Multiple N-Acyl Homoserine Lactone Signals of Rhizobium leguminosarum Are Synthesized in a Distinct Temporal Pattern

Quorum sensing is a type of intercellular communication found in many gram-negative bacteria. Communication between cells by quorum sensing is mediated by the production and release of specific N-acyl homoserine lactone (AHL) signal metabolites. The individual cells in a population are able to sense the population density through extracellular accumulation of these signals (7, 8, 10, 21). Two families of transcriptional regulators, called the LuxI and LuxR protein families, control this communication system. LuxI proteins catalyze the synthesis of the AHL signals, which then interact with LuxR proteins to activate or repress specific target genes. This process was first identified as a process that controls bioluminescence in the marine bacterium Vibrio fischeri but has since been found to regulate a variety of functions in many different bacteria (21). Many bacteria produce more than one type of AHL signal, thus creating the opportunity for interactions among different quorum sensing systems in the same organism (1, 16). One such organism is the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum, which produces at least six different AHLs (13, 17). These molecules have been identified as N-hexanoyl homoserine lactone (C6HSL), N-(3-oxo)hexanoyl homoserine lactone [(3-O)C6HSL], N-heptanoyl homoserine lactone (C7HSL), N-octanoyl homoserine lactone (C8HSL), N-(3-oxo)octanoyl homoserine lactone [(3-O)C8HSL], and N-(3-hydroxy-7-cis)tetradecanoyl homoserine lactone [(3-OH)C14:1HSL] (9, 13, 17, 19). The abilities to produce these different signals are encoded by at least four luxI homologs in this species. The chromosomal gene cinI encodes a protein that synthesizes (3-OH)C14:1HSL, while the rhiI gene of the symbiosis (Sym) plasmid pRL1JI encodes an enzyme that produces both C6HSL and C8HSL (13, 17). Two additional loci for AHL synthesis, one each on pRL1JI and the chromosome, have been identified but have not been described yet (13). The only R. leguminosarum genes known to be regulated directly at the transcriptional level by quorum sensing are those composing the rhiABC operon. This operon is found on the Sym plasmid pRL1JI and is controlled by the LuxR homolog RhiR. The RhiR protein activates rhiABC transcription in response to the presence of C6HSL and other AHLs (4, 17). It was previously found that addition of the chromosomally encoded signal (3-OH)C14:1HSL also resulted in increased expression of the rhiABC operon (9), but subsequent work showed that the activation is indirect, possibly a result of increased synthesis of other AHLs (13, 17). Because (3-OH)C14:1HSL also activates genes that inhibit the growth of cells containing pRL1JI (9), uncharacterized regulatory functions encoded by the Sym plasmid typically suppress (3-OH)C14:1HSL synthesis to levels that are about 5% of normal levels (13). These low levels of (3-OH)C14:1HSL still appear to be required for normal production of the other AHLs made by cells containing pRL1JI (13). The complex effects of (3-OH)C14:1HSL suggest that there may be communication among different quorum sensing loci and thus a hierarchy of AHL signals within R. leguminosarum. The purpose of this research was to examine possible signal interactions in R. leguminosarum by measuring the temporal pattern of AHL synthesis at a global level and the response of this pattern to exogenous addition of individual AHLs.