In gram-negative bacterial pathogens, such asPseudomonas aeruginosaandYersinia pseudotuberculosis, cell-to-cell communication via theN-acylhomoserine lactone (AHL) signal molecules is involved in the cell population density-dependent control of genes associated with virulence. This phenomenon, termed quorum sensing, relies upon the accumulation of AHLs to a threshold concentration at which target structural genes are activated. By using biosensors capable of detecting a range of AHLs we observed that, in cultures ofY. pseudotuberculosisandP. aeruginosa, AHLs accumulate during the exponential phase but largely disappear during the stationary phase. When added to late-stationary-phase, cell-free culture supernatants of the respective pathogen, the majorP. aeruginosa[N-butanoylhomoserine lactone (C4-HSL) andN-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL)] andY. pseudotuberculosis[N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) andN-hexanoylhomoserine lactone (C6-HSL)] AHLs were inactivated. Short-acyl-chain compounds (e.g., C4-HSL) were turned over more extensively than long-chain molecules (e.g., 3-oxo-C12-HSL). Little AHL inactivation occurred with cell extracts, and no evidence for inactivation by specific enzymes was apparent. This AHL turnover was discovered to be due to pH-dependent lactonolysis. By acidifying the growth media to pH 2.0, lactonolysis could be reversed. By using carbon-13 nuclear magnetic resonance spectroscopy, we found that the ring opening of homoserine lactone (HSL),N-propionyl HSL (C3-HSL), and C4-HSL increased as pH increased but diminished as theN-acyl chain was lengthened. At low pH levels, the lactone rings closed but not via a simple reversal of the ring opening reaction mechanism. Ring opening of C4-HSL, C6-HSL, 3-oxo-C6-HSL, andN-octanoylhomoserine lactone (C8-HSL), as determined by the reduction of pH in aqueous solutions with time, was also less rapid for AHLs with more electron-donating longer side chains. Raising the temperature from 22 to 37°C increased the rate of ring opening. Taken together, these data show that (i) to be functional under physiological conditions in mammalian tissue fluids, AHLs require anN-acyl side chain of at least four carbons in length and (ii) that the longer the acyl side chain the more stable the AHL signal molecule.

For many years, bacterial cells were considered primarily as selfish individuals, but, in recent years, it has become evident that, far from operating in isolation, they coordinate collective behaviour in response to environmental challenges using sophisticated intercellular communication networks. Cell-to-cell communication between bacteria is mediated by small diffusible signal molecules that trigger changes in gene expression in response to fluctuations in population density. This process, generally referred to as quorum sensing (QS), controls diverse phenotypes in numerous Gram-positive and Gram-negative bacteria. Recent advances have revealed that bacteria are not limited to communication within their own species but are capable of ‘listening in’ and ‘broadcasting to’ unrelated species to intercept messages and coerce cohabitants into behavioural modifications, either for the good of the population or for the benefit of one species over another. It is also evident that QS is not limited to the bacterial kingdom. The study of two-way intercellular signalling networks between bacteria and both uni- and multicellular eukaryotes as well as between eukaryotes is just beginning to unveil a rich diversity of communication pathways.

Cell-to-cell communication (quorum sensing (QS)) co-ordinates bacterial behaviour at a population level. Consequently the behaviour of a natural multi-species community is likely to depend at least in part on co-existing QS and quorum quenching (QQ) activities. Here we sought to discover novel N-acylhomoserine lactone (AHL)-dependent QS and QQ strains by investigating a bacterial community associated with the rhizosphere of ginger (Zingiber officinale) growing in the Malaysian rainforest.

TheYersinia enterocoliticaLuxI homologue YenI directs the synthesis ofN-3-(oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) andN-hexanoylhomoserine lactone (C6-HSL). In aY. enterocolitica yenImutant, swimming motility is temporally delayed while swarming motility is abolished. Since both swimming and swarming are flagellum dependent, we purified the flagellin protein from the parent andyenImutant. Electrophoresis revealed that in contrast to the parent strain, theyenImutant grown for 17 h at 26°C lacked the 45-kDa flagellin protein FleB. Reverse transcription-PCR indicated that while mutation ofyenIhad no effect onyenR,flhDC(the motility master regulator) orfliA(the flagellar sigma factor) expression,fleB(the flagellin structural gene) was down-regulated. Since 3-oxo-C6-HSL and C6-HSL did not restore swimming or swarming in theyenImutant, we reexamined theN-acylhomoserine lactone (AHL) profile ofY. enterocolitica. Using AHL biosensors and mass spectrometry, we identified three additional AHLs synthesized via YenI:N-(3-oxodecanoyl)homoserine lactone,N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL), andN-(3-oxotetradecanoyl)homoserine lactone. However, none of the long-chain AHLs either alone or in combination with the short-chain AHLs restored swarming or swimming in theyenImutant. By investigating the transport of radiolabeled 3-oxo-C12-HSL and by introducing an AHL biosensor into theyenImutant we demonstrate that the inability of exogenous AHLs to restore motility to theyenImutant is not related to a lack of AHL uptake. However, both AHL synthesis and motility were restored by complementation of theyenImutant with a plasmid-borne copy ofyenI.