Differential assay for high-throughput screening of antibacterial compounds.

The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed beta-gal in the periplasm, suggesting leakage of beta-gal as the means by which this assay detects compound activities. A model is proposed according to which beta-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-beta-D-galactoside as a single reagent. Cell wall inhibitors release beta-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielded 8 compounds that cause beta-gal release. These results validate the BAU-102 assay as an effective tool in antimicrobial drug discovery.

Effects of heat treatment of electron-transport particles on bacterial oxidative phosphorylation.

The electron-transport particles from Mycobacterium phlei exhibit low levels of phosphorylation unless supplemented with soluble coupling proteins. Heat treatment of the electron transport particles for 15 min at 50 degrees C was found to result in a slight loss of oxidation and an activation of phosphorylation with NADH as substrate, while with succinate as substrate both activities increased. The heat-activated particles do not require the addition of soluble coupling factors and their level of oxidative phosphorylation is similar to that of the regular particles supplemented with the soluble coupling factors. In contrast to the lack of a requirement for the soluble coupling factors after heat activation, the heat-treated electron-transport particles require the presence of a particulate-bound coupling factor for phosphorylation. The heat-activated system, like the untreated system, was found to be sensitive to uncoupling agents.