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.

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).

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.

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.