TEXT

Top Abstract Text References

Transcription and translation have proven to be fruitful targets for antibiotic discovery. A number of marketed antibiotics inhibit bacterial growth through inhibition of prokaryotic RNA transcription and protein translation. Rifampin is a potent inhibitor of bacterial RNA polymerase, and the macrolides, lincosamides, aminoglycosides, tetracyclines and oxazolidinones all have protein translation as their site of action. Unfortunately, the increase in the antibiotic resistance of gram-positive bacteria threatens to reduce the effectiveness of these and other antibiotics. These concerns act as an incentive to discover new and more effective transcription and protein translation inhibitors. The development of a nonradioactive Staphylococcus aureus cell extract transcription-translation (T/T) assay will provide a means to rapidly evaluate new inhibitors of prokaryotic transcription and translation in a gram-positive-bacterium-specific system.

S. aureus RN4220 (ATCC 35556), a restriction-deficient strain, was used in the development of the T/T assay (3). The procedure for making S. aureus S30 extracts that was described by R. Mahmood et al. (1) was followed, with some modifications. Six liters of brain heart infusion medium was inoculated with 250 ml of an S. aureus overnight culture and grown at 37°C for 4 to 5 h to an optical density at 600 nm of 2 to 4. Cells were pelleted and washed successively with 500 ml of cold S30-buffer A (10 mM Tris-acetate, pH 8.0, 14 mM Mg-acetate, 1 mM dithiothreitol [DTT]) containing 1 M KCl and then 250 ml of buffer A containing 50 mM KCl. Cell pellets (~50 g [wet weight]) were stored at 70°C. Frozen cell pellets were thawed on ice for 30 to 60 min. The slurry was resuspended up to a final volume of 99 ml of buffer B (10 mM Tris-acetate, pH 8.0, 20 mM Mg-acetate, 50 mM KCl, 1 mM DTT). A 1.5-ml volume of lysostaphin (0.8 mg/ml) in buffer B was added to the bottom of three precooled, 35-ml polyallomer SS-34 centrifuge tubes. Thirty-three milliliters of cell slurry was transferred to each of the three tubes containing lysostaphin solution (final concentration, 35 µg/ml), capped, and gently mixed by inversion. The slurry was incubated at 37°C for 45 to 60 min, and the tubes were inverted periodically. After incubation, 150 µl of 0.5 M DTT was added to each tube and mixed gently. The lysed cells were spun at 4°C in an SS-34 rotor (16,000 rpm; 30,000 × g for 30 min). The supernatant was saved, and the cell pellet was recentrifuged under the same conditions. The supernatants were pooled and recentrifuged to remove cellular debris. The top two-thirds of the supernatant was collected, and 0.25 volume of preincubation buffer was added (670 mM Tris-acetate, pH 8.0, 20 mM Mg-acetate, 7 mM Na₃-phosphoenolpyruvate, 7 mM DTT, 5.5 mM ATP, 70 µM amino acids, complete [Promega], 75 µg of pyruvate kinase [Sigma]/ml). The mixture was incubated at 37°C for 30 min. The preincubated supernatant was dialyzed overnight (Spectra-Por 7; molecular weight cutoff, 3,500) at 4°C against 2 liters of dialysis buffer (10 mM Tris-acetate, pH 8.0, 14 mM Mg-acetate, 60 mM K-acetate, 1 mM DTT) with one buffer change. The dialysate was gently concentrated to ~10 mg/ml by covering a dialysis bag (Spectra-Por 7; molecular weight cutoff, 3,500) containing the extract with precooled polyethylene glycol 8000 powder (Sigma) at 4°C. The extract was aliquoted, flash frozen, and stored in the vapor phase of a liquid nitrogen freezer.

Construction of the S. aureus luciferase reporter plasmid. The pBESTluc plasmid (Promega Corporation) contains the firefly luciferase gene under the control of the Escherichia coli tac promoter. The E. coli tac promoter was exchanged with the cap1A promoter and corresponding Shine-Dalgarno site from the S. aureus type 1 capsule polysaccharide biosynthesis gene cluster to produce the pSAluc plasmid as shown in Fig. 1 (4). The cap1A promoter was selected because it has been shown to be a strong promoter in S. aureus (4). The cap1A-luc gene construct was assembled by synthesizing a long oligonucleotide containing the cap1A promoter, the Shine-Dalgarno site, and a 5' region of the luc gene. PCR primers were designed using the software Oligo 5.0 (Molecular Biology Insights), which amplified a DNA fragment containing the promoter region fused with the first 1,419 bp of the firefly luciferase gene sequence. The amplified fragment was cloned into pBESTluc as a HindIII-ClaI fragment. The pSAluc plasmid can be easily propagated in E. coli but can be used as the template to drive the S. aureus in vitro coupled T/T assay.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   pSAluc cloning strategy. (A) pSAluc upper primer sequence. The S. aureus cap1A promoter, the Shine-Dalgarno (S-D) sequence, and the first 20 bp of the firefly luciferase gene sequence are shown with relevant restriction sites. (B) Plasmid map of pSAluc. The region between the HindIII and ClaI restriction sites indicates the area of the gene amplified which replaced the tac promoter-luc gene construct in the pBESTluc plasmid (Promega) with the S. aureus cap1A promoter-luc gene construct in pSAluc. The sequence of the pSAluc lower primer used in this cloning strategy is as follows: GTCATCGTCGGGAAGACCTG. The S. aureus cap1A promoter and corresponding Shine-Dalgarno sequences are as those published by S. Ouyang and C. Y. Lee (4).

S. aureus in vitro coupled T/T assay. The reagents (amino acid mixture; Premix) in the commercially available kit for E. coli in vitro coupled T/T assay (Promega Corporation) can be used interchangeably with the S. aureus S30 assay. Each new preparation of S. aureus S30 extract or pSAluc DNA is titrated in the luciferase assay to determine optimal concentration (Fig. 2). For trichloroacetic acid (TCA) precipitation, the reaction volume was 50 µl, and for the luciferase assay, the reaction volume was 25 µl. In each case, 2.5× Premix (500 mM potassium acetate, 87.5 mM Tris-acetate [pH 8.0], 67.5 mM ammonium acetate, 50 µg of folinic acid/ml, 5 mM DTT, 87.5 mg of polyethylene glycol 8000/ml, 5.0 mM ATP, 1.25 mM [each] additional ribonucleoside triphosphate, 0.02 mM amino acids, 50 mM phosphoenolpyruvate [trisodium salt], 2.5 mM cyclic AMP, 250 µg of each E. coli tRNA/ml), 10× amino acid mix (1.25 mM concentrations of each amino acid), pSAluc plasmid, S. aureus strain RN4220 S30 extract, and inhibitor were added. Inhibitor compounds were dissolved in 100% dimethyl sulfoxide and diluted such that the final dimethyl sulfoxide concentration was 3% or less. For TCA precipitation, 10× amino acid minus methionine mix plus 10 µCi of [³⁵S]methionine (1,000 Ci/mmol; Amersham) was used in the reaction mixture. (The reagent concentrations are essentially as described by Zubay [5]). Reaction mixtures were incubated at 37°C for 1 h. A 10-µl aliquot of the reaction mixture was used for the determination of either TCA-precipitable counts or luciferase activity. For TCA precipitation, the reaction aliquot was hydrolyzed in 250 µl of 1 N NaOH at 37°C for 10 min. This mixture was acid precipitated by the addition of 1 ml of 25% cold TCA with 2% Casamino Acids. The filtrate was collected on glass fiber filters (GF/A; Whatman) and counted by liquid scintillation spectrometry. For the luciferase assay, 50 µl of Luciferase Assay Reagent (Promega Corporation) was added and the readout was taken on a Trilux scintillation/luminescence reader (Wallac) according to the manufacturer's instructions.

View larger version (11K): [in this window] [in a new window]   FIG. 2.   S. aureus in vitro coupled T/T luciferase assay in the presence of increasing concentrations of reaction components: pSAluc plasmid DNA (A) and S. aureus RN4220 S30 extract (B). Reaction conditions were as described in the text for the luciferase assay. Activity is expressed in arbitrary light units.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Inhibitory activity profiles for various known antibiotics in the S. aureus and E. coli in vitro coupled T/T luciferase assays. Error bars indicate standard deviation.