Escherichia coli strains bearing single-copy fusions between the lacZ reporter gene and the cspA, ibp, or P3rpoH stress promoters offer a simple means to detect sublethal concentrations of antibacterial agents interfering with prokaryotic translation or cell envelope integrity while simultaneously providing information on the mechanism of action of the test compound (A. A. Bianchi and F. Baneyx, Appl. Environ. Microbiol. 65:5023-5027, 1999). Here, we expand the usefulness of this system by (i) demonstrating that a fusion between the SOS-inducible sulA promoter and lacZ is a highly specific probe for the detection of antimicrobial agents that ultimately interfere with DNA replication, (ii) showing that inactivation of the tolC gene allows efficient detection of very low concentrations of model antibiotics (including aminoglycosides) whereas polymyxin B-mediated outer membrane permeabilization facilitates the identification of intermediate concentrations of hydrophobic compounds, and (iii) validating the potential of detector strains and sensitization strategies for high-throughput screening using a reproducible and internally consistent 96-well microplate assay.

Error-prone PCR was used to isolate Photinus pyralis luciferase mutants producing bright light in the red-orange region of the spectrum. All mutations were clustered in the beta5-alpha10-beta6 region of N-terminal subdomain B and appear to affect bioluminescence color by modulating the position of the Ser314-Leu319 mobile loop with respect to the putative active site. Two red variants (Q283R and S284G) and one orange mutant (S293P) contained a single substitution. Although the remaining orange variant contained two mutations, L287I mainly contributed to the color change. Emission spectra collected on whole cells at pH 7.0 revealed that while a single peak of lambdamax approximately 605 nm accounts for red light production by the Q283R and S284G variants, orange light results from the contribution of two peaks of lambdamax approximately 560 and 600 nm. All spectra underwent a red-shift when cells were assayed under acidic conditions, whereas a blue-shift was observed at pH 8.0, indicating that the internal pH of Escherichia coli is close to the external pH shortly after imposition of acid or alkaline stress. In addition, changes in assay pH led to bimodal emission spectra, lending support to the idea that bioluminescence color is determined by the relative contribution of yellow-green and red-orange peaks. The set of multicolored luciferase mutants described here may prove useful for a variety of applications including biosensing, pH monitoring, and tissue and animal imaging.

No abstract