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GENE REGULATION

Enterococcus faecalis rnjB Is Required for Pilin Gene Expression and Biofilm Formation

Peng Gao, Kenneth L. Pinkston, Sreedhar R. Nallapareddy, Ambro van Hoof, Barbara E. Murray, Barrett R. Harvey
Peng Gao
1Center for Immunology and Autoimmune Diseases, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, Texas 77030
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Kenneth L. Pinkston
1Center for Immunology and Autoimmune Diseases, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, Texas 77030
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Sreedhar R. Nallapareddy
2Division of Infectious Diseases, Department of Internal Medicine
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Ambro van Hoof
3Department of Microbiology and Molecular Genetics
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Barbara E. Murray
2Division of Infectious Diseases, Department of Internal Medicine
3Department of Microbiology and Molecular Genetics
4Center for the Study of Emerging and Reemerging Pathogens, University of Texas Medical School at Houston, Houston, Texas 77030
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Barrett R. Harvey
1Center for Immunology and Autoimmune Diseases, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, Texas 77030
2Division of Infectious Diseases, Department of Internal Medicine
4Center for the Study of Emerging and Reemerging Pathogens, University of Texas Medical School at Houston, Houston, Texas 77030
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  • For correspondence: Barrett.Harvey@uth.tmc.edu
DOI: 10.1128/JB.00725-10
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  • FIG. 1.
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    FIG. 1.

    Cell surface binding of anti-EbpC MAb 69 to E. faecalis. Wild-type OG1RF (wt) and ebp deletion mutant (ΔebpABC) cells grown in BHI medium to early exponential phase, exponential phase, early stationary phase, and stationary phase were labeled with anti-EbpC MAb 69 and a secondary antibody-phycoerythrin conjugate, and 10,000 cells were analyzed by flow cytometry. Wild-type OG1RF cells grown to stationary phase and labeled with secondary antibody only (wild-type control) served as a negative control.

  • FIG. 2.
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    FIG. 2.

    Identification of genes involved in the surface expression or regulation of EbpC. Each E. faecalis gene has a systematic name beginning with “ef,” and the 540 clones in the Tn library are represented by the number in their name on the x axis. Relative whole-cell ELISA signals are represented on the y axis. Each clone was labeled with our anti-EbpC antibody followed by a goat anti-mouse IgG-HRP conjugate and developed with the TMB substrate. Tn insertions in ef1090 (ebpR), ef1091 (ebpA), ef1184, and ef1579 (as indicated by arrows) were identified in two independent evaluations as clones which produced the lowest ELISA signal.

  • FIG. 3.
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    FIG. 3.

    Flow cytometry analysis of surface-exposed EbpC in E. faecalis wild-type and mutant strains. Each population was labeled with anti-EbpC MAb 69. Mean fluorescence intensities are compared in the OG1RF (A), ΔebpABC (ebpABC deletion) (B), ebpR (ef1090 insertion mutant) (C), ebpA (ef1091 insertion mutant) (D), ef1184 (dapA insertion mutant) (E), ΔdapA (dapA deletion mutant) (F), and ΔrnjB (rnjB deletion mutant) (G) strains. The MFI value of each sample is indicated. ▾, Tn insertion mutant.

  • FIG. 4.
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    FIG. 4.

    Western blotting results of Ebp pilus formation of the wild-type OG1RF and mutant strains. Mutanolysin surface extractions were probed with anti-EbpC MAb 69. Lanes (left to right): EZ-run protein marker (M), OG1RF, ebpR mutant, ebpA mutant, ef1184 mutant, ΔebpABC mutant, ΔrnjB mutant, and ΔdapA mutant. High-molecular-weight Ebp pilus polymers and EbpC monomers are indicated. ▾, Tn insertion mutant.

  • FIG. 5.
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    FIG. 5.

    E. faecalis rnjB encodes an RNase J2 ortholog. (A) Comparison of the E. faecalis OG1RF ef1183-ef1185 operon with the B. subtilis asd-rnjB operon. The red arrow indicates the Tn insertion site in the ef1184 mutant (dapA insertion mutant). (B) Phylogram of six RNase J1s and six RNase J2s from the bacterial order Bacilli and several other bacterial RNase Js based on multiple-sequence alignment generated by ClustalW2 with default parameters.

  • FIG. 6.
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    FIG. 6.

    Complementation of the rnjB deletion in OG1RF. Flow cytometry analysis of OG1RF (wild type) (B), the ΔrnjB mutant (C), the ΔrnjB mutant with the vector control (D), and the ΔrnjB mutant with the rnjB complement vector (E). Cells grown to the stationary phase in BHI medium with 25 ng/ml nisin were labeled with anti-EbpC MAb 69 followed by a secondary antibody-phycoerythrin conjugate. Mean fluorescence intensities are compared to that of the control, OG1RF with secondary antibody only (A).

  • FIG. 7.
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    FIG. 7.

    Comparison of transcript levels of ebp-related genes in wild-type OG1RF and the ΔrnjB mutant. (A) Northern blot of RNA isolated from mid-log-phase wild-type OG1RF and ΔrnjB cells. Blots were hybridized with 32P-labeled 5′-UTR-ebpA (top panel), ebpC (middle panel), and 23S rRNA (loading control) (bottom panel) probes. (B) qRT-PCR analysis of RNA isolated from mid-log-phase wild-type OG1RF and ΔrnjB cells. Each column represents the gene of interest shown in the x axis. The fold decrease in gene expression in the ΔrnjB mutant corresponds to the ratio of the transcript level of each gene in the wild-type strain to that of each gene in the ΔrnjB mutant. The level of each gene transcript is tested in triplicate and normalized using 23S rRNA transcript levels.

  • FIG. 8.
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    FIG. 8.

    rnjB affects the expression of an ebpA::lacZ reporter but not an ebpR::lacZ reporter. Wild-type OG1RF and the ΔrnjB mutant containing either PebpR::lacZ or PebpA::lacZ were grown in BHI and collected at log phase and stationary phase for β-galactosidase (B-gal) assay. The y axis represents the β-galactosidase units (OD420/total protein concentration in mg/ml). Error bars represent the standard errors of the measurements from three individual cultures.

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    FIG. 9.

    Biofilm formation of OG1RF gene deletion mutants. Cells grown for 24 h on polystyrene microtiter plates were evaluated for biofilm formation, expressed as a mean crystal violet absorbance of a biofilm biomass in four independent microtiter wells. Error bars represent the standard errors of the means.

Tables

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  • TABLE 1.

    Strains and plasmids used in this study

    Strain (GenBank accession no.) or plasmidRelevant characteristic(s)Source or reference
    Strains
    E. faecalis
        OG1RFWild-type strain, Fusr Rifr, p-Cl-Pher 34
        OG1RF Tn libraryOG1RF Tn insertion mutants 17
        TX5608 (ΔebpABC mutant)OG1RF ebpABC deletion mutant 35
        ΔrnjB mutantOG1RF rnjB in-frame deletionThis study
    E. coli
        XL1-BlueCloning hostStratagene
        EC1000Cloning host, provides RepA in trans 25
    Plasmids
        pQE30:rEbpCRecombinant EbpC expression vector 35
        pMSP3535Nisin-inducible expression vector, Emr 8
        pCJK47Delivery plasmid with pheS* counterselectable marker 8
        pMSP:rnjB rnjB cloned into pMSP3535This study
        pTEX5585PebpA::lacZ fusion in pTCV-lacZ 6
        pTEX5586PebpR::lacZ fusion in pTCV-lacZ 5
        pCJK47:dapA Intermediate construct carrying dapA-flanking sequenceThis study
        pCJK47:rnjB Intermediate construct carrying rnjB-flanking sequenceThis study
  • TABLE 2.

    Primers used in this study

    Construction or test and primer or probeSequence
    dapA deletion construction
        1184del-1F5′-GCGCGGATCCTGAGGTTATTGGCGTCTAAG-3′
        1184del-1R5′-GGCCCTGCAGCCTATTCCCTCCCGAGTATA-3′
        1184del-2F5′-AAAACTGCAGAAGAACGTGTAATCATAGAGAG-3′
        1184del-2R5′-GCCGGAATTCTAAATCGCCTTCTTCAATTC-3′
        1184delUpF5′-TAAGTCAAAAGTCAATGGAT-3′
        1184delDnR5′-AATCATAATATTTTCGGCCG-3′
    rnjB deletion construction
        1185del-1F5′-GCGCGGATCCTGGTTACGCCGTTTCAAGAATC-3′
        1185del-1R5′-GGCCCTGCAGATTTTTTCCATTTTCACGAACGCC-3′
        1185del-2F5′-AAAACTGCAGTTCGTCCTGATCAAAGCAGG-3′
        1185del-2R5′-GCCGGAATTCTCAGTTGCTTGCTCTTCTAAAC-3′
        1185delUpF5′-ATATAGAAGTGAAACAAGCAGATGC-3′
        1185delDnR5′-TTCACGAATCAAACGGCTCA-3′
    ebpA qRT-PCR
        Forward primerCAACAACACCAGGGCTTTTTG
        Reverse primerACCGGACCAGTCAACGACTAAG
    ebpB qRT-PCR
        Forward primerCGTACAGGCGGCAAGTCTTT
        Reverse primerAGGTATTCCCCCGCTTGATTT
    ebpC qRT-PCR
        Forward primerGCGGCACACTAAAATTCGTTTA
        Reverse primerGTCGTCGGTATGACCGTTATCA
    23S rRNA qRT-PCR
        Forward primerGTGATGGCGTGCCTTTTGTA
        Reverse primerCGCCCTATTCAGACTCGCTTT
    dapA cloning
        Forward primerGCGCGGATCCGGAGGGAATAGGATGGA
        Reverse primerCCGGCTCGAGTTAAATCTCTAACGTGC
    rnjB cloning
        Forward primerGCGCGGATCCGGTGAAAAGAGTGAG
        Reverse primerCCGGCTCGAGCTATGCGTTATTTTTGG
    Northern blot probes
        ebpA 5-UTR probeCAGTTAAATTAGAATTGCCTAGCACG
        ebpC probeGTCGTCGGTATGACCGTTATCA
        23S rRNA probeCGCCCTATTCAGACTCGCTTT
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Enterococcus faecalis rnjB Is Required for Pilin Gene Expression and Biofilm Formation
Peng Gao, Kenneth L. Pinkston, Sreedhar R. Nallapareddy, Ambro van Hoof, Barbara E. Murray, Barrett R. Harvey
Journal of Bacteriology Sep 2010, 192 (20) 5489-5498; DOI: 10.1128/JB.00725-10

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Enterococcus faecalis rnjB Is Required for Pilin Gene Expression and Biofilm Formation
Peng Gao, Kenneth L. Pinkston, Sreedhar R. Nallapareddy, Ambro van Hoof, Barbara E. Murray, Barrett R. Harvey
Journal of Bacteriology Sep 2010, 192 (20) 5489-5498; DOI: 10.1128/JB.00725-10
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KEYWORDS

biofilms
Enterococcus faecalis
Fimbriae Proteins

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