Staphylococcus aureus Cell Extract Transcription-Translation Assay: Firefly Luciferase Reporter System for Evaluating Protein Translation Inhibitors

Abstract

The promoter for the Staphylococcus aureus capsule polysaccharide synthesis gene (cap1A) was cloned in front of the firefly luciferase gene for use in a cell extract S. aureus transcription-translation system. The assay is rapid, reproducible, and sensitive and has a lower background level than the radiolabeled amino acid incorporation translation assays. We present data evaluating a transcription inhibitor and a number of protein translation inhibitors in this system.

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.

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.

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.

Figure 2 demonstrates the dependence of the production of luciferase on the amount of pSAluc in the assay and on the amount of S. aureus S30 extract. The amount of S30 and pSAluc plasmid used should be optimized each time a new S30 extract is prepared due to slight variations in the activity in each new S30 preparation.

Translation was measured either by monitoring radiolabeled amino acid incorporation or by measuring luminescence. The pSAluc plasmid was used in both assays as a template. Table 1 shows that the background is lower and, consequently, that the signal-to-noise ratio is much higher when luminescence is measured rather than TCA-precipitable radiolabeled protein. The high background in the radiolabeled assay is thought to be caused by the presence of the endogenous mRNAs or mRNA fragments in the S30 that result in the translation of nonspecific protein products. In contrast, the luminescence assay detects only full-length functional firefly luciferase protein.

TABLE 1.

Comparison of background values of in vitro coupled T/T assay^a

Assay	Total counts (SD)	Background (SD)	Signal/noise ratio
S. aureus T/T TCA	84,522 (9,185)	4,476 (20)	19
S. aureus T/T luciferase	122,104 (3,271)	240 (32)	509

^aTotal and background values are expressed as either [³⁵S]methionine TCA precipitable counts or arbitrary light units. 

Since a commercially available E. coli T/T assay (Promega) which uses luciferase as the indicator to monitor translation is available, the question of whether the S. aureus T/T assay would offer any advantage over the E. coli system could be asked. Table 2 shows the results of testing several antibiotics in both systems. With the exception of streptomycin, the inhibition of translation by the gram-positive-bacteria spectrum antibiotics in the S. aureus T/T assay more closely agreed with the S. aureus MICs. The dose-response inhibition profiles for chloramphenicol, erythromycin, streptomycin, tetracycline, two oxazolidinones, and rifampin in the S. aureus T/T assay can be seen in Fig. 3. The reason for the different shapes of the inhibition profiles is not completely understood but is likely associated with the mechanism of action.

TABLE 2.

Comparison of results of in vitro coupled T/T luciferase assay

Inhibitor	MIC (μg/ml) for S. aureusa	IC50 [μM (μg/ml)] for:
		S. aureus	E. coli
Chloramphenicol	8	11 (3.6)	5 (1.6)
Erythromycin	0.25	0.4 (0.3)	0.3 (0.2)
Streptomycin	8	0.2 (0.3)	0.07 (0.1)
Tetracycline	0.25	0.5 (0.2)	30 (13)
Linezolid	2	7 (2.4)	2.5 (0.8)
Eperezolid	4	3 (1.2)	2.5 (1.0)
Rifampin	0.03	0.03 (0.02)	0.02 (0.02)

^aAntibiotic susceptibilities were determined for antibiotics and oxazolidinones by a broth microdilution method previously described (2). 

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.

The S. aureus T/T assay is a simple nonradioactive method that can be used to examine transcription-coupled translation in a medically important gram-positive organism. The assay may also be used to characterize mutants resistant to inhibitors of translation or transcription. By fractionating S30 into the soluble S100 fraction and the ribosomal pellet, ribosomes or S100 from resistant organisms can be mixed with S100 or ribosomes from sensitive organisms to determine if the resistance resides with the ribosome or with some factor in the soluble S100 fraction. The assay makes use of commercially available reagents, a modified plasmid DNA, and an S. aureus S30 extract. The assay provides a facile, rapid, high throughput method for evaluating putative inhibitors of prokaryotic transcription and translation. We have demonstrated an association between MIC and luciferase activity inhibition in the presence of known antibiotics as well as two oxazolidinones—linezolid and eperezolid.