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GENETICS AND MOLECULAR BIOLOGY

A Protein Important for Antimicrobial Peptide Resistance, YdeI/OmdA, Is in the Periplasm and Interacts with OmpD/NmpC

M. Carolina Pilonieta, Kimberly D. Erickson, Robert K. Ernst, Corrella S. Detweiler
M. Carolina Pilonieta
1Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado 80309
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Kimberly D. Erickson
1Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado 80309
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Robert K. Ernst
2University of Maryland-Baltimore, Department of Microbial Pathogenesis, School of Dentistry, Baltimore, Maryland 21201
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Corrella S. Detweiler
1Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado 80309
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  • For correspondence: detweile@colorado.edu
DOI: 10.1128/JB.00688-09
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  • FIG. 1.
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    FIG. 1.

    ydeI mRNA accumulation requires PhoP and PmrA. Real-time quantitative PCR was used to evaluate ydeI mRNA levels and samples were normalized to a gapA control. (A) ydeI mRNA levels after exposure to polymyxin B for 40 min in LB medium using strains with the indicated genes deleted. (B) ydeI mRNA levels in early stationary phase cells in LB medium. WT, wild type. The data are representative of three independent experiments.

  • FIG. 2.
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    FIG. 2.

    LPS and lipid A from ydeI mutant strains and the wild type are indistinguishable. (A) LPS was isolated from wild-type (WT) and strains with deletions of the genes indicated, separated by SDS-PAGE, and silver stained. (B) MALDI-TOF mass spectra of lipid A from wild-type and phoP and ydeI mutant strains. The mass/charge (m/z) ratios and related structures are as follows: 1,796 unmodified hexa-acyl lipid A; 1,813, hexa-acyl lipid A with 2-OH myristate; 1,937, hexa-acyl lipid A with Ara4N; 2,036, unmodified hepta-acyl lipid A; and 2,167, hepta-acyl lipid A with Ara4N.

  • FIG. 3.
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    FIG. 3.

    OmpD specifically copurifies with YdeI. (A) Bound proteins were eluted from YdeI-HA using an HA dipeptide, resolved by SDS-PAGE, and analyzed by silver staining. Eluted fractions (E1 to E3) from a pYdeI-HA strain (left) and from a wild-type (WT) strain (right). The 40-kDa band was isolated and submitted for mass spectrometric analysis. Lanes shown are from the same gel, but the image was cropped to remove unloaded lanes. (B) Bound proteins were eluted from YdeI-HA or GST-HA with low pH, resolved by SDS-PAGE, and analyzed by silver staining. Each strain harbored endogenous OmpD or chromosomal OmpD-3xFLAG, as marked. GST-HA (28 kDa) is marked with an asterisk. Lanes shown are from the same gel, but the image was cropped to remove unloaded lanes. (C) The flowthrough (FT) and eluate from the GST-HA sample (B) were subject to immunoblotting with anti-FLAG and anti-HA antibodies.

  • FIG. 4.
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    FIG. 4.

    OmpD-3xFLAG coimmunoprecipitates with chromosomally expressed YdeI-HA. Strains encoding chromosomally expressed, epitope-tagged YdeI-HA and OmpD-3xFLAG were grown overnight in LB medium, harvested, and lysed, and YdeI-HA was immunoprecipitated with mouse anti-HA antibody coupled to protein G-agarose beads. Supernatant (SN) or agarose bead immunoprecipitate (IP) from equivalent numbers of bacteria was resolved by SDS-PAGE and analyzed by immunoblotting with (A) an anti-FLAG antibody or (B) an anti-HA antibody. Molecular weight markers are indicated by bars to the left; the heavy and light chains of the immunoprecipitating antibody are indicated by arrows to the right of (B).

  • FIG. 5.
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    FIG. 5.

    ompD mutant strains are sensitive to polymyxin B and cathelicidin antimicrobial peptide. Strains harboring deletions of ydeI, ompD, or both genes were incubated in the presence of 4 μg of polymyxin B/ml (A) or 50 μM cathelicidin antimicrobial peptide (B) and plated for CFU. Controls included wild-type (WT) and a phoP insertion mutant. The data are reported as percent survival relative to the wild type. Asterisks represent P values on triplicate samples, as determined by a Student t test (P < 0.001).

  • FIG. 6.
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    FIG. 6.

    YdeI is in the periplasm. Immunoblotting of whole-cell (WC) lysates and fractions from an YdeI-HA strain (KDE686). The upper blot was probed with an anti-HA antibody, and the lower with anti-DnaK and anti-maltose binding protein (MBP) antibodies to identify cytoplasmic (C) and periplasmic (PP) fractions.

  • FIG. 7.
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    FIG. 7.

    ygiW and ompF mutants are sensitive to polymyxin B. Strains with the indicated gene deleted were incubated with 8 μg of polymyxin B/ml and plated for CFU. The data are reported as percent survival relative to the wild type (WT). Single and double asterisks represent P values on triplicate samples, as determined by a Student t test (P < 0.01 and P < 0.001, respectively).

Tables

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  • TABLE 1.

    S. enterica serovar Typhimurium strains

    StrainGenotype and/or descriptionSource or reference
    SL1344 hisG xyl rpsL (wild-type serovar Typhimurium) 58
    CSD22114028s pKD46Charlie Kim
    CSD257 galE::Tn10 Bruce Stocker
    FL416SL1344 phoP::Cm 20
    KDE445SL1344 ydeI::Cm 20
    KDE517SL1344 rcsB::CmThis study
    KDE565SL1344 invA::CmThis study
    KDE602SL1344 ydeIΔThis study
    KDE612SL1344 ydeI::HA::KanThis study
    KDE642SL1344 ydeI::HAThis study
    KDE662SL1344 phoP::Cm, rcsBΔThis study
    KDE666SL1344 pmrA::CmThis study
    KDE686SL1344 with pYdeI-HAThis study
    KDE735SL1344 ompD::CmThis study
    KDE737SL1344 yddG::CmThis study
    NMM748SL1344 ompD::3xFLAG::KanThis study
    KDE755SL1344 ydeIΔ ompD::CmThis study
    KDE759SL1344 ydeI::HA ompD::3xFLAG::KanThis study
    NMM764SL1344 ompD::3xFLAGThis study
    KDE769SL1344 ydeI::HA ompD::3xFLAGThis study
    NMM800SL1344 ompE/phoE::CmThis study
    NMM801SL1344 ompF::CmThis study
    NMM802SL1344 ompN::CmThis study
    NMM803SL1344 STM1530::CmThis study
    NMM805SL1344 ompC::CmThis study
    NMM806SL1344 ygiW::CmThis study
    MCP847SL1344 ompD::3xFLAG, with pGEX-3xThis study
    MCP848SL1344 ompD::3xFLAG, with pYdeI-HAThis study
  • TABLE 2.

    PCR primers used in this study

    PrimeraSequence (5′→3′)b
    invA-P1TGAAAAGCTGTCTTAATTTAATATTAACAGGATACCTATAGTGTAGGCTGGAGCTGCTTC
    invA-P4ATATCCAAATGTTGCATAGATCTTTTCCTTAATTAAGCCCATGGGAATTAGCCATGGTCC
    invA-5′PCRGCAGAACAGCGTCGTACTAT
    invA-3′PCRCGGAACGAACTAATTCAGCG
    ompC-P1ATAAAAAAGCAATAAAGGCATATAACAGAGGGTTAATAAC GTGTAGGCTGGAGCTGCTTC
    ompC-P4AAAAAGGGCCCGCAGGCCCTTTAGCAACATCTTTTGCTGA ATGGGAATTAGCCATGGTCC
    ompC-5′PCRGTTAACCAGTAAGCAGTGGC
    ompC-3′PCRTACGCCGGAATAAGGCATGA
    ompD-P1GTTGAGGAAACACGCTAAGAAAATTATAAGGATTATTAAAGTGTAGGCTGGAGCTGCTTC
    ompD-P2GCCCTGAAAGGACTGGCTTTGTATTCAGACTACAACAAAAATGGGAATTAGCCATGGTCC
    ompD-5′PCRAAACGCCTCGTTTAACAATG
    ompD-3′PCRTACATCAAGAGAAAAAGCCA
    ompD-3xFLAG-5′ACCGACAACATCGTTGCTGTTGGTCTGAACTACCAGTTC GACTACAAAGACCATGACGGT
    ompD-3xFLAG-3′CCAGTGAACGTCTGCACGGCATACTCCTTATGACCGAGTC CATATGAATATCCTCCTTAG
    ompF-P1GCAGGTGTCATATAAAAAAACCAATGAGGGTAATAAATAGTGTAGGCTGGAGCTGCTTC
    ompF-P4AAGTCCTGTTTTTGAGGCATAAAACAAAGGGGTCTGCTGAATGGGAATTAGCCATGGTCC
    ompF-5′PCRCGGAATTTATTGACGGCAGT
    ompF-3′PCR GAGATAAAAAAACAGGACCG
    ompN-P1CAATCTTTTGCAAATAAGTTAAGTTTTTAAGGATAAAAAA GTGTAGGCTGGAGCTGCTTC
    ompN-P4GCCCGCCGAAACGGCGGGCTTGAGAAGAATTAATGAATAA ATGGGAATTAGCCATGGTCC
    ompN-5′PCRTCAACGAATCTGTAGAAGTT
    ompN-3′PCRGTGGTGATGAAAAAAGAAAA
    phoE-P1TCCCGACAAATCATAGCGCGTAATTAAAACAGGAATGGAAGTGTAGGCTGGAGCTGCTTC
    phoE-P4ATGCCTGATGGCGCAGCGCCATCAGGCACAATGCGACTTAATGGGAATTAGCCATGGTCC
    phoE-5′PCRTTCCTGTTTTTTACCGGGTT
    phoE-3′PCRGTCGGCATAACCCTGCTGCC
    pmrA-P1CCGCAGATGATATTCTGCAACCGTGCAGGAGACTAAGCGA GTGTAGGCTGGAGCTGCTTC
    pmrA-P4GAAGGGTCATCGCTCTTCGCTGAAAACGCATCAGGCTCAC ATGGGAATTAGCCATGGTCC
    pmrA-5′PCRACAAACGACGTATTACCAGG
    pmrA-3′PCRTGTCAGCATTAAACGCTGGC
    STM1530-P1TTGCGGAAAGTCCAAAAATAAGACAAATAAGGCATATAAAGTGTAGGCTGGAGCTGCTTC
    STM1530-P4AAGGAGGGTATCCCTAAACTGTCTTAATCAGCAAGTTTTAATGGGAATTAGCCATGGTCC
    STM1530-5′PCRGACAGTTCCGCGAGCAGGCT
    STM1530-3′PCRCAGTGACACAGTTTTGGGAA
    yddG-P1AACGCTAATAGGGCTTGTTGCCATCGTTCTGTGGAGCACG GTGTAGGCTGGAGCTGCTTC
    yddG-P4GCGGGTCGATTTAATATCAATATCGGCCCGCCCGTCGCTA ATGGGAATTAGCCATGGTCC
    yddG-5′PCRAGCATGACATCACAAAAAGC
    yddG-3′PCRATGAAGAGTGTCAAGGTACA
    ydeI-HA-5′GCGAAGGAACCGCTTGTTCGCGTGAACCGACTGCAAAAATATCCGTATGATGTTCCT
    ydeI-HA-3′CGCTTTTACAAGACCCATGGTATCAGGCCAGCCGCGTGCCCATATGAATATCCTCCTTAG
    ygiW-P1ATTAAATGGATCTGAAACGACATGAAAGGGAAAAGTAATCGTGTAGGCTGGAGCTGCTTC
    ygiW-P4GGGCGGCTGTGAATCCTGACCGATCTTGCGCAATGTGGGA ATGGGAATTAGCCATGGTCC
    ygiW-5′PCRAAGTTGTTAAGGATTACCTT
    ygiW-3′PCRTGTGGCCTATTCTGCCCACC
    • ↵ a The primer name indicates the gene or operon deleted or the epitope tag, followed by the primer binding site or epitope tag. P1, P2, and P4 indicate priming sites in pKD3 or pKD4 plasmids from Datsenko and Wanner (15). 5′PCR and 3′PCR refer to PCR primers used to screen deletion mutants and correspond to serovar Typhimurium genomic sequences. Primer families are divided by lines of space.

    • ↵ b Underlined sequences denote priming sequences for P1, P2, or P4 sites (15) or epitope tag sites in pSU315 (HA) or pSUB11 (3xFLAG) for 5′ epitope tag primers (65).

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A Protein Important for Antimicrobial Peptide Resistance, YdeI/OmdA, Is in the Periplasm and Interacts with OmpD/NmpC
M. Carolina Pilonieta, Kimberly D. Erickson, Robert K. Ernst, Corrella S. Detweiler
Journal of Bacteriology Nov 2009, 191 (23) 7243-7252; DOI: 10.1128/JB.00688-09

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A Protein Important for Antimicrobial Peptide Resistance, YdeI/OmdA, Is in the Periplasm and Interacts with OmpD/NmpC
M. Carolina Pilonieta, Kimberly D. Erickson, Robert K. Ernst, Corrella S. Detweiler
Journal of Bacteriology Nov 2009, 191 (23) 7243-7252; DOI: 10.1128/JB.00688-09
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KEYWORDS

Antimicrobial Cationic Peptides
Bacterial Proteins
Drug Resistance, Multiple, Bacterial
periplasm
porins
Salmonella enterica

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