Acylated homoserine lactones (AHLs) regulate a wide variety of phenotypes in Gram-negative bacteria. Most research suggests that AHL-mediated phenotypes are not expressed in populations until late logarithmic phase or stationary phase. Here, we model how the concentration of AHLs inside bacterial cells and in a biofilm changes over time as a function of population growth rate, diffusion of AHLs and the rate of autoinduction. Our theoretical results show that the concentration of AHLs inside a single bacterium (and by implication induction of a phenotype) has a non-trivial behaviour over time, and often exhibits a rapid increase early in population growth. This rapid increase is followed by a plateau, followed by another rise in the concentration of AHLs, to a second plateau. High concentrations of AHLs inside the bacterial cell early in population growth are positively affected by slow diffusion rates out of the cell and the biofilm, slow bacterial growth rates and fast autoinduction. In contrast, fast growth rates, slow autoinduction rates and high diffusion rates result in a high concentration plateau in stationary phase. More generally, the density-dependent nature of AHL regulation can be viewed as a trade-off between factors that dilute intracellular concentrations of AHLs (diffusion out of the cell, cell division), and those that increase concentrations (a slowing or restriction of diffusion or growth, or autoinduction). These results suggest that expression of AHL-mediated phenotypes can occur at relatively low cell densities and low external/environmental AHL concentrations.

In cell‐free Pseudomonas aeruginosa culture supernatants, we identified two compounds capable of activating an N ‐acylhomoserine lactone (AHL) biosensor. Mass spectrometry and NMR spectroscopy revealed that these compounds were not AHLs but the diketopiperazines (DKPs), cyclo(ΔAla‐ l ‐Val) and cyclo( l ‐Pro‐ l ‐Tyr) respectively. These compounds were also found in cell‐free supernatants from Proteus mirabilis , Citrobacter freundii and Enterobacter agglomerans [cyclo(ΔAla‐ l ‐Val) only]. Although both DKPs were absent from Pseudomonas fluorescens and Pseudomonas alcaligenes , we isolated, from both pseudomonads, a third DKP, which was chemically characterized as cyclo( l ‐Phe‐ l ‐Pro). Dose–response curves using a LuxR‐based AHL biosensor indicated that cyclo(ΔAla‐ l ‐Val), cyclo( l ‐Pro‐ l ‐Tyr) and cyclo( l ‐Phe‐ l ‐Pro) activate the biosensor in a concentration‐dependent manner, albeit at much higher concentrations than the natural activator N ‐(3‐oxohexanoyl)‐ l ‐homoserine lactone (3‐oxo‐C6‐HSL). Competition studies showed that cyclo(ΔAla‐ l ‐Val), cyclo( l ‐Pro‐ l ‐Tyr) and cyclo( l ‐Phe‐ l ‐Pro) antagonize the 3‐oxo‐C6‐HSL‐mediated induction of bioluminescence, suggesting that these DKPs may compete for the same LuxR‐binding site. Similarly, DKPs were found to be capable of activating or antagonizing other LuxR‐based quorum‐sensing systems, such as the N ‐butanoylhomoserine lactone‐dependent swarming motility of Serratia liquefaciens . Although the physiological role of these DKPs has yet to be established, their activity suggests the existence of cross talk among bacterial signalling systems.