Panel of Bacillus subtilis Reporter Strains Indicative of Various Modes of Action

Abstract

In a recent project, we collected the transcriptional profiles of Bacillus subtilis 168 after treatment with a large set of diverse antibacterial agents. One result of the data analysis was the identification of marker genes that are indicative of certain compounds or compound classes. We cloned these promoter regions in front of a luciferase reporter gene and reintroduced the constructs individually into the B. subtilis chromosome. Strains were analyzed for their responsiveness after treatment with a set of 37 antibacterials. Twelve functional reporter strains were generated that were selectively and significantly upregulated by the compounds. The selectivity of the reporter strains ranged from generic pathways like protein biosynthesis, cell wall biosynthesis, and fatty acid biosynthesis to compound classes (quinolones and glycopeptides) and individual compounds (rifampin, cycloserine, and clindamycin). Five of the strains are amenable for high-throughput applications, e.g., pathway-specific screening. In summary, we successfully generated B. subtilis reporter strains that are indicative of the mechanisms of action of various classes of antibacterials. The set of reporter strains presented herein can be used for mode-of-action analyses and for whole-cell screening of compound libraries in a mode-of-action-specific manner.

Staphylococcus aureus promoter-lux reporters for drug discovery

“…Similar promoter-lux systems have been used to study the multiple effects of subinhibitory levels of antibiotics, individually or in combination, on gene expression in Salmonella typhimurium and S. aureus; 14,[17][18][19]23 the sensitive and specific nature of transcription responses to bioactive compounds make them useful as a convenient and robust whole-cell screening system for antimicrobials. The light response readout of a lux-based system has an advantage over firefly luciferase (luc) or b-galactosidase (lacZ) reporter systems for this purpose [35][36][37] in that additional substrates are not required for the assay. Promoter-reporters have been used for the discovery of antimicrobials active against Bacillus subtilis 20,35,36,38 and E. coli, 37,39-41 but not against S. aureus until now.…”

“…The light response readout of a lux-based system has an advantage over firefly luciferase (luc) or b-galactosidase (lacZ) reporter systems for this purpose [35][36][37] in that additional substrates are not required for the assay. Promoter-reporters have been used for the discovery of antimicrobials active against Bacillus subtilis 20,35,36,38 and E. coli, 37,39-41 but not against S. aureus until now.…”

The Bacillus subtilis GntR Family Repressor YtrA Responds to Cell Wall Antibiotics

“…However, the ytrA operon was not induced by acetoin (45), and subsequent studies indicate that acetoin catabolism is determined by the carbon metabolite-repressed and acetoin-inducible acoABCL operon (1,35,41). Conversely, induction of the ytrA operon was noted in previous global analyses of antibiotic stress responses, and ytrA was even proposed as a reporter for glycopeptide antibiotics (18).…”

A High-Throughput, Homogeneous, Bioluminescent Assay for Pseudomonas aeruginosa Gyrase Inhibitors and Other DNA-Damaging Agents

“…A similar approach has been used to build B. subtilis reporter screening strains, but use of those strains for HTS required an additional pipetting step for the addition of luciferase substrate prior to measuring luminescence. 2 We used the entire luciferase operon from P. luminescens to build strain MBX-623. The resulting reporter strain requires no reagent addition for production of bioluminescence, is genetically stable, and produces high levels of luminescence in 4 to 7 h of incubation.…”

“…Several reporter assays of this type have been described in Bacillus subtilis based on the response of cells to antibiotic treatments and using firefly luciferase as a reporter. 2 We have extended this approach to a less permeable species, P. aeruginosa, and eliminated the need for luciferase substrate addition while achieving excellent sensitivity and highly positive Z′ scores. The resulting screen is simple, inexpensive, and homogeneous.…”