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Listeria monocytogenes (Lm) is an important food-borne human pathogen that is able to strive under a wide range of environmental conditions. Its accessory gene regulator (agr) system was shown to impact on biofilm formation and virulence and has been proposed as one of the regulatory mechanisms involved in adaptation to these changing environments. The Lm agr operon is homologous to the Staphylococcus aureus system, which includes an agrD-encoded autoinducing peptide that stimulates expression of the agr genes via the AgrCA two-component system and is required for regulation of target genes. The aim of the present study was to identify the native autoinducing peptide (AIP) of Lm using a luciferase reporter system in wildtype and agrD deficient strains, rational design of synthetic peptides and mass spectrometry. Upon deletion of agrD, luciferase reporter activity driven by the PII promoter of the agr operon was completely abolished and this defect was restored by co-cultivation of the agrD-negative reporter strain with a producer strain. Based on the sequence and structures of known AIPs of other organisms, a set of potential Lm AIPs was designed and tested for PII-activation. This led to the identification of a cyclic pentapeptide that was able to induce PII-driven luciferase reporter activity and restore defective invasion of the agrD deletion mutant into Caco-2 cells. Analysis of supernatants of a recombinant Escherichia coli strain expressing AgrBD identified a peptide identical in mass and charge to the cyclic pentapeptide. The Lm agr system is specific for this pentapeptide since the AIP of Lactobacillus plantarum, which also is a pentapeptide yet with different amino acid sequence, did not induce PII activity. In summary, the presented results provide further evidence for the hypothesis that the agrD gene of Lm encodes a secreted AIP responsible for autoregulation of the agr system of Lm. Additionally, the structure of the native Lm AIP was identified.

Listeria monocytogenes is able to form biofilms on various surfaces and this ability is thought to contribute to persistence in the environment and on contact surfaces in the food industry. Extracellular DNA (eDNA) is a component of the biofilm matrix of many bacterial species and was shown to play a role in biofilm establishment of L. monocytogenes. In the present study, the effect of DNaseI treatment on biofilm formation of L. monocytogenes EGD-e was investigated under static and dynamic conditions in normal or diluted complex medium at different temperatures. Biofilm formation was quantified by crystal violet staining or visualized by confocal laser scanning microscopy. Biomass of surface-attached L. monocytogenes varies depending on temperature and dilution of media. Interestingly, L. monocytogenes EGD-e forms DNase-sensitive biofilms in diluted medium whereas in full strength medium DNaseI treatment had no effect. In line with these observations, eDNA is present in the matrix of biofilms grown in diluted but not full strength medium and supernatants of biofilms grown in diluted medium contain chromosomal DNA. The DNase-sensitive phenotype could be clearly linked to reduced ionic strength in the environment since dilution of medium in PBS or saline abolished DNase sensitivity. Several other but not all species of the genus Listeria display DNase-sensitive and -resistant modes of biofilm formation. These results indicate that L. monocytogenes biofilms are DNase-sensitive especially at low ionic strength, which might favor bacterial lysis and release of chromosomal DNA. Since low nutrient concentrations with increased osmotic pressure are conditions frequently found in food processing environments, DNaseI treatment represents an option to prevent or remove Listeria biofilms in industrial settings.