No abstract

Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of <25 g/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis.

The X-ray crystal structures of two complexes of the composition trans-[Pt(NH3)2(9-EtG-N7)(1-MeC-N3‘)]X·nH2O (1) with 9-EtG = 9-ethylguaninate and 1-MeC = 1-methylcytosine are reported. 1b (X = picrate, n = 1) crystallizes to produce a dimetalated base quartet, held together by H-bonding interactions between pairs of cations. This feature essentially corresponds to the solution structure previously proposed by us on the basis of 1H NMR and ESI-MS, with a H-bonding interaction between the aromatic H5‘ proton of 1-MeC and the deprotonated N1 position of 9-EtG. 1c (X = trifluoromethanesulfonate, n = 0) crystallizes in a radically different fashion as a consequence of nucleobase rotation about the Pt−N bond, leading to a reversed Hoogsteen arrangement without any intracomplex H bonding between the two bases. In the solid-state structure of 1b short intermolecular H bonds exist between the exocyclic NH2 group of 1-MeC and O6 of 9-EtG (2.715(7) Å). Considerably longer intra- (N4‘(1-MeC)···O6(9-EtG), 3.229(6) Å) and intermolecular (C5‘(1-MeC)···N1(9-EtG), 3.548(7) Å) H bonds are primarily a consequence of considerable base twisting, presumably caused by stacking between the guanine residues and the picrate anions. In DMSO-d 6 solution, the cyclic base quartet structure is favored, regardless of the nature of the anion X (X = picrate, trifluoromethanesulfonate, perchlorate, nitrate). An association constant K D = 44.1 ± 3.2 M-1 for the dimerization has been determined.