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.

Bacterial communication signals, acylated homoserine lactones (AHLs), were extracted from samples of commercial bean sprouts undergoing soft-rot spoilage. Bean sprouts produced in the laboratory did not undergo soft-rot spoilage and did not contain AHLs or AHL-producing bacteria, although the bacterial population reached levels similar to those in the commercial sprouts, 10 8 to 10 9 CFU/g. AHL-producing bacteria (Enterobacteriaceae and pseudomonads) were isolated from commercial sprouts, and strains that were both proteolytic and pectinolytic were capable of causing soft-rot spoilage in bean sprouts. Thin-layer chromatography and liquid chromatography-high-resolution mass spectrometry revealed the presence of N-3-oxohexanoyl-L-homoserine lactone in spoiled bean sprouts and in extracts from pure cultures of bacteria. During normal spoilage, the pH of the sprouts increased due to proteolytic activity, and the higher pH probably facilitated the activity of pectate lyase. The AHL synthetase gene (I gene) from a spoilage Pectobacterium was cloned, sequenced, and inactivated in the parent strain. The predicted amino acid sequence showed 97% homology to HslI and CarI in Erwinia carotovora. Spoilage of laboratory bean sprouts inoculated with the AHL-negative mutant was delayed compared to sprouts inoculated with the wild type, and the AHL-negative mutant did not cause the pH to rise. Compared to the wild-type strain, the AHL-negative mutant had significantly reduced protease and pectinase activities and was negative in an iron chelation (siderophore) assay. This is the first study demonstrating AHL regulation of iron chelation in Enterobacteriaceae. The present study clearly demonstrates that the bacterial spoilage of some food products is influenced by quorum-sensingregulated phenotypes, and understanding these processes may be useful in the development of novel food preservation additives that specifically block the quorum-sensing systems.Many gram-negative bacteria use chemical signal molecules, acylated homoserine lactones (AHLs), to regulate the expression of particular phenotypes as a function of cell density. This phenomenon, known as quorum sensing (QS), is involved in the expression of virulence factors in several pathogenic bacteria and in the expression of factors important for the bacterial colonization of higher organisms (35,52,59,62,63). The growth and activity of gram-negative bacteria are also important in food microbiology, where spoilage of fresh foods, such as fish, meat, milk, and vegetables, is mostly a consequence of the degradative activity of gram-negative bacteria growing to high densities (10 8 to 10 9 CFU per gram). Food spoilage is a major economic and societal problem, and large amounts of foods are lost due to bacterial growth and spoilage (9). Understanding of the microbial processes leading to spoilage would facilitate development of novel preservation techniques and reduce loss of food. We hypothesized that bacterial fooddegradative processes could be regulated by QS, and if QS systems...

Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected in Aeromonas spp., but not in Shewanella strains. Thin-layer chromatographic profiles of cod extracts and P. phosphoreum culture supernatants identified a molecule similar in relative mobility (R f value) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain of P. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain of P. phosphoreum appeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains of P. phosphoreum. The phenotypes of nonbioluminescent P. phosphoreum strains regulated by AHLs remains to be elucidated.Photobacterium phosphoreum is a marine psychrotolerant bacterium commonly found free living in temperate waters or in high cell densities in fish gastrointestinal tracts and in specific light organelles in deep-sea fish (30,47,48). P. phosphoreum is a bioluminescent bacterium (17, 46, 48) which can also be found in dim and dark versions; loss of bioluminescence among bioluminescent species is frequently observed (16,30,31,53). It is unknown if all nonbioluminescent P. phosphoreum strains have ever been able to produce light, although it has been shown that at least part of the essential lux genes is present in both the luminous and nonluminous strains (17). P. phosphoreum belongs to the Vibrionaceae, which harbor several bioluminescent species, including Vibrio fischeri and Vibrio harveyi. These two species regulate bioluminescence through a circuit popularly referred to as quorum sensing (22,50,54). In brief, quorum sensing involves the production and release of communication molecules, which accumulate in the surrounding environment and allow the bacteria to coregulate specific phenotypes. The communication molecules regulating bioluminescence in V. fischeri are N-acylated homoserine lactones (AHLs). Quorum sensing enables V. fischeri to up-regulate bioluminescence when situated in its symbiont Euprymna scolopes and abolish light production when in the free-living state in t...

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