title: N-acylhomoserine lactones undergo lactonolysis in a pH-, temperature-, and acyl chain length-dependent manner during growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa.
abstract: In gram-negative bacterial pathogens, such as Pseudomonas aeruginosa and Yersinia pseudotuberculosis, cell-to-cell communication via the N-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 of Y. pseudotuberculosis and P. 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 major P. aeruginosa [N-butanoylhomoserine lactone (C4-HSL) and N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL)] and Y. pseudotuberculosis [N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-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 the N-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, and N-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 degrees 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 an N-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.
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom.
authors.forename: Edwin A
authors.initials: EA
authors.lastname: Yates
authors.forename: Bodo
authors.initials: B
authors.lastname: Philipp
authors.forename: Catherine
authors.initials: C
authors.lastname: Buckley
authors.forename: Steve
authors.initials: S
authors.lastname: Atkinson
authors.forename: Siri Ram
authors.initials: SR
authors.lastname: Chhabra
authors.forename: R Elizabeth
authors.initials: RE
authors.lastname: Sockett
authors.forename: Morris
authors.initials: M
authors.lastname: Goldner
authors.forename: Yves
authors.initials: Y
authors.lastname: Dessaux
authors.forename: Miguel
authors.initials: M
authors.lastname: Cámara
authors.forename: Harry
authors.initials: H
authors.lastname: Smith
authors.forename: Paul
authors.initials: P
authors.lastname: Williams
mesh_terms: D001826:Body Fluids / Q000378:metabolism / Q000382:microbiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007783:Lactones / Q000737:chemistry* / Q000378:metabolism*; D015394:Molecular Structure; D011550:Pseudomonas aeruginosa / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity; D013696:Temperature; D014774:Virulence; D015011:Yersinia pseudotuberculosis / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity
chemical_list: D007783:Lactones