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Research Article | Molecular Biology and Physiology

Transcriptional Regulation of Carnitine Catabolism in Pseudomonas aeruginosa by CdhR

Jamie A. Meadows, Matthew J. Wargo
Gary Sawers, Editor
Jamie A. Meadows
aDepartment of Microbiology and Molecular Genetics, University of Vermont Larner College of Medicine, Burlington, Vermont, USA
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Matthew J. Wargo
aDepartment of Microbiology and Molecular Genetics, University of Vermont Larner College of Medicine, Burlington, Vermont, USA
bThe Vermont Lung Center, University of Vermont Larner College of Medicine, Burlington, Vermont, USA
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Gary Sawers
Martin Luther University of Halle-Wittenberg
Roles: Editor
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DOI: 10.1128/mSphere.00480-17
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  • FIG 1
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    FIG 1

    Diagram of the P. aeruginosa PAO1 carnitine catabolism operon and the catabolic pathway. (A) Arrows represent the individual open reading frames of the carnitine catabolism operon and the regulator chdR. Below the arrow is the designated gene name. (B) Diagram of the cdhR-caiX intragenic region organized such that caiX transcription occurs left to right. The orange box denotes the cdhR 5′ UTR, the dark green box marks the position of the CdhR and GbdR binding sites (CdhR binding sequence listed below), and the light green box denotes the caiX 5′ UTR. (C) Diagram of the converging carnitine and choline catabolism pathways. Black arrows represent an enzymatic step in the catabolic pathway, and the gene names are italicized below. The blue arrows represent positive regulation by either CdhR or GbdR, and the T-bar represents repression by BetI.

  • FIG 2
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    FIG 2

    Mapping the caiX promoter and its CdhR binding site. (A) Transcriptional fusions of lacZ to the upstream region of caiX start at bp +3 from the caiX transcriptional start site and end at base pairs marked in the figure. P. aeruginosa PA14 strains carrying each construct were grown in MOPS with 20 mM pyruvate and 20 µg·ml−1 gentamicin, with (black bars) or without (white bars) 1 mM carnitine. β-Galactosidase activities for these caiX transcriptional fusions are reported in Miller units. Error bars represent standard deviation from three biological replicates, and results are representative of three independent experiments. (B) EMSA with biotin-labeled caiX promoter DNA probe alone (lane 1) or with increasing concentrations of purified MBP-CdhR (lanes 2 and 3). An unlabeled (cold) caiX probe was used to compete for binding of MBP-CdhR from the labeled probe (lane 4). An unrelated dhc DNA probe was used to show specificity of MBP-CdhR binding to caiX (lanes 5 to 8).

  • FIG 3
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    FIG 3

    The CdhR binding site and key residues for CdhR-dependent induction. (A) A DNase I footprinting assay was performed by taking the caiX UAS end labeled with 32P and adding increasing concentrations of MBP-CdhR, followed by DNase I treatment and nondenaturing 5% polyacrylamide TBE gel. The first lane of the gel is the A+G sequencing ladder, and the nanomolar concentration of MBP-CdhR is marked. (B) The caiX enhancer site was mutated by changing two bases at a time (in red and underlined) in the caiX distal binding site to adenosines and fused to lacZ. The P. aeruginosa PA14 wild type carrying each of the plasmids was grown in MOPS with 20 mM pyruvate at 20 µg·ml−1, with or without 1 mM carnitine for 4 h, and then β-galactosidase activity was reported as Miller units. Error bars represent standard deviations from three biological replicates, and results are representative of three independent experiments. Data were analyzed using a two-way analysis of variance (ANOVA) with a Sidak’s multiple-comparison posttest comparing each mutant’s pyruvate to carnitine. Abbreviations: P, pyruvate; C, carnitine; n.s., not significant; *, P < 0.05; ***, P < 0.001.

  • FIG 4
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    FIG 4

    caiX transcription is repressed by glucose and glycine betaine. P. aeruginosa PA14 with PcaiX-lacZ (pJAM22) was grown in MOPS medium with 20 mM pyruvate and 20 µg·ml−1 gentamicin, with or without 1 mM carnitine. Glucose or glycine betaine was added at the millimolar concentrations noted. Cultures were induced for 4 h prior to measurement of β-galactosidase activity. Error bars represent standard deviations from three biological replicates, and results are representative of three independent experiments. Data were analyzed using a one-way ANOVA with a Dunnett’s multiple-comparison posttest comparing each condition to the condition with carnitine alone. Bars denote that all data underneath are different from carnitine alone with the same statistical certainty. Abbreviations: Glu, glucose; GB, glycine betaine; ***, P < 0.001; ****, P < 0.0001.

  • FIG 5
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    FIG 5

    CdhR binds but does not regulate cbcXWV expression. (A) EMSA with a biotin-labeled (biot-) cbcX upstream region and purified MBP-CdhR in increasing concentrations. (B) Relative expression of cbcX was calculated based on the expression in WT pyruvate normalized to the rplU transcript using qRT-PCR. Three biological samples were run in triplicate, and the graph represents the mean values and standard deviation. Data were analyzed using a two-way ANOVA with a Tukey’s multiple comparison posttest comparing all strains and conditions to each other. Ends of the bars denote the comparison groups shown. Abbreviations: Pyr, pyruvate; Carn, carnitine; n.s., not significant; ***, P < 0.001.

  • FIG 6
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    FIG 6

    GbdR binds the caiX-cdhR intergenic region but does not induce transcription of caiX. (A) EMSAs were performed with increasing concentrations of purified MBP-GbdR with either the biotin-labeled cdhR wild-type probe or a mutant binding site probe. The mutated probe has the distal half-site CG residues (in relation to cdhR) changed to AA. (B) β-Galactosidase assay with a caiX-lacZ reporter plasmid (pJAM22) in wild-type, ΔcdhR, ΔgbdR, or ΔcdhR ΔgbdR strains of both PA14 (14) and PAO1 (1) grown in MOPS, 20 mM pyruvate, and 20 µg·ml−1 gentamicin. Induced cultures have an additional 1 mM carnitine. Error bars represent standard deviations from three biological replicates, and results are representative of three independent experiments. Data were analyzed with two-way ANOVA with a Tukey’s multiple-comparison test comparing all mutants and conditions within a given strain. (PA14 was not compared to PAO1.) Abbreviations: ***, P < 0.001 compared to WT with pyruvate; #, P < 0.01 compared to WT with carnitine.

  • FIG 7
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    FIG 7

    CdhR promotes cdhR expression, and GbdR dampens basal repression. (A) WT, ΔcdhR ΔgbdR, and ΔcdhR ΔgbdR strains in both PA14 (14) and PAO1 (1) backgrounds carrying a cdhR-lacZ translational plasmid reporter (pJAM135) were grown in MOPS with 20 mM pyruvate and 20 µg·ml−1 gentamicin, with or without 1 mM carnitine, and β-galactosidase activity was reported as fold change over WT pyruvate. Data were analyzed using a two-way ANOVA with a Sidak’s multiple-comparison posttest comparing to the WT pyruvate condition within each strain. (PA14 was not compared to PAO1.) *, P < 0.05. (B) PAO1 WT, ΔcdhR, and ΔgbdR strains, all with the translational fusion cdhR-yfp integrated at the attTn7 site, were grown on MOPS agar pads with 20 mM pyruvate and with or without 1 mM carnitine. Cells were imaged under phase-contrast and YFP fluorescence every 10 min at 32°C. Data were analyzed using a one-way ANOVA with a Dunnett’s multiple-comparison posttest comparing each time point within a strain to pyruvate at time zero (t = 0). ***, P < 0.001.

  • FIG 8
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    FIG 8

    Regulation of caiX and cdhR by CdhR and GbdR. Shown is a genetic network diagram of the interactions known and proposed in this study. Arrows indicate positive interactions (induction or activation), and T-bars indicate negative interactions (repression or other inhibition). The regulatory steps are black for CdhR and green or blue for interactions between other members in the network. The dotted blue line from carnitine to glycine betaine (GB) marks the metabolic conversion noted in Fig. 1C.

Tables

  • Figures
  • TABLE 1

    Strains and plasmids used in this study

    Strain or plasmidGenotype or descriptionReference
    or source
    Strains
        P. aeruginosa PAO1
            MJ79Wild type14
            MJ80ΔgbdR15
            JM236ΔcdhRThis study
            JM253Wild type attTn7::88-89intYFPCFP-2This study
            JM339ΔcdhR attTn7::88-89intYFPCFP-2This study
            JM340ΔgbdR attTn7::88-89intYFPCFP-2This study
            MJ784ΔgbdR ΔcdhRThis study
        P. aeruginosa PA14
            MJ101Wild type42
            MJ11ΔcdhR11
            MJ26ΔgbdR15
            MJ262ΔcdhCA11
            JM179ΔcdhR ΔgbdRThis study
        E. coli
            MJ340Wild-type S17λpir
            DH5αNEB C2987NEB
            T7ExpressNEB C3016NEB
    Plasmids
        pMQ30Suicide vector, Gmr36
        pMQ80High-copy-no. Pseudomonas vector, Gmr36
        pMal-C2XT7-expressing vector, MBP N-terminal tag, AmprNEB
        pTNS2Plasmid carrying attTn7 transposase43
        pUC18-mini-Tn7T-GmGmr on mini-Tn7T35
        pUC18-mini-Tn7T-Gm-eyfpGmr on mini-Tn7T with YFP35
        pUCP22High-copy-no. Pseudomonas stabilization vector, Gmr44
        pMW5lacZYA in pUCP2232
        pMW79PA14 genomic clone of PA5380-PA5389 in pMQ8011
        pPA5380KOgbdR deletion construct in pEX18-Gm15
        pJAM22Promoter caiX-lacZYA transcriptional fusion AThis study
        pJAM23Promoter caiX-lacZYA transcriptional fusion BThis study
        pJAM24Promoter caiX-lacZYA transcriptional fusion CThis study
        pJAM25Promoter caiX-lacZYA transcriptional fusion DThis study
        pJAM50PA5389 in pMal-C2XThis study
        pJAM76YFP-CFP in pMQ80This study
        pJAM86CFP PA5388-PA5389 intergenic region YFP in pUC18mini, DR2This study
        pJAM90PA5389 deletion construct in pMQ30This study
        pJAM122Promoter caiX-lacZYA transcriptional fusionThis study
        pJAM123Promoter caiX-lacZYA mut 1 transcriptional fusionThis study
        pJAM124Promoter caiX-lacZYA mut 2 transcriptional fusionThis study
        pJAM125Promoter caiX-lacZYA mut 3 transcriptional fusionThis study
        pJAM126Promoter caiX-lacZYA mut 4 transcriptional fusionThis study
        pJAM127Promoter caiX-lacZYA mut 5 transcriptional fusionThis study
        pJAM130Promoter caiX-lacZYA mut 6 transcriptional fusionThis study
        pJAM131C terminus of lacZ in pUCP22This study
        pJAM135PcdhRlacZYA in pUCP22This study
  • TABLE 2

    Primers used in this study

    PrimerSequence (5′ to 3′)a
    Deletion constructs
        5389GOIF1KpnIATAGGGTACCGAAGAACACCACCCACTGCT
        5389SOEGOIR1AAGTACGAAGGCGACTCGACCATGGAGAAGCCCATTACCGAGAAGC
        5389SOEGOIF1GCTTCTCGGTAATGGGCTTCTCCATGGTCGAGTCGCCTTCGTACTT
        5389GOIR1BamHIATCGTCTTCGCTGTTTTTCC
    Protein expression construct
        5389Mal-c2xFGCATCAGAATTCTCCCAGGACTTCTGGTTTCT
        5389Mal-c2xRGCATCAAAGCTTTCAGCCTCGCTCAGCTCGA
    Primer extension
        5388primerextension5′-Fluorescein 6-FAM-ACTGGCCAGGATCAGCAGG
        5389primerextension5′-Fluorescein 6-FAM-AGACAGTATCGGCCTCAGGAA
    EMSA probes
        PA5388promF3AAGCTTGTGCCAGCGGTAGAGGTC
        PA5388promRTGAGGTACCTTGATTGTTTTTCTGCGAGGT
        PA5388promRbiotBiotin-TTGATTGTTTTTCTGCGAGGT
        5389EMSA-FATGAAAGCTTGCAGCAGGAGAAACCAGAAG
        5389EMSA-R-biotBiotin-TTGATTGTTTTTCTGCGAGGT
        5389EMSA-Mut3FGGACGGCGGCGAAGCGCACTGCGAAGACC
        cbcXprom-FCCGGCAAAGACCACTATGAT
        cbcXprom-R-biotBiotin-GAACTCCTCTGCAGGGTAAGG
        dhcprom-F-biotBiotin-GAGGCTTTCCTCCAGGCTCT
        dhcprom-RGGATGGTACCCTCTTCCGGCTCTTGTGATT
        dhcprom-FGAACTCCTCTGCAGGGTAAGG
    Transcriptional reporters
        PA5388promRTGAGGTACCTTGATTGTTTTTCTGCGAGGT
        PA5388promF1ATGAAAGCTTACAGCAGGTCGCCTTTCTT
        PA5388promF3AAGCTTGTGCCAGCGGTAGAGGTC
        PA5388promF2ATGAAAGCTTGCAGCAGGAGAAACCAGAAG
        PA5388promF4AAGCTTCTGCAGTGCAAGAGCTGGT
        P5388posATGAAAGCTTCGCTTGGCAATGGCCAGGTCGCT
        P5388mut1ATGAAAGCTTCGCTTGGCAATGGCCAAATCGCT
        P5388mut2ATGAAAGCTTCGCTTGGCAATGGCCAGAACGCT
        P5388mut3ATGAAAGCTTCGCTTGGCAATGGCCAGGAAGCT
        P5388mut4ATGAAAGCTTCGCTTGGCAATGGCCAGGTAACT
        P5388mut5ATGAAAGCTTCGCTTGGCAATGGCCAGGTCAAT
        P5388mut6ATGAAAGCTTCGCTTGGCAATGGCCAGATCGCT
    Translational reporters
        2-lacZCtermFhindclaGCAAGCTTATTATCGATGAGCGTGGTGGTTATGC
        2-lacZCtermRsmakpnCGGTACCCGGGGATCCTTATTTTTGACACCAGACC
        YFP R HindIIIGCATCAAAGCTTATTACTTGTACAGCTCGTCCA
        YFP F Kpn SalGACAGCGGTACCAATCGTCGACCATATGCTGAGCAAGGGCGAGG
        88-89YC-DR#2ycFGGGCACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCAGCATGGGGCGCTCCGGGGTTGA
        88-89YC-DR#2ycRCGGCACCACGCCGGTGAACAGCTCCTCGCCCTTGCTCAGCATCGGTCTCCCCTCGTGCGG
    • ↵a 6-FAM, 6-carboxyfluorescein.

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Transcriptional Regulation of Carnitine Catabolism in Pseudomonas aeruginosa by CdhR
Jamie A. Meadows, Matthew J. Wargo
mSphere Feb 2018, 3 (1) e00480-17; DOI: 10.1128/mSphere.00480-17

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Transcriptional Regulation of Carnitine Catabolism in Pseudomonas aeruginosa by CdhR
Jamie A. Meadows, Matthew J. Wargo
mSphere Feb 2018, 3 (1) e00480-17; DOI: 10.1128/mSphere.00480-17
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KEYWORDS

metabolism
osmoprotectant
quaternary amine
transcriptional regulation

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