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Virtanen, P., Wäneskog, M. & Koskiniemi, S. (2019). Class II contact‐dependent growth inhibition (CDI) systems allow for broad‐range cross‐species toxin delivery within the Enterobacteriaceae family. Molecular Microbiology, 111(4), 1109-1125
Open this publication in new window or tab >>Class II contact‐dependent growth inhibition (CDI) systems allow for broad‐range cross‐species toxin delivery within the Enterobacteriaceae family
2019 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 111, no 4, p. 1109-1125Article in journal (Refereed) Published
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.

National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-382983 (URN)10.1111/mmi.14214 (DOI)000464655800017 ()30710431 (PubMedID)
Funder
Swedish Foundation for Strategic Research Swedish Research CouncilÅke Wiberg FoundationWenner-Gren Foundations
Available from: 2019-05-13 Created: 2019-05-13 Last updated: 2019-12-19Bibliographically approved
Ghosh, A., Baltekin, Ö., Wäneskog, M., Elkhalifa, D., Larsson, D., Elf, J. & Koskiniemi, S. (2018). Contact-dependent growth inhibition induces high levels of antibiotic-tolerant persister cells in clonal bacterial populations. EMBO Journal, 37(9), Article ID UNSP e98026.
Open this publication in new window or tab >>Contact-dependent growth inhibition induces high levels of antibiotic-tolerant persister cells in clonal bacterial populations
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2018 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 37, no 9, article id UNSP e98026Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
WILEY, 2018
Keywords
bet-hedging, contact-dependent growth inhibition, persisters, toxin
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-356400 (URN)10.15252/embj.201798026 (DOI)000431279400003 ()29572241 (PubMedID)
Funder
Swedish Foundation for Strategic Research Swedish Research CouncilEU, European Research Council
Note

Anirban Ghosh and Özden Baltekin contributed equally to this work.

Available from: 2018-07-25 Created: 2018-07-25 Last updated: 2019-12-19Bibliographically approved
Wistrand-Yuen, E., Knopp, M., Hjort, K., Koskiniemi, S., Berg, O. G. & Andersson, D. I. (2018). Evolution of high-level resistance during low-level antibiotic exposure. Nature Communications, 9(1), Article ID 1599.
Open this publication in new window or tab >>Evolution of high-level resistance during low-level antibiotic exposure
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2018 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, no 1, article id 1599Article in journal (Refereed) Published
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.

National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-353487 (URN)10.1038/s41467-018-04059-1 (DOI)000430541900015 ()29686259 (PubMedID)
Funder
Swedish Research CouncilSwedish Research Council Formas
Available from: 2018-06-13 Created: 2018-06-13 Last updated: 2018-06-30Bibliographically approved
Michalska, K., Nhan, D. Q., Willett, J. L. E., Stols, L. M., Eschenfeldt, W. H., Jones, A. M., . . . Hayes, C. S. (2018). Functional plasticity of antibacterial EndoU toxins. Molecular Microbiology, 109(4), 509-527
Open this publication in new window or tab >>Functional plasticity of antibacterial EndoU toxins
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2018 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 109, no 4, p. 509-527Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
WILEY, 2018
National Category
Microbiology in the medical area Microbiology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-368051 (URN)10.1111/mmi.14007 (DOI)000446522100008 ()29923643 (PubMedID)
Available from: 2018-12-07 Created: 2018-12-07 Last updated: 2018-12-07Bibliographically approved
Ruhe, Z. C., Nguyen, J. Y., Xiong, J., Koskiniemi, S., Beck, C. M., Perkins, B. R., . . . Hayes, C. S. (2017). CdiA Effectors Use Modular Receptor-Binding Domains To Recognize Target Bacteria. mBio, 8(2), Article ID e00290-17.
Open this publication in new window or tab >>CdiA Effectors Use Modular Receptor-Binding Domains To Recognize Target Bacteria
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2017 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 2, article id e00290-17Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
AMER SOC MICROBIOLOGY, 2017
Keywords
bacterial competition, cell-cell adhesion, self/nonself discrimination, toxin immunity proteins, type V secretion system
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-323668 (URN)10.1128/mBio.00290-17 (DOI)000400575700055 ()
Available from: 2017-06-20 Created: 2017-06-20 Last updated: 2017-06-20Bibliographically approved
Amlinger, L., Hoekzema, M., Wagner, G. E. H., Koskiniemi, S. & Lundgren, M. (2017). Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition. Scientific Reports, 7, Article ID 10392.
Open this publication in new window or tab >>Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 10392Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-335401 (URN)10.1038/s41598-017-10876-z (DOI)000408997700091 ()28871175 (PubMedID)
Funder
Swedish Research CouncilWenner-Gren FoundationsThe Royal Swedish Academy of SciencesScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2017-12-05 Created: 2017-12-05 Last updated: 2020-02-04Bibliographically approved
Koskiniemi, S. & Elf, J. (2016). Arming the Neighborhood. Developmental Cell, 39(1), 5-6
Open this publication in new window or tab >>Arming the Neighborhood
2016 (English)In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 39, no 1, p. 5-6Article in journal, Editorial material (Other academic) Published
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.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-309477 (URN)10.1016/j.devcel.2016.09.024 (DOI)000385471100003 ()27728781 (PubMedID)
Available from: 2016-12-05 Created: 2016-12-05 Last updated: 2018-01-13Bibliographically approved
Koskiniemi, S. (2010). Dynamics of the Bacterial Genome: Rates and Mechanisms of Mutation. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Dynamics of the Bacterial Genome: Rates and Mechanisms of Mutation
2010 (English)Doctoral 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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. p. 56
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 509
Keywords
bacteria, bacterial evolution, genome reduction, gene loss, serial passage, DNA homology, tranlesion DNA polymerase, stress
National Category
Microbiology in the medical area Microbiology in the medical area
Research subject
Evolutionary Genetics; Microbiology
Identifiers
urn:nbn:se:uu:diva-111428 (URN)978-91-554-7687-8 (ISBN)
Public defence
2010-02-05, C10:305, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2010-01-14 Created: 2009-12-14 Last updated: 2018-01-12
Koskiniemi, S., Hughes, D. & Andersson, D. I. (2010). Effect of the translesion DNA polymerases, endonucleases and RpoS on mutation rates in Salmonella typhimurium. Genetics, 185(3), 783-795
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
Koskiniemi, S. & Andersson, D. I. (2009). Translesion DNA polymerases are required for spontaneous deletion formation in Salmonella typhimurium. Proceedings of the National Academy of Sciences of the United States of America, 106(25), 10248-10253
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
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-3275-0936

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