificity. A survey of E. coli CdiA proteins reveals two additional effector classes, which presumably recognize distinct receptors. Using a genetic approach, we identify the outer membrane nucleoside transporter Tsx as the receptor for a third class of CdiA effectors. Thus, CDI systems exploit multiple outer membrane proteins to identify and engage target cells. These results underscore the modularity of CdiA proteins and suggest that novel effectors can be constructed through genetic recombination to interchange different receptor-binding domains and toxic payloads. IMPORTANCE CdiB/CdiA two-partner secretion proteins mediate interbacterial competition through the delivery of polymorphic toxin domains. This process, known as contact-dependent growth inhibition (CDI), requires stable interactions between the CdiA effector protein and specific receptors on the surface of target bacteria. Here, we localize the receptor-binding domain to the central region of E. coli CdiA. Receptor-binding domains vary between CdiA proteins, and E. coli strains collectively encode at least four distinct effector classes. Further, we show that receptor specificity can be altered by exchanging receptor-binding regions, demonstrating the modularity of this domain. We propose that novel CdiA effectors are naturally generated through genetic recombination to interchange different receptor-binding domains and toxin payloads.

  • 11.
    Virtanen, Petra
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wäneskog, Marcus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Class II contact‐dependent growth inhibition (CDI) systems allow for broad‐range cross‐species toxin delivery within the Enterobacteriaceae family2019In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 111, no 4, p. 1109-1125Article in journal (Refereed)
    Abstract [en]

    Contact‐dependent growth inhibition (CDI) allows bacteria to recognize kin cells in mixed bacterial populations. In Escherichia coli, CDI mediated effector delivery has been shown to be species‐specific, with a preference for the own strain over others. This specificity is achieved through an interaction between a receptor‐binding domain in the CdiA protein and its cognate receptor protein on the target cell. But how conserved this specificity is has not previously been investigated in detail. Here, we show that class II CdiA receptor‐binding domains and their Enterobacter cloacae analog are highly promiscuous, and can allow for efficient effector delivery into several different Enterobacteriaceae species, including Escherichia, Enterobacter, Klebsiella and Salmonella spp. In addition, although we observe a preference for the own receptors over others for two of the receptor‐binding domains, this did not limit cross‐species effector delivery in all experimental conditions. These results suggest that class II CdiA proteins could allow for broad‐range and cross‐species growth inhibition in mixed bacterial populations.

  • 12.
    Wistrand-Yuen, Erik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Knopp, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hjort, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Evolution of high-level resistance during low-level antibiotic exposure2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, no 1, article id 1599Article in journal (Refereed)
    Abstract [en]

    It has become increasingly clear that low levels of antibiotics present in many environments can select for resistant bacteria, yet the evolutionary pathways for resistance development during exposure to low amounts of antibiotics remain poorly defined. Here we show that Salmonella enterica exposed to sub-MIC levels of streptomycin evolved high-level resistance via novel mechanisms that are different from those observed during lethal selections. During lethal selection only rpsL mutations are found, whereas at sub-MIC selection resistance is generated by several small-effect resistance mutations that combined confer high-level resistance via three different mechanisms: (i) alteration of the ribosomal RNA target (gidB mutations), (ii) reduction in aminoglycoside uptake (cyoB, nuoG, and trkH mutations), and (iii) induction of the aminoglycoside-modifying enzyme AadA (znuA mutations). These results demonstrate how the strength of the selective pressure influences evolutionary trajectories and that even weak selective pressures can cause evolution of high-level resistance.

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