Screening of DNA-Encoded Small Molecule Libraries inside a Living Cell.

DNA-encoded small molecule libraries (DELs) have facilitated the discovery of novel modulators of many different therapeutic protein targets. We report the first successful screening of a multimillion membered DEL inside a living cell. We demonstrate a novel method using oocytes from the South African clawed frog Xenopus laevis. The large size of the oocytes of 1 µL, or 100 000 times bigger than a normal somatic cell, permits simple injection of DELs, thus resolving the fundamental problem of delivering DELs across cell membranes for in vivo screening. The target protein was expressed in the oocytes fused to a prey protein, to allow specific DNA labeling and hereby discriminate between DEL members binding to the target protein and the endogenous cell proteins. The 194 million member DEL was screened against three pharmaceutically relevant protein targets, p38α, ACSS2, and DOCK5. For all three targets multiple chemical clusters were identified. For p38α, validated hits with single digit nanomolar potencies were obtained. This work demonstrates a powerful new approach to DEL screening, which eliminates the need for highly purified active target protein and which performs the screening under physiological relevant conditions and thus is poised to increase the DEL amenable target space and reduce the attrition rates.

Effects of iron on DNA release and biofilm development by Pseudomonas aeruginosa.

Extracellular DNA is one of the major matrix components in Pseudomonas aeruginosa biofilms. It functions as an intercellular connector and plays a role in stabilization of the biofilms. Evidence that DNA release in P. aeruginosa PAO1 biofilms is controlled by the las-rhl and pqs quorum-sensing systems has been previously presented. This paper provides evidence that DNA release in P. aeruginosa PAO1 biofilms is also under iron regulation. Experiments involving cultivation of P. aeruginosa in microtitre trays suggested that pqs expression, DNA release and biofilm formation were favoured in media with low iron concentrations (5 microM FeCl(3)), and decreased with increasing iron concentrations. Experiments involving cultivation of P. aeruginosa in a flow-chamber system suggested that a high level of iron (100 microM FeCl(3)) in the medium suppressed DNA release, structural biofilm development, and the development of subpopulations with increased tolerance toward antimicrobial compounds. Experiments with P. aeruginosa strains harbouring fluorescent reporters suggested that expression of the pqs operon was induced in particular subpopulations of the biofilm cells under low-iron conditions (1 microM FeCl(3)), but repressed in the biofilm cells under high-iron conditions (100 microM FeCl(3)).

Quorum-sensing-directed protein expression in Serratia proteamaculans B5a.

N-Acyl-L-homoserine-lactone-producing Serratia species are frequently encountered in spoiling foods of vegetable and protein origin. The role of quorum sensing in the food spoiling properties of these bacteria is currently being investigated. A set of luxR luxI homologous genes encoding a putative quorum sensor was identified in the N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL)-producing Serratia proteamaculans strain B5a. The 3-oxo-C6-HSL synthase SprI showed 79 % similarity with EsaI from Pantoea stewartii and the putative regulatory protein SprR was 86 % similar to the SpnR of Serratia marcescens. Proteome analysis suggested that the presence of at least 39 intracellular proteins was affected by the 3-oxo-C6-HSL-based quorum sensing system. The lipB-encoded secretion system was identified as one target gene of the quorum sensing system. LipB was required for the production of extracellular lipolytic and proteolytic activities, thus rendering the production of food-deterioration-relevant exoenzymes indirectly under the control of quorum sensing. Strain B5a caused quorum-sensing-controlled spoilage of milk. Furthermore, chitinolytic activity was controlled by quorum sensing. This control appeared to be direct and not mediated via LipB. The data presented here demonstrate that quorum-sensing-controlled exoenzymic activities affect food quality.

Food spoilage--interactions between food spoilage bacteria.

Food spoilage is a complex process and excessive amounts of foods are lost due to microbial spoilage even with modern day preservation techniques. Despite the heterogeneity in raw materials and processing conditions, the microflora that develops during storage and in spoiling foods can be predicted based on knowledge of the origin of the food, the substrate base and a few central preservation parameters such as temperature, atmosphere, a(w) and pH. Based on such knowledge, more detailed sensory, chemical and microbiological analysis can be carried out on the individual products to determine the actual specific spoilage organism. Whilst the chemical and physical parameters are the main determining factors for selection of spoilage microorganisms, a level of refinement may be found in some products in which the interactive behavior of microorganisms may contribute to their growth and/or spoilage activity. This review gives three such examples. We describe the competitive advantage of Pseudomonas spp. due to the production of iron-chelating siderophores, the generation of substrates for spoilage reactions by one organism from another microorganism (so-called metabiosis) and the up-regulation of phenotypes potentially involved in spoilage through cell-to-cell communication. In particular, we report for the first time the widespread occurrence of N-acyl homoserine lactones (AHL) in stored and spoiling fresh foods and we discuss the potential implications for spoilage and food preservation.