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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. INTRODUCTIONThe bacterial phenomenon of quorum sensing enables bacteria to communicate and thereby coordinate the expression of specific target genes in response to the population density. In a minimum of 30 Gram-negative bacterial species, quorum sensing is mediated by small diffusible signal molecules, N-acylated derivatives of L-homoserine lactone, which may differ in the length and substitution of their respective acyl side chains. N-Acyl-Lhomoserine lactone (AHL) molecules with N-acyl side chains ranging from 4 to 14 carbons and with an oxo-, hydroxy-or no substituent at the C3 position have been identified [for recent reviews see Whitehead et al. (2001), Fuqua et al. (2001) and Miller & Bassler (2001)]. One of the best characterized quorum sensing systems is present in the marine bacterium Vibrio fischeri, in which LuxI synthesizes N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL). It is believed that the signal molecule 3-oxo-C6-HSL at a certain threshold concentration binds to a regulatory protein, LuxR, and thereby renders it active. LuxR hereafter activates transcription of the luxICDABEG operon. As a consequence, the bacterial culture expresses a bioluminescent phenotype when it colonizes the light organs of certain fish and squids and a non-bioluminescent phenotype when the bacteria live in a planktonic state in sea water [(for reviews see Sitnikov et al. (1995) and Dunlap (1999)]. This is one of the few known examples of quorumsensing-regulated gene expression in relation to a symbiotic relationship. In most other cases, quorum sensing is related to pathogenicity traits such as conjugal transfer of the Ti plasmids from Agrobacterium tumefaciens ...

Quorum-sensing (QS) signals (N-acyl homoserine lactones [AHLs]) were extracted and detected from five commercially produced vacuum-packed meat samples. Ninety-six AHL-producing bacteria were isolated, and 92 were identified as Enterobacteriaceae. Hafnia alvei was the most commonly identified AHL-producing bacterium. Thin-layer chromatographic profiles of supernatants from six H. alvei isolates and of extracts from spoiling meat revealed that the major AHL species had an R f value and shape similar to N-3-oxo-hexanoyl homoserine lactone (OHHL). Liquid chromatography-mass spectrometry (MS) (high-resolution MS) analysis confirmed the presence of OHHL in pure cultures of H. alvei. Vacuum-packed meat spoiled at the same rate when inoculated with the H. alvei wild type compared to a corresponding AHL-lacking mutant. Addition of specific QS inhibitors to the AHL-producing H. alvei inoculated in meat or to naturally contaminated meat did not influence the spoilage of vacuum-packed meat. An extracellular protein of approximately 20 kDa produced by the H. alvei wild-type was not produced by the AHL-negative mutant but was restored in the mutant when complemented by OHHL, thus indicating that AHLs do have a regulatory role in H. alvei. Coinoculation of H. alvei wild-type with an AHL-deficient Serratia proteamaculans B5a, in which protease secretion is QS regulated, caused spoilage of liquid milk. By contrast, coinoculation of AHL-negative strains of H. alvei and S. proteamaculans B5a did not cause spoilage. In conclusion, AHL and AHL-producing bacteria are present in vacuumpacked meat during storage and spoilage, but AHL does not appear to influence the spoilage of this particular type of conserved meat. Our data indicate that AHL-producing H. alvei may induce food quality-relevant phenotypes in other bacterial species in the same environment. H. alvei may thus influence spoilage of food products in which Enterobacteriaceae participate in the spoilage process.

A DNA-encoded small-molecule library was prepared using yoctoReactor technology followed by binder trap enrichment to identify selective inhibitors with nanomolar potencies against p38α MAP kinase.

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.

Swarming motility of Serratia liquefaciens MG1 requires the expression of two genetic loci, flhDC andswrI. Here we demonstrate that the products of theflhDC operon (the flagellar master regulator) and theswrI gene (the extracellular signal moleculeN-butanoyl-l-homoserine lactone) are global regulators which control two separate regulons.