We have generated a database of expression profiles carrying the transcriptional responses of the model organism Bacillus subtilis following treatment with 37 well-characterized antibacterial compounds of different classes. The database was used to build a predictor for the assignment of the mechanisms of action (MoAs) of antibacterial compounds by the use of support vector machines. This predictor was able to correctly classify the MoA class for most compounds tested. Furthermore, we provide evidence that the in vivo MoA of hexachlorophene does not match the MoA predicted from in vitro data, a situation frequently faced in drug discovery. A database of this kind may facilitate the prioritization of novel antibacterial entities in drug discovery programs. Potential applications and limitations are discussed.

The bacterial cell wall has been a celebrated target for antibiotics and holds real promise for the discovery of new antibacterial chemical matter. In addition to peptidoglycan, the walls of Gram-positive bacteria contain large amounts of the polymer teichoic acid, covalently attached to peptidoglycan. Recently, wall teichoic acid was shown to be essential to the proper morphology of Bacillus subtilis and an important virulence factor for Staphylococcus aureus. Additionally, recent studies have shown that the dispensability of genes encoding teichoic acid biosynthetic enzymes is paradoxical and complex. Here, we report on the discovery of a promoter (P(ywaC)), which is sensitive to lesions in teichoic acid synthesis. Exploiting this promoter through a chemical-genetic approach, we revealed surprising interactions among undecaprenol, peptidoglycan, and teichoic acid biosynthesis that help explain the complexity of teichoic acid gene dispensability. Furthermore, the new reporter assay represents an exciting avenue for the discovery of antibacterial molecules.

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.

Gradual depletion of oxygen causes the shift-down of aerobic growing Mycobacterium bovis BCG to an anaerobic synchronized state of nonreplicating persistence. The persistent culture shows induction of glycine dehydrogenase and α-crystallin-like protein and is sensitive to metronidazole.

Upon depletion of oxygen, the obligate aerobe mycobacteria switch from growth to a state of non-replicating persistence or dormancy. Here, we report the first functional analysis of a dormancy-dependent mycobacterial promoter in Mycobacterium bovis BCG. Promoter probing using a PlacZ reporter detected a dormancy-inducible promoter activity upstream of the coding sequence for the putative nitrite extrusion protein NarK2. Primer extension analysis mapped a transcriptional start point 47 bp upstream of the narK2 start codon. Deletion analysis revealed that the sequence 3222 to 3133 bp upstream from the transcriptional start point was required for basal and dormancyinducible reporter expression. The sequence +1 to +47 downstream of the transcriptional start point had a strong inhibitory effect on the level of dormancy-induced L-galactosidase activity. The identification of apparent activating and inhibiting regions suggests that the narK2 promoter is at least under dual control. ß