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Genetics and Molecular Biology

Transcription of the Subtilase Cytotoxin Gene subAB1 in Shiga Toxin-Producing Escherichia coli Is Dependent on hfq and hns

Laura Heinisch, Katharina Zoric, Maike Krause, Herbert Schmidt
Maia Kivisaar, Editor
Laura Heinisch
aInstitute of Food Science and Biotechnology, Department of Food Microbiology and Hygiene, University of Hohenheim, Stuttgart, Germany
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Katharina Zoric
aInstitute of Food Science and Biotechnology, Department of Food Microbiology and Hygiene, University of Hohenheim, Stuttgart, Germany
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Maike Krause
aInstitute of Food Science and Biotechnology, Department of Food Microbiology and Hygiene, University of Hohenheim, Stuttgart, Germany
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Herbert Schmidt
aInstitute of Food Science and Biotechnology, Department of Food Microbiology and Hygiene, University of Hohenheim, Stuttgart, Germany
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Maia Kivisaar
University of Tartu
Roles: Editor
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DOI: 10.1128/AEM.01281-19
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  • FIG 1
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    FIG 1

    Growth curves of E. coli DH5α wild-type and mutant strains. Cultivation was conducted for 5 h in LB at 37°C with aeration. The OD600 was determined over a period of 5 h for E. coli DH5α/pKMD3, DH5α Δhns/pKMD3, and DH5α Δhfq/pKMD3 strains. Data represent mean values and standard deviations from three biological replicates.

  • FIG 2
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    FIG 2

    Impact of deletion of genes hfq and hns on growth of STEC O113:H21 strain TS18/08. Optical density of wild-type strain (TS18/08), TS18/08/pKMD3 + pBR322 (TS18/08/pBR322) empty vector backbone control, TS18/08 Δhns/pKMD3 (TS18/08 Δhns) and TS18/08 Δhfq/pKMD3 (TS18/08 Δhfq) deletion mutants, and TS18/08 Δhns/pKMD3 + pLH02 (TS18/08 Δhns + hns) and TS18/08 Δhfq/pKMD3 + pLH01 (TS18/08 Δhfq + hfq) complemented strains is shown for measurements over 5 h. Data from three biological replicates are shown; error bars indicate standard deviations from mean values. Asterisks indicate statistical significance (P < 0.05) compared to values for TS18/08.

  • FIG 3
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    FIG 3

    Comparison of PsubAB1 promoter activities in DH5α and TS18/08. Relative reporter gene activity (RLU/OD600) is shown. Bars represent data for E. coli DH5α (dark blue) and an STEC strain (TS18/08, light blue). OD600 is shown as squares. Cultivation was conducted for 5 h in LB at 37°C with aeration. Data from three biological replicates are shown; error bars indicate standard deviations from mean values.

  • FIG 4
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    FIG 4

    Impact of Hfq on PsubAB1 activity in E. coli DH5α (A) and STEC E. coli TS18/08 (B). Relative reporter gene activity (RLU/OD600) is shown. Bars represent data from the wild-type strain (DH5α/pKMD3 [A] or TS18/08/pKMD3 [B], solid blue) and an empty vector control (DH5α/pKMD3 + pBR322 [A] or TS18/08/pKMD3 + pBR322 [B], dashed blue). Additionally, data from E. coli DH5α Δhfq/pKMD3 (DH5α Δhfq) (A) and E. coli TS18/08 Δhfq/pKMD3 (TS18/08 Δhfq) (B) (solid green) deletion mutants and DH5α Δhfq/pKMD3 + pLH01 (DH5α Δhfq + hfq) (A) and TS18/08 Δhfq/pKMD3 + pLH01 (TS18/08 Δhfq + hfq) (B) (dashed green) complemented strains are depicted. Data are presented for measurements over 5 h of cultivation in LB at 37°C with aeration. Data from three biological replicates are shown; error bars indicate standard deviations from mean values. Statistical significance compared to the wild-type strain is indicated (*, P < 0.05; **, P < 0.01).

  • FIG 5
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    FIG 5

    Impact of H-NS on PsubAB1 activity in E. coli DH5α (A) and STEC TS18/08 (B). Relative (rel.) reporter gene activity (RLU/OD600) is shown. Bars represent data from the wild-type strain (DH5α/pKMD3 [A] or TS18/08/pKMD3 [B], blue) and an empty vector control (DH5α/pKMD3 + pBR322 [A] or TS18/08/pKMD3 + pBR322 [B], dashed blue). Additionally, data from E. coli DH5α Δhns/pKMD3 (DH5α Δhns) (A) and E. coli TS18/08 Δhns/pKMD3 (TS18/08 Δhns) (B) (solid red) deletion mutants and DH5α Δhns/pKMD3 + pLH01 (DH5α Δhns + hns) (A) and TS18/08 Δhns/pKMD3 + pLH01 (TS18/08 Δhns + hns) (B) (dashed red) complemented strains are depicted. Data are presented for measurements over 5 h of cultivation in LB at 37°C with aeration. Data from three biological replicates are shown; error bars indicate standard deviations from mean values. Statistical significance compared to the wild-type strain is indicated (**, P < 0.01).

  • FIG 6
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    FIG 6

    Expression of subAB1, stx2, and cdt-V in E. coli TS18/08 Δhfq (A) and TS18/08 Δhns (B) strains. Transcription is shown as fold change relative to the level of the control (E. coli TS18/08). Gene expression was measured under standard batch conditions (LB medium, 37°C, with aeration) using quantitative real-time PCR. Quantification was conducted using rrsB as a reference gene. Error bars indicate standard deviations from the mean values. Data from three biological replicates are shown. Asterisks indicate significance in fold change compared to the level for the control (P < 0.05).

  • FIG 7
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    FIG 7

    Plasmid map of the luciferase reporter gene plasmid pKMD3. The putative promoter region of subAB1 (405 bp; PsubAB1) was cloned upstream of the luciferase reporter gene luc (1,652 bp) in the pWSK29 backbone restricted with PvuII (pWSK29_PvuII).

  • FIG 8
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    FIG 8

    Cloning strategy of complementation vectors pLH01 (A) and pLH02 (B). Positions of hns and hfq genes refer to the genome sequence of E. coli DH5α (NCBI accession number NZ_JRYM01000001).

Tables

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  • Additional Files
  • TABLE 1

    Strains and plasmids used in this study

    Strain or plasmidRelevant geno- and phenotypeaReference or source
    E. coli
        DH5αtonA lacZΔM15 endA1 recA1 thi-1 supE44 phoA gyrA96 hsdR17 Δ(lacZYA-argF)U169 relA1Invitrogen
        DH5α ΔhnstonA lacZΔM15 endA1 recA1 thi-1 supE44 phoA gyrA96 hsdR17 Δ(lacZYA-argF)U169 relA1 ΔhnsThis study
        DH5α ΔhfqtonA lacZΔM15 endA1 recA1 thi-1 supE44 phoA gyrA96 hsdR17 Δ(lacZYA-argF)U169 relA1 ΔhfqThis study
        HB101F− mcrB mrr hsdS20(rB− mB−) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20 (Smr) supE44 λ−52
        TS18/08O113:H21, cdt-V+ subAB1+ stx2+26
        TS18/08 ΔhnsO113:H21, cdt-V+ subAB1+ stx2+ ΔhnsThis study
        TS18/08 ΔhfqO113:H21, cdt-V+ subAB1+ stx2+ ΔhfqThis study
    Plasmids
        pKD46Contains genes of red recombinase system under control of araB promoter, Ampr46
        pKD4Template plasmid for kanamycin resistance cassette, Ampr, Kanr46
        pCP20Contains genes for FLP recombinase, Ampr46
        pKMD3Luciferase reporter gene plasmid containing the subAB1 promoter regionThis study
        pBR322Cloning vector, pMB1 origin of replication, Ampr, Tetr53
        p3121Luciferase reporter gene (luc) template vector, Ampr, Kanr54
        pWSK29Cloning vector (low copy number), Ampr55
        pLH01Complementation plasmid, pBR322 backbone, with hfq gene under control of its own promoter, TetrThis study
        pLH02Complementation plasmid, pBR322 backbone, with hns gene under control of its own promoter, TetrThis study
    • ↵a Ampr, ampicillin resistance gene; Kanr, kanamycin resistance gene; Tetr, tetracycline resistance gene.

  • TABLE 2

    Oligonucleotide primers used in this study

    Primer and/or categoryUseNucleotide sequencea (5′ to 3′)Source
    Hfq_forDetection of hfq geneATGGCTAAGGGGCAATCTTTACAAGThis study
    Hfq_revDetection of hfq geneTTATTCGGTTCTTCGCTGTCCTGThis study
    Hns_forDetection of hns geneTTATTGCTTGATCAGGAAATCGTCGThis study
    Hns_revDetection of hns geneATGAGCGAAGCACTTAAAATTCTGThis study
    Mutagenesis
        Del_hfq_forDeletion of hfq gene, creation of resistance cassetteAATGTGTACAATTGAGACGTATCGTGCGCATTTTTTCAGAATCGCGATTGTGTAGGCTGGAGCThis study
        Del_hfq_revDeletion of hfq gene, creation of resistance cassetteAGCGTATAACCCTCTAAATAGATCAGCGGGGAACGCAGGATCCATGGTCCATATGAATATCCTCCThis study
        Hfq + 186up_forVerification of deletion of hfqCCTGGCTGCGTGGTTGGGAAGThis study
        Hfq-176down_revVerification of deletion of hfqACCAGAGATTCAAACTCCTGGAGGTCThis study
        Del-hns_forDeletion of hns gene, creation of resistance cassetteACATCCGTATCGGTGTTACCACGAAACGGCGTTGAGCAATCGCGATTGTGTAGGCTGGAGCThis study
        Del-hns_revDeletion of hns gene, creation of resistance cassetteATAGGGAATTCTCGTAAACACAACTAATACAGAAGACTGAAAGGCATGGTCCATATGAATATCCTCCThis study
        Hns + 244up_forVerification of deletion of hnsTAGGTTACATGCAGGCCTTCGTTGThis study
        Hns-705down_revVerification of deletion of hnsACAGTGCGCATGCCGCGTTCCThis study
    Construction of complementation plasmids
        Hfq + 86up_AseI_forComplementation studiesGGGGGGGGGGattaatAATGTGTACAATTGAGACGTATCGTGCGCAATTTTTTCAGAATCThis study
        Hfq-76down_PvuI_revComplementation studiesGGGGGcgatcgAGCGTATAACCCTCTAAATAGATCAGCGGGGAACGCAGGATCThis study
        Hns + 144up_AseI_forComplementation studiesCCCCCCaattaatACATCCGTATCGGTGTTATCCACGAAACGGCGTTGAGCAATCThis study
        Hns-300down_PvuI_revComplementation studiesCCCCCCcgatcgATAGGGAATTCTCGTAAACACAACTAATACAGAAGACTGAAAGGThis study
    Construction of luciferase reporter gene plasmid pKMD3
        subAB-gibson_forCloning of subAB1 promoter region for pKMD3TTCGCTATTACGCCAGCTGAACGGTATCGATCCGThis study
        subAB-gibson_revCloning of subAB1 promoter region for pKMD3GTCTTCCATAAGCTCCTCCAGTTAGTACThis study
        luc-gibson_forCloning of luc for pKMD3AGGAGCTTATGGAAGACGCCAAAAACThis study
        luc-gibson_revCloning of luc for pKMD3CCGATTCATTAATGCAGTCACAATTTGGACTTTCCGThis study
        Gibson-1_forVerification of pKMD3ACACCCGCCGCGCTTAATGCThis study
        Seq 2-gibson_revVerification of pKMD3GATACCGCTCGCCGCAGCCGThis study
        Seq 3-gibson_forVerification of pKMD3GGTTGTGGATCTGGATGCCGThis study
        Seq 4-gibson_revVerification of pKMD3AGGGCGTATCTCTTCATAGCThis study
    • ↵a Underlined bases depict homologous regions in mutagenesis, and underlined lowercase letters depict restriction sites for cloning strategy.

  • TABLE 3

    Oligonucleotide primers used for transcription analysis by qRT-PCR

    PrimerNucleotide sequence (5’ to 3’ direction)PCR product size and conditionsReference
    rrsB-forGCATAACGTCGCAAGACCAAA91 bp; 95°C, 10 s; 60.0°C, 30 s; 72°C, 10 s51
    rrsB-revGCCGTTACCCCACCTACTAGCT
    RTsubAB-2-forGCAGGTATTATGGGATGTCT177 bp; 95°C, 15 s; 60.3°C, 20 s; 72°C, 10 s12
    RTsubABrevGCAGGCACTTATGGAGGAG
    Upper-stx2TCATATCTGGCGTTAATGGAGTTC99 bp; 95°C, 10 s; 52.4°C, 30 s; 72°C, 10 s51
    Lower-stx2GCGTAAGGCTTCTGCTGTG
    RTCDTA-forCCGATGGTAACACGCAATG184 bp; 95°C, 10 s; 53.0°C, 30 s; 72°C, 10 s12
    RTCDTA-revGCCTTAATGGTTCGCTTATGG

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    • Supplemental file 1 -

      Fig. S1 and S2

      PDF, 158K

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Transcription of the Subtilase Cytotoxin Gene subAB1 in Shiga Toxin-Producing Escherichia coli Is Dependent on hfq and hns
Laura Heinisch, Katharina Zoric, Maike Krause, Herbert Schmidt
Applied and Environmental Microbiology Oct 2019, 85 (20) e01281-19; DOI: 10.1128/AEM.01281-19

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Transcription of the Subtilase Cytotoxin Gene subAB1 in Shiga Toxin-Producing Escherichia coli Is Dependent on hfq and hns
Laura Heinisch, Katharina Zoric, Maike Krause, Herbert Schmidt
Applied and Environmental Microbiology Oct 2019, 85 (20) e01281-19; DOI: 10.1128/AEM.01281-19
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    • ABSTRACT
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KEYWORDS

STEC
Shiga toxin-producing Escherichia coli
gene expression
global regulator
hfq
hns
subtilase cytotoxin

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