Recently, several light receptors have been identified in non-phototrophic bacteria, but their physiological roles still remain rather elusive. Here we show that colonies of the saprophytic bacterium Listeria monocytogenes undergo synchronized multicellular behaviour on agar plates, in response to oscillating light/dark conditions, giving rise to alternating ring formation (opaque and translucent rings). On agar plates, bacteria from opaque rings survive increased levels of reactive oxygen species (ROS), as well as repeated cycles of light and dark, better than bacteria from translucent rings. The ring formation is strictly dependent on a blue-light receptor, Lmo0799, acting through the stress-sigma factor, σB. A transposon screening identified 48 mutants unable to form rings at alternating light conditions, with several of them showing a decreased σB activity/level. However, some of the tested mutants displayed a varied σB activity depending on which of the two stress conditions tested (light or H2O2 exposure). Intriguingly, the transcriptional regulator PrfA and the virulence factor ActA were shown to be required for ring formation by a mechanism involving activation of σB. All in all, this suggests a distinct pathway for Lmo0799 that converge into a common signalling pathway for σB activation. Our results show that night and day cycles co-ordinate a reversible differentiation of a L. monocytogenes colony at room temperature, by a process synchronized by a blue-light receptor and σB.

Background: PARP13 contains a divergent PARP homology ADP-ribosyltransferase domain of unknown function. Results: The consensus NAD ϩ pocket of PARP13 is occluded by interacting protein side chains.

SummaryThe transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes cellular uptake by reducing the expression of virulence genes. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA-binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds at two separate sites on the protein: one within a hydrophobic pocket or tunnel, located between the C- and N-terminal domains of PrfA, and the second in the vicinity of the DNA-binding helix-turn-helix motif. At both sites the compound interacts with residues important for PrfA activation and helix-turn-helix formation. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes.

Inhibiting ADP-ribosyl transferases with PARP-inhibitors is considered a promising strategy for the treatment of many cancers and ischemia, but most of the cellular targets are poorly characterized. Here, we describe an inhibitor of ADP-ribosyltransferase-3/poly(ADP-ribose) polymerase-3 (ARTD3), a regulator of DNA repair and mitotic progression. In vitro profiling against 12 members of the enzyme family suggests selectivity for ARTD3, and crystal structures illustrate the molecular basis for inhibitor selectivity. The compound is active in cells, where it elicits ARTD3-specific effects at submicromolar concentration. Our results show that by targeting the nicotinamide binding site, selective inhibition can be achieved among the closest relatives of the validated clinical target, ADP-ribosyltransferase-1/poly(ADP-ribose) polymerase-1.

Acetylcholinesterase (AChE) is an essential enzyme that terminates cholinergic transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Compounds inhibiting this enzyme can be used (inter alia) to treat cholinergic deficiencies (e.g. in Alzheimer's disease), but may also act as dangerous toxins (e.g. nerve agents such as sarin). Treatment of nerve agent poisoning involves use of antidotes, small molecules capable of reactivating AChE. We have screened a collection of organic molecules to assess their ability to inhibit the enzymatic activity of AChE, aiming to find lead compounds for further optimization leading to drugs with increased efficacy and/or decreased side effects. 124 inhibitors were discovered, with considerable chemical diversity regarding size, polarity, flexibility and charge distribution. An extensive structure determination campaign resulted in a set of crystal structures of protein-ligand complexes. Overall, the ligands have substantial interactions with the peripheral anionic site of AChE, and the majority form additional interactions with the catalytic site (CAS). Reproduction of the bioactive conformation of six of the ligands using molecular docking simulations required modification of the default parameter settings of the docking software. The results show that docking-assisted structure-based design of AChE inhibitors is challenging and requires crystallographic support to obtain reliable results, at least with currently available software. The complex formed between C5685 and Mus musculus AChE (C5685•mAChE) is a representative structure for the general binding mode of the determined structures. The CAS binding part of C5685 could not be structurally determined due to a disordered electron density map and the developed docking protocol was used to predict the binding modes of this part of the molecule. We believe that chemical modifications of our discovered inhibitors, biochemical and biophysical characterization, crystallography and computational chemistry provide a route to novel AChE inhibitors and reactivators.