Article Figures & Data
Figures
- 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.
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.
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.
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.
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.
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.
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.
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.
- 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
- TABLE 1.
Strains and plasmids used in this study
Strain (GenBank accession no.) or plasmid Relevant characteristic(s) Source or reference Strains E. faecalis OG1RF Wild-type strain, Fusr Rifr, p-Cl-Pher 34 OG1RF Tn library OG1RF Tn insertion mutants 17 TX5608 (ΔebpABC mutant) OG1RF ebpABC deletion mutant 35 ΔrnjB mutant OG1RF rnjB in-frame deletion This study E. coli XL1-Blue Cloning host Stratagene EC1000 Cloning host, provides RepA in trans 25 Plasmids pQE30:rEbpC Recombinant EbpC expression vector 35 pMSP3535 Nisin-inducible expression vector, Emr 8 pCJK47 Delivery plasmid with pheS* counterselectable marker 8 pMSP:rnjB rnjB cloned into pMSP3535 This study pTEX5585 PebpA::lacZ fusion in pTCV-lacZ 6 pTEX5586 PebpR::lacZ fusion in pTCV-lacZ 5 pCJK47:dapA Intermediate construct carrying dapA-flanking sequence This study pCJK47:rnjB Intermediate construct carrying rnjB-flanking sequence This study - TABLE 2.
Primers used in this study
Construction or test and primer or probe Sequence dapA deletion construction 1184del-1F 5′-GCGCGGATCCTGAGGTTATTGGCGTCTAAG-3′ 1184del-1R 5′-GGCCCTGCAGCCTATTCCCTCCCGAGTATA-3′ 1184del-2F 5′-AAAACTGCAGAAGAACGTGTAATCATAGAGAG-3′ 1184del-2R 5′-GCCGGAATTCTAAATCGCCTTCTTCAATTC-3′ 1184delUpF 5′-TAAGTCAAAAGTCAATGGAT-3′ 1184delDnR 5′-AATCATAATATTTTCGGCCG-3′ rnjB deletion construction 1185del-1F 5′-GCGCGGATCCTGGTTACGCCGTTTCAAGAATC-3′ 1185del-1R 5′-GGCCCTGCAGATTTTTTCCATTTTCACGAACGCC-3′ 1185del-2F 5′-AAAACTGCAGTTCGTCCTGATCAAAGCAGG-3′ 1185del-2R 5′-GCCGGAATTCTCAGTTGCTTGCTCTTCTAAAC-3′ 1185delUpF 5′-ATATAGAAGTGAAACAAGCAGATGC-3′ 1185delDnR 5′-TTCACGAATCAAACGGCTCA-3′ ebpA qRT-PCR Forward primer CAACAACACCAGGGCTTTTTG Reverse primer ACCGGACCAGTCAACGACTAAG ebpB qRT-PCR Forward primer CGTACAGGCGGCAAGTCTTT Reverse primer AGGTATTCCCCCGCTTGATTT ebpC qRT-PCR Forward primer GCGGCACACTAAAATTCGTTTA Reverse primer GTCGTCGGTATGACCGTTATCA 23S rRNA qRT-PCR Forward primer GTGATGGCGTGCCTTTTGTA Reverse primer CGCCCTATTCAGACTCGCTTT dapA cloning Forward primer GCGCGGATCCGGAGGGAATAGGATGGA Reverse primer CCGGCTCGAGTTAAATCTCTAACGTGC rnjB cloning Forward primer GCGCGGATCCGGTGAAAAGAGTGAG Reverse primer CCGGCTCGAGCTATGCGTTATTTTTGG Northern blot probes ebpA 5-UTR probe CAGTTAAATTAGAATTGCCTAGCACG ebpC probe GTCGTCGGTATGACCGTTATCA 23S rRNA probe CGCCCTATTCAGACTCGCTTT
