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Invertebrate Microbiology

CpxRA Regulates Mutualism and Pathogenesis in Xenorhabdus nematophila

Erin E. Herbert, Kimberly N. Cowles, Heidi Goodrich-Blair
Erin E. Herbert
Department of Bacteriology, University of Wisconsin—Madison, Madison, Wisconsin 53706
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Kimberly N. Cowles
Department of Bacteriology, University of Wisconsin—Madison, Madison, Wisconsin 53706
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Heidi Goodrich-Blair
Department of Bacteriology, University of Wisconsin—Madison, Madison, Wisconsin 53706
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  • For correspondence: hgblair@bact.wisc.edu
DOI: 10.1128/AEM.01586-07
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  • FIG. 1.
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    FIG. 1.

    CpxR shows inverse growth phase-dependent control of cpxR expression. (A) Transcript levels of cpxR, cpxA, and cpxP analyzed by qPCR in early log phase in the WT (Tn7) (normalized to 1.0 [not shown]), ΔcpxR1 (Tn7) (open bars, not visible), ΔcpxR1 (Tn7/cpxRA) (striped bars), and WT (Tn7/cpxRA) (gray bars) strains. qPCR was performed using the primers indicated in Table 2. The bars indicate averages of three or more replicates. An asterisk indicates a value significantly different from the WT (Tn7) transcript level. Fold-WT, fold increase compared with the wild type. (B) cpxR promoter activity in wild-type X. nematophila (□) and the ΔcpxR1 mutant (○), both carrying a cpxRp-lacZ fusion, measured by the β-galactosidase assay. Growth of the wild type (▪) and the ΔcpxR1 mutant (•) in LB broth is also shown. The symbols indicate the averages for three replicates. The error bars indicate standard errors.

  • FIG. 2.
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    FIG. 2.

    ΔcpxR1 mutant is attenuated in virulence for M. sexta insects and mutualistic nematode colonization. (A) Fourth-instar M. sexta larvae were injected with log-phase X. nematophila cells, and insect survival was monitored over time. The strains included in this analysis were the WT (Tn7) (□), WT (Tn7/cpxRA) (⋄), ΔcpxR1 (Tn7) (○), and ΔcpxR1 (Tn7/cpxRA) (•) strains. The symbols indicate the averages of six to eight experiments (10 insects per experiment). The error bars indicate standard errors. The letters b and c indicate values significantly different from the WT (Tn7) strain value (a). (B) X. nematophila bacteria were isolated from colonized S. carpocapsae nematodes, and the average number of CFU/infective juvenile (IJ) for each strain was normalized to wild-type values, which were defined as 100%. Nematodes were colonized with the strains indicated at the bottom. The values are averages of three or more experiments. An asterisk indicates the number of CFU/infective juvenile is statistically different from the wild-type value (WT) (P < 0.01).

  • FIG. 3.
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    FIG. 3.

    ΔcpxR1 mutant has altered morphology and phenotypic properties. (A) Wild-type X. nematophila (WT) and ΔcpxR1 mutant colony and cell morphologies. Images were obtained using phase-contrast microscopy at magnifications of ×100 (colony morphology) and ×1,000 (cell morphology). Cell and colony morphologies were analyzed using X. nematophila strains grown in plates containing liquid LB and LB with 0.1% pyruvate, respectively. The scale bars indicate 5 and 100 μm in the cell and colony morphology images, respectively. (B) Phenotypes inhibited (antibiotics, hemolysin, and protease) and activated (lipase and motility) by CpxR. The values under the antibiotic, hemolysin, and protease images indicate the halo area/colony area (averages ± standard errors). The values under the lipase images indicate the precipitate density, and the values under the motility images are growth diameters. An asterisk indicates a value that is significantly different (P < 0.01) from the WT (Tn7) strain value.

  • FIG. 4.
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    FIG. 4.

    CpxR positively regulates nilA, nilB, and nilC expression. lacZ reporter fusions were used to measure expression of nilA (A), nilB (B), and nilC (C) in the ΔcpxR1 mutant by the β-galactosidase assay. Assays were performed using stationary-phase cultures (averages of three replicates are indicated by the bars). The error bars indicate standard errors. An asterisk indicates a value significantly different from the wild-type value (WT) (P < 0.001).

  • FIG. 5.
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    FIG. 5.

    Model of CpxR regulation in X. nematophila. CpxR positively regulates motility and lipase activity and nematode colonization factors, potentially through transcriptional activation or derepression of lrhA and nilA, nilB, and nilC. No genes have been associated yet with antibiotic production in X. nematophila, but negative regulation of protease and hemolysin activities by CpxR may be attributed to transcriptional repression of prtA and xaxA. mrxA is also repressed by CpxR. The designations of genes associated with the activities are indicated. The arrows indicate positive regulation, and negative regulation is indicated by lines ending with a perpendicular line.

Tables

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  • TABLE 1.

    Strains and plasmids used in this study

    Strain or plasmidDescriptionReference or source
    X. nematophila strains
        HGB800 X. nematophila ATCC 19061, wild typeAmerican Type Culture Collection
        HGB1227HGB800 (Tn7)This study
        HGB1228HGB800 (Tn7/cpxRA)This study
        HGB1230HGB800 ΔcpxR1This study
        HGB1231HGB800 ΔcpxR1 (Tn7)This study
        HGB1232HGB800 ΔcpxR1 (Tn7/cpxRA)This study
        HGB1236HGB800 (Tn7/cpxRp-lacZ)This study
        HGB1237HGB800 ΔcpxR1 (Tn7/cpxRp-lacZ)This study
        HGB1117HGB800 (Tn7/nilA-lacZ) 11
        HGB1121HGB800 (Tn7/nilAB-lacZ) 11
        HGB1101HGB800 (Tn7/nilC-lacZ) 10
        HGB1239HGB800 ΔcpxR1 (Tn7/nilA-lacZ)This study
        HGB1240HGB800 ΔcpxR1 (Tn7/nilAB-lacZ)This study
        HGB1241HGB800 ΔcpxR1 (Tn7/nilC-lacZ)This study
    E. coli strains
        S17-1(λpir)Donor strain for conjugations 72
        SM10Donor strain for conjugations of Strr plasmids 72
    Plasmids
        pCR2.1-TOPOCloning vector, Ampr KanrInvitrogen (Carlsbad, CA)
        pKR100oriR6K, mobRP4 suicide vector, CmrK. Visick, Loyola University
        pKJN102Source of aad (Strr) geneK. Cowles, University of Wisconsin—Madison
        pKRcpxRuppKR100 plus 1,058-bp cpxR 5′ flanking regionThis study
        pKRcpxRupStrpKRcpxRup plus aad (Strr) geneThis study
        pKRcpxRStrpKRcpxRupStr plus 1,288-bp cpxR 3′ flanking regionThis study
        pEVS107Tn7 transposon delivery vector, oriR6K, mobRP4, Ermr Kanr 46
        pEVSCmDerivative of pEVS107; Tn7 transposon vector, Ermr CmrE. Martens, University of Wisconsin—Madison
        pEVSCmKanDerivative of pEVS107; Tn7 transposon vector, Cmr KanrE. Martens, University of Wisconsin—Madison
        pUX-BF13Triparental mating helper plasmid expressing Tn7 transposase, Ampr 4
        pTn7/nilA-lacZpEVS107 plus nilA 5′ flanking region fused to lacZ, Ermr Kanr 11
        pTn7/nilAB-lacZpEVS107 plus nilB 5′ flanking region fused to lacZ, Ermr Kanr 11
        pTn7/nilC-lacZpEVS107 plus nilC 5′ flanking region fused to lacZ, Ermr Kanr 10
        pTOPOcpxRAcomppCR2.1-TOPO plus cpxRA, including 152 bp of 5′ regionThis study
        pTn7cpxRAcomppEVSCm plus cpxRA and 152 bp of cpxRA 5′ regionThis study
        pKV124Source of promoterless lacZ for mini-Tn7 fusions, Cmr 76
        pTOPOcpxRppCR2.1-TOPO plus 152 bp of cpxR 5′ flanking regionThis study
        pKVTOPOcpxRppKV124 fused to pTOPOcpxRp, placing ′lacZ downstream of cpxRpThis study
        pTn7/cpxRp-lacZpEVSCmKan plus cpxRp-lacZ fragmentThis study
  • TABLE 2.

    Primers used in this study

    PrimerSequence (5′ to 3′)aUse
    EH079NNNGTCGACGAGTGATCTACTTGGATGΔcpxR1 mutant construction
    EH078NNNTCTAGAGCATTAATTCTTCCTCCAAGΔcpxR1 mutant construction; creation of cpxRp-lacZ fusion
    EH080NNNGAGCTCGTAAAATGATCAATAGCCΔcpxR1 mutant construction
    EH081NNNCCCGGGCGTTTCAACAATCGCTAATCΔcpxR1 mutant construction
    EH146TAAATTTCTCCGCTTTCTCGAATAGCΔcpxR1 mutant complementation; creation of cpxRp-lacZ fusion
    EH121GTCTAGAAATTTGGCTTATCCTCTTAGΔcpxR1 mutant complementation
    EH145ACGCTTAACACCGTTTGATCGRT-PCR for cpxRA cotranscription
    EH013CATTAAATATCCACGACCACGRT-PCR for cpxRA cotranscription
    EH014TTCTGGACAGTGAGTATCAGCRT-PCR for cpxRA cotranscription
    EH015CACTGGTAACAAGGAGTAAGCRT-PCR for cpxRA cotranscription
    recAminForTGTCCGTTTGGATATCCGCCqPCR (recA)
    recAminRevCCCAGAGTATTAATACCTTCCCCAqPCR (recA)
    EH102CGCAGATGACTATCTCCCTAAACCqPCR (cpxR)
    EH025CCGTCAAAACTGGCCTCCqPCR (cpxR)
    EH014TTCTGGACAGTGAGTATCAGCqPCR (cpxA)
    EH015CACTGGTAACAAGGAGTAAGCqPCR (cpxA)
    EH147GCCAGCAGATGTGGGATTTAGqPCR (cpxP)
    EH103CTCAAGTTGCAGCCGAACCqPCR (cpxP)
    lrhAintCFCGCTGGAAACGCTGGATATqPCR (lrhA)
    lrhAintCRGGCAGACGTGGTAATCCTTqPCR (lrhA)
    xlpAintFCGCTGCATTGGCAACAGGAAAqPCR (xlpA)
    xlpAintRGCCAATCGTGCTGAACGGTATqPCR (xlpA)
    EH035GCGGAGCACTATAGTTAAATGTGqPCR (flgE)
    EH036CAACGGCGAAATTCAGATAGGCqPCR (flgE)
    flhDintFGCGATGTTCCGTTTAGGTAqPCR (flhD)
    flhDintRGCGATTCCTTCGTTAACTGqPCR (flhD)
    fliAintFGGCACGAAATTCACGGTGAAqPCR (fliA)
    fliAintRCCGTTCAGGCAATGACTCAAqPCR (fliA)
    fliCintFGCGAACGTTAAAGGCCTGACTqPCR (fliC)
    fliCintRGCCGTTGAACTGAGTTTGTGTqPCR (fliC)
    prot 1TCTTGGTCTGATGTTGCCqPCR (prtA)
    prot 2ACCCTGAGGCAGATTTGCqPCR (prtA)
    xhlBquant1GCTAAAGGCTATATTACGGCGqPCR (xhlB)
    xhlBquant2GCTGATTCAGGTTGAGTGGCTqPCR (xhlB)
    EH124CTGATAAGCAGCTGGCTGqPCR (xaxA)
    EH125CCATCTGATAACTCACCGCCqPCR (xaxA)
    EH068CTATGCCAGGCTTATTAGCGCqPCR (mrxA)
    EH069CGATGGCGGTAAATTCACCCqPCR (mrxA)
    • ↵ a Engineered restiction enzyme sites are underlined. N = G, A, T, or C.

  • TABLE 3.

    Relative expression of potential CpxR-regulated genes in the ΔcpxR1 mutant

    ClassGeneTranscript level in ΔcpxR (fold increase compared with the wild type)a
    OD600, ∼0.5OD600, ∼0.8OD600, ∼5.0Plate
    Hemolysin related xaxA 6.96 (5)b0.38 (3)1.17 (4)ND
    xhlB 1.71 (9)1.72 (2)0.41 (7)ND
    Protease related prtA 3.05 (5)dND0.58 (7)ND
    Lipase related lrhA 0.60 (9)0.29 (7)c0.77 (5)ND
    xlpA 1.06 (8)0.75 (5)1.05 (4)ND
    Flagellum related fliA 1.06 (3)1.51 (3)NDND
    flhD 1.57 (8)0.84 (6)0.77 (3)ND
    flgE 1.05 (9)1.21 (6)0.84 (6)ND
    fliC 1.06 (9)1.46 (6)0.61 (3)ND
    Pilus related mrxA NDNDND3.36 (5)b
    • ↵ a The numbers in parentheses are the numbers of replicates. ND, not determined.

    • ↵ b The value is significantly different from the wild-type value, which was defined as 1.0 (P < 0.01).

    • ↵ c The value is significantly different from the wild-type value, which was defined as 1.0 (P < 0.05).

    • ↵ d The value is significantly different from the wild-type value, which was defined as 1.0 (P < 0.001).

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CpxRA Regulates Mutualism and Pathogenesis in Xenorhabdus nematophila
Erin E. Herbert, Kimberly N. Cowles, Heidi Goodrich-Blair
Applied and Environmental Microbiology Dec 2007, 73 (24) 7826-7836; DOI: 10.1128/AEM.01586-07

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CpxRA Regulates Mutualism and Pathogenesis in Xenorhabdus nematophila
Erin E. Herbert, Kimberly N. Cowles, Heidi Goodrich-Blair
Applied and Environmental Microbiology Dec 2007, 73 (24) 7826-7836; DOI: 10.1128/AEM.01586-07
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KEYWORDS

Bacterial Proteins
Gene Expression Regulation, Bacterial
Protein Kinases
Xenorhabdus

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