Analysis of a gene cluster of Enterococcus faecalis involved in polysaccharide biosynthesis - PubMed (original) (raw)

FIG. 1

(A) Southern blot analysis of OG1RF, TX5179, and TX5180. Genomic DNA of OG1RF, TX5179, and TX5180 was digested with _Eco_RI and _Xba_I, separated by electrophoresis in a 0.6% agarose gel and transferred onto a Hybond-N+ nylon membrane. The insert fragments of pTEX5177 and pTEX5178 were labeled with [32P]dCTP and used as probes. Lanes 1-4 were probed with the insert of pTEX5177, and lanes 5-8 were probed with that of pTEX5178. Hybridization was performed under high-stringency conditions at 65°C. Lane 1, OG1RF plus _Eco_RI; lane 2, TX5179 plus _Eco_RI; lane 3, OG1RF plus _Xba_I; lane 4, TX5179 plus _Xba_I; lane 5, OG1RF plus _Eco_RI; lane 6, TX5180 plus _Eco_RI; lane 7, OG1RF plus _Xba_I; lane 8, TX5180 plus _Xba_I. (B) Illustration of the local organizations of orfde4, orfde6, and the flanking regions in OG1RF, TX5179, and TX5180. RI, _Eco_RI; Xb, _Xba_I. The numbers in the parentheses indicate the nucleotide positions within the epa region in OG1RF. In TX5179, orfde4 was interrupted by pTEX5177, resulting in two partial copies, while in TX5180, orfde6 was interrupted by pTEX5178, resulting in two partial copies of orfde6. T3 and T7 are promoter regions on pTEX4577, and GW378 and GW379 are primers to the ends of the KM resistance gene. The arrows were not drawn to scale. The striped boxes underneath the disrupted ORFs indicate where the probes hybridized.

FIG. 1

FIG. 2

Growth curves of OG1RF, TX5179, and TX5180. Klett units were measured every hour until stationary phase. Circles, OG1RF; triangles, TX5180; boxes, TX5179.

FIG. 3

(A). Comparison of the levels of mRNA transcripts of TX5180 and OG1RF. The target ORF is labeled next to each panel. The primers used for each ORF are as follows: panel I, GW337 and GW338; panel II, GW303 and GW304; panel III, GW365 and GW366; panel IV, GW316 and GW317; panel V, GW318 and GW319; panel VI, GW320 and GW321; panel VII, GW323 and GW324; panel VIII, GW367 and GW368; and panel IX, GW331 and GW332. The arrows next to the orfde5 and orfde10 panels indicate the specific bands (of expected sizes). The final concentration of each primer in the reaction was 400 nM for the reverse primers and 200 nM for the forward primers. A total of 25 ng of total RNA or 5 ng of chromosomal DNA was used as the template in each reaction. Lanes 1 to 3, OG1RF RNA; lanes 4 to 6, TX5180 RNA; and lanes 7, OG1RF chromosomal DNA. In addition, AmpliTaq DNA Polymerase (Taq) and/or MuLV RT, or neither, was used in each different reaction, as follows: lanes 1 and 4, RT and Taq; lanes 2, 5, and 7, Taq only; and lanes 3 and 6, neither. Reverse transcription was performed at 42°C for 40 min and then the mixture was heated at 95°C for 5 min. The PCR was carried out in a Perkin-Elmer 9600 thermal cycler using the following conditions: 94°C, 30 s; 55°C, 30 s; and 72°C, 30 s for 30 cycles. (B) RT-PCR of serial dilutions of OG1RF total RNA. Total RNA (25 ng/μl) was diluted 10-, 100-, 1,000-, and 10,000-fold with DEPC-treated H2O, and 1 μl of each dilution was used as the template for RT-PCR. GW301 and GW302 were used as primers. Lanes 1 to 3, undiluted RNA; lanes 4 to 6, 10-fold dilution; lanes 7 to 9, 100-fold dilution; lanes 10 to 12, 1,000-fold dilution; and lanes 13 to 15, 10,000-fold dilution. AmpliTaq DNA Polymerase (Taq) and/or MuLV RT, or neither, was used in each different reaction, as follows: lanes 1, 4, 7, 10, and 13, RT and Taq; lanes 2, 5, 8, 11, and 14, Taq only; and lanes 3, 6, 9, 12, and 15, neither. Reverse transcription and PCR were carried out using the same conditions as those described for panel A.

FIG. 3

FIG. 4

Percentages of surviving mice injected with OG1RF, TX5179, or TX5180 over time. Circles, OG1RF; squares, TX5179; triangles, TX5180. The inoculum sizes and the number of mice used for each strain were as follows: OG1RF, 3.5 × 108 CFU (6 mice); TX5179, 4.0 × 108 CFU (16 mice): and TX5180, 5.0 × 108 CFU (16 mice). See Table 4 for statistical significance.

FIG. 5

Immunoblot of two mucoid E. faecalis isolates with the eluted antibody. Strains were grown on BHI agar for 24 h at either room temperature or 37°C and then lifted onto nitrocellulose filters. Immunoblotting was carried out with the eluted antibody as described in Materials and Methods.

FIG. 6

Hypothetical transcriptional organization of the epa region. Line 1, open reading frames in epa (open boxes) and their direction of transcription (arrows below the boxes). Line 2, putative promoters identified by homology to the E. coli −35 and −10 sequences recognized by ς70 (27); vertical lines indicate the position of each putative promoter, and arrows indicate their directions. Line 3, summary of possible promoters in epa that were supported by experimental data. P5_6 and P6 cannot be distinguished based on current data.