Journal:

Article Title: Involvement of PhoP-PhoS Homologs in Enterococcus faecalis Virulence

doi: 10.1128/IAI.70.4.1991-1996.2002

Figure Lengend Snippet: Percentages of surviving mice injected with OG1RF, TX10293, or TX37200 over time. (A) The inoculum size and the number of mice used for each strain were as follows: OG1RF, 5.0 × 107 CFU (six mice); TX10293, 5.0 × 107 CFU (six mice); and TX37200, 8.0 × 107 CFU (six mice). (B) The inoculum size and the number of mice used for each strain were as follows: OG1RF, 2.7 × 107 (12 mice); TX10293, 3.0 × 107 CFU (12 mice); TX37200, 3.2 × 107 CFU (12 mice). The LD50 values were calculated based on this experiment (see the text).

Article Snippet: An internal fragment of the first gene of each of the potential two-component gene pairs was amplified from OG1RF genomic DNA (oligonucleotides used are listed in Table ) and cloned into the TOPO TA cloning vector (Invitrogen, Carlsbad, Calif.).

Techniques: Injection

Journal:

Article Title: Involvement of PhoP-PhoS Homologs in Enterococcus faecalis Virulence

doi: 10.1128/IAI.70.4.1991-1996.2002

Figure Lengend Snippet: (A) Survival of OG1RF, TX10293, and TX37200 in BHI, pH 3.4, adjusted with lactic acid. (B) Survival of OG1RF, TX10293, and TX37200 in BHI at 55°C. The percent surviving 40 min from time zero is shown (see Materials and Methods for details).

Article Snippet: An internal fragment of the first gene of each of the potential two-component gene pairs was amplified from OG1RF genomic DNA (oligonucleotides used are listed in Table ) and cloned into the TOPO TA cloning vector (Invitrogen, Carlsbad, Calif.).

Techniques:

Journal: Infection and Immunity

Article Title: AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

doi: 10.1128/IAI.01210-12

Figure Lengend Snippet: Enterococcus faecalis strains and oligonucleotides used in this study

Article Snippet: EF0999 is annotated as a conserved hypothetical protein in strain V583 and a hypothetical protein in the E. faecalis OG1RF genome sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP002621.1","term_id":"327533853","term_text":"CP002621.1"}} CP002621.1 ) housed at the National Center for Biotechnology Information (NCBI), but contains a conserved region of homology in its C-terminal half that is found in the sepF loci of other Gram-positive microbes.

Techniques: Sequencing, Mutagenesis, Disruption, Plasmid Preparation, Expressing

Journal: Infection and Immunity

Article Title: AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

doi: 10.1128/IAI.01210-12

Figure Lengend Snippet: Characterization of the role of biofilm determinants in biofilm biomass accumulation at 6 and 24 h. Biofilm biomass accumulation after 6 (black bars) and 24 (white bars) h in a microtiter plate biofilm assay was measured for E. faecalis wild-type strain OG1RF and seven strains with either mariner transposon insertions (designated by Ω in the strain name) or in-frame markerless deletions (designated by Δ in the strain name) in previously identified biofilm determinant genes (20, 25). The ΩahrC+ahrC strain carries plasmid pCJK124 (20), a pMSP3535-based vector with the ahrC open reading frame cloned downstream of a nisin-inducible promoter. The ΩahrC+p23ahrCKI strain constitutively expresses the ahrC open reading frame from the p23 promoter at an ectopic chromosomal location, as described in the text. The mean absorbance for n = 2 to 6 biological replicates per strain is graphed as percent biofilm biomass relative to OG1RF, which was set to 100%. Error bars represent standard deviations (SD). The mean absorbances ± SD for OG1RF were 0.092 ± 0.012 for 6 h and 0.126 ± 0.010 for 24 h (n = 6 for both time points). The dotted line at 75% represents the level used to define reduced biofilm accumulation in our previous genetic screen for biofilm-defective transposon insertion mutants (20).

Article Snippet: EF0999 is annotated as a conserved hypothetical protein in strain V583 and a hypothetical protein in the E. faecalis OG1RF genome sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP002621.1","term_id":"327533853","term_text":"CP002621.1"}} CP002621.1 ) housed at the National Center for Biotechnology Information (NCBI), but contains a conserved region of homology in its C-terminal half that is found in the sepF loci of other Gram-positive microbes.

Techniques: Biofilm Production Assay, Plasmid Preparation, Clone Assay

Journal: Infection and Immunity

Article Title: AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

doi: 10.1128/IAI.01210-12

Figure Lengend Snippet: Role of biofilm determinants in experimental endocarditis virulence. Aortic valve leaflets and vegetations from rabbits in which E. faecalis endocarditis had been induced were harvested, homogenized, and plated to enumerate bacteria at 4 days postinoculation. Each symbol represents the log10 valve bacterial load for an individual animal. Thick horizontal bars show the arithmetic mean of the log10-transformed values. For comparison, the arithmetic mean log10 valve bacterial loads for the ΔebrA, ΔproB, and Δeep strains tested in the same experimental endocarditis model in a previous study (13) are shown at the far right (thin horizontal bars). OG1RF, n = 8; ΔopuBC, n = 6; ΩargR, n = 5; ΩatlA, n = 3; ΩahrC, n = 6; ΩahrC+ahrC, n = 9; ΩahrC-p23ahrCKI, n = 5; ΔrecN, n = 6; ΩsepF, n = 6; ΩpyrC, n = 5. ##, P < 0.05 by one-way ANOVA followed by Bonferroni's multiple comparison test.

Article Snippet: EF0999 is annotated as a conserved hypothetical protein in strain V583 and a hypothetical protein in the E. faecalis OG1RF genome sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP002621.1","term_id":"327533853","term_text":"CP002621.1"}} CP002621.1 ) housed at the National Center for Biotechnology Information (NCBI), but contains a conserved region of homology in its C-terminal half that is found in the sepF loci of other Gram-positive microbes.

Techniques: Bacteria, Transformation Assay, Comparison

Journal: Infection and Immunity

Article Title: AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

doi: 10.1128/IAI.01210-12

Figure Lengend Snippet: Enterococcal biofilm determinants important for endocarditis contribute to virulence in an experimental model of CAUTI. The graphs indicate the log10 CFU recovered from retrieved catheter implants (A), homogenized bladders (B), and homogenized kidneys (C) of mice implanted with silicone catheter pieces and subsequently infected with E. faecalis OG1RF (n = 12), the Δeep strain (EF2380; n = 15), or the ΩahrC strain (EF0983; n = 14) for 24 h. Each symbol represents an individual animal, and only animals from which catheter implants were retrieved were included in the analysis. Horizontal bars represent the median, and the dashed lines indicate the limits of detection. P values were determined with the Mann-Whitney U test: n.s., not significant (P > 0.05); *, P = 0.01; **, P = 0.0003; and ***, P < 0.0001.

Article Snippet: EF0999 is annotated as a conserved hypothetical protein in strain V583 and a hypothetical protein in the E. faecalis OG1RF genome sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP002621.1","term_id":"327533853","term_text":"CP002621.1"}} CP002621.1 ) housed at the National Center for Biotechnology Information (NCBI), but contains a conserved region of homology in its C-terminal half that is found in the sepF loci of other Gram-positive microbes.

Techniques: Infection, MANN-WHITNEY

Journal: Infection and Immunity

Article Title: AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

doi: 10.1128/IAI.01210-12

Figure Lengend Snippet: ΩahrC biofilms display a profound biomass defect in early biofilms. OG1RF and ΩahrC biofilms were grown on Aclar fluoropolymer coupons for 6 h. Biofilms were stained with an Alexa Fluor 594-wheat germ agglutinin conjugate and imaged as described in Materials and Methods. Simple maximum intensity projections of mean image stacks are shown for the ΩahrC strain (A) and a matched OG1RF sample (B). Biomass stack surface projections of the ΩahrC strain (C) and OG1RF (D) provide a modicum of depth information to the two-dimensional images. Scale bars, 20 μm.

Article Snippet: EF0999 is annotated as a conserved hypothetical protein in strain V583 and a hypothetical protein in the E. faecalis OG1RF genome sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP002621.1","term_id":"327533853","term_text":"CP002621.1"}} CP002621.1 ) housed at the National Center for Biotechnology Information (NCBI), but contains a conserved region of homology in its C-terminal half that is found in the sepF loci of other Gram-positive microbes.

Techniques: Staining

Journal: Infection and Immunity

Article Title: AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

doi: 10.1128/IAI.01210-12

Figure Lengend Snippet: E. faecalis OG1RF ahrC expression at the RNA and protein levels. (A) Gene expression in batch culture. Aliquots of OG1RF cells growing in BHI were collected at the indicated time points and processed for quantitation by serial dilution and plating or for RNA extraction. Cell counts (circles connected by dashed line) correspond to the left y axis. cDNA copy numbers per μl for ahrC (squares) and gelE (triangles), included as a control for a gene known to be expressed in the stationary phase after cells reach an fsr-dependent quorum, correspond with values on the right y axis. Symbols are the mean of n = 3 biological replicates. Error bars represent standard deviations (SD). (B) Gene expression in biofilms. OG1RF planktonic and biofilm cells grown in plastic dishes in TSB-dex were harvested for quantitation and RNA extraction after growth for 6 or 22 h. Values are the means ± SD of n = 5 to 6 biological replicates collected on 2 separate days. Relative expression of ahrC and gelE was calculated by dividing the copies of detected transcript per μl of cDNA by the corresponding CFU/ml count from the harvested biofilm cells. b.d., below detectable limit of the qPCR assay. (C) Protein expression in early exponential and stationary phases. Whole-cell lysates of exponential (E)- or stationary (S)-phase cells were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-AhrC polyclonal antisera. His-tagged AhrC is approximately 18 kDa. The upper band is a cross-reactive protein of unknown identity that was used as a loading control in densitometry measurements, which revealed that the relative amount of AhrC detected in stationary-phase OG1RF lysates was 25% less than the amount detected in exponential-phase lysates. Due to the strong overexpression of AhrC in the ΩahrC-p23ahrCKI strain (labeled “ΩahrC-KI” on the figure for clarity), a shorter exposure of the same blot is shown in the bottom image. The top image was adjusted for brightness and contrast after densitometry in order to make the loading control bands visible for publication.

Article Snippet: EF0999 is annotated as a conserved hypothetical protein in strain V583 and a hypothetical protein in the E. faecalis OG1RF genome sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP002621.1","term_id":"327533853","term_text":"CP002621.1"}} CP002621.1 ) housed at the National Center for Biotechnology Information (NCBI), but contains a conserved region of homology in its C-terminal half that is found in the sepF loci of other Gram-positive microbes.

Techniques: Expressing, Gene Expression, Quantitation Assay, Serial Dilution, RNA Extraction, Control, SDS Page, Over Expression, Labeling