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A palette of fluorophores that are differentially accumulated by wild-type and mutant strains of Escherichia coli: surrogate ligands for bacterial membrane transporters

Jesus Enrique Salcedo-Sora, Srijan Jindal, Steve O’Hagan, View ORCID ProfileDouglas B. Kell
doi: https://doi.org/10.1101/2020.06.15.152629
Jesus Enrique Salcedo-Sora
1Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Crown St, Liverpool L69 7ZB, UK
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Srijan Jindal
1Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Crown St, Liverpool L69 7ZB, UK
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Steve O’Hagan
2Department of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess St, Manchester M1 7DN, UK
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Douglas B. Kell
1Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Crown St, Liverpool L69 7ZB, UK
3Novo Nordisk Foundation Centre for Biosustainability, Technical University of Denmark, Building 220, Kemitorvet, 2800 Kgs. Lyngby, Denmark
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  • ORCID record for Douglas B. Kell
  • For correspondence: dbk@liv.ac.uk doukel@biosustain.dtu.dk
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Abstract

Our previous work had demonstrated that two commonly used fluorescent dyes that were accumulated by wild-type E. coli MG1655 were accumulated differentially in single-gene knockout strains, and also that they might be used as surrogates in flow cytometric transporter assays. We summarise the desirable properties of such stains, and here survey 143 candidate dyes. We triage them eventually (on the basis of signal, accumulation levels, and cost) to a palette of 39 commercially available and affordable fluorophores that are accumulated significantly by wild-type cells of the ‘Keio’ strain BW25113, as measured flow cytometrically. Cheminformatic analyses indicate both their similarities and their (much more considerable) structural differences. We describe the effects of pH and of the efflux pump inhibitor chlorpromazine on the accumulation. Even the ‘wild-type’ MG1655 and BW25113 strains can differ significantly in their ability to take up such dyes. We illustrate the highly differential uptake of our dyes into strains with particular lesions in, or overexpressed levels of, three particular transporters or transporter components (yhjV, yihN, and tolC). The relatively small collection of dyes described offers a rapid, inexpensive, convenient and valuable approach to the assessment of microbial physiology and transporter function.

Competing Interest Statement

DBK and SJ are named inventors on a vaguely connected patent application relating to the use [17] of flow cytometry in antimicrobial detection. The authors declare that they otherwise have no conflicts of interest beyond the funding of their projects given in the Acknowledgments section.

  • Abbreviations

    ANS
    8-Anilinonaphthalene-1-sulfonic acid
    BODIPY
    Difluoro{2-[(3,5-dimethyl-2H-pyrrol-2-ylidene-N)methyl]-3,5-dimethyl-1H-pyrrolato-N}boron
    CCCP
    Carbonyl cyanide m-chlorophenyl hydrazone
    CPDPP
    3,6-Bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione
    CPZ
    Chlorpromazine
    ASP
    4-(4-(Dimethylamino)styryl-N-methylpyridinium (ASP+)
    diS-C3(5)
    3-Propyl-2-{(1E,3E,5E)-5-(3-propyl-1,3-benzothiazol-2(3H)-ylidene)-1,3-pentadien-1-yl}-1,3-benzothiazol-3-ium iodide
    DMSO
    Dimethyl sulfoxide
    DPDPP
    3,6-Diphenyl-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione
    H2FDA
    Dihydrofluorescein diacetate
    IPTG
    Isopropyl β-d-1-thiogalactopyranoside
    NBDG
    2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose
    SYBR Green I
    N,N-dimethyl-N′-[(4E)-4-[(3-methyl-1,3-benzothiazol-3-ium-2-yl)methylidene]-1-phenyl-2,3-dihydroquinolin-2-yl]-N′-propylpropane-1,3-diamine
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    Posted June 15, 2020.
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    A palette of fluorophores that are differentially accumulated by wild-type and mutant strains of Escherichia coli: surrogate ligands for bacterial membrane transporters
    Jesus Enrique Salcedo-Sora, Srijan Jindal, Steve O’Hagan, Douglas B. Kell
    bioRxiv 2020.06.15.152629; doi: https://doi.org/10.1101/2020.06.15.152629
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    A palette of fluorophores that are differentially accumulated by wild-type and mutant strains of Escherichia coli: surrogate ligands for bacterial membrane transporters
    Jesus Enrique Salcedo-Sora, Srijan Jindal, Steve O’Hagan, Douglas B. Kell
    bioRxiv 2020.06.15.152629; doi: https://doi.org/10.1101/2020.06.15.152629

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