In cell-free Yersinia pseudotuberculosis culture supernatants, we have chemically characterized three N-acyl homoserine lactone (AHL) molecules, N-octanoyl homoserine lactone (C8-HSL), N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoyl homoserine lactone (C6-HSL). We have identified, cloned and sequenced two pairs of LuxR/I homologues termed YpsR/I and YtbR/I. In Escherichia coli at 37 degrees C, YpsI and YtbI both synthesize C6-HSL, although YpsI is responsible for 3-oxo-C6-HSL and YtbI for C8-HSL synthesis respectively. However, in a Y. pseudotuberculosis ypsI-negative background, YtbI appears capable of adjusting the AHL profile from all three AHLs at 37 degrees C and 22 degrees C to the absence of 3-oxo-C6-HSL at 28 degrees C. Insertion deletion mutagenesis of ypsR leads to the loss of C8-HSL at 22 degrees C, which suggests that at this temperature the YpsR protein is involved in the hierarchical regulation of the ytbR/I locus. When compared with the parent strain, the ypsR and ypsI mutants exhibit a number of phenotypes, including clumping (ypsR mutant), overexpression of a major flagellin subunit (ypsR mutant) and increased motility (both ypsR and ypsI mutants). The clumping and motility phenotypes are both temperature dependent. These data are consistent with a hierarchical quorum-sensing cascade in Y. pseudotuberculosis that is involved in the regulation of clumping and motility.