Multiple N-acyl homoserine lactone signals of Rhizobium leguminosarum are synthesized in a distinct temporal pattern

A common form of bacterial quorum sensing involves the production and release of acyl homoserine lactone (AHL) signal metabolites. The nitrogen-fixing symbiont Rhizobium leguminosarum reportedly produces at least six different AHLs, but little is known about the regulation of biosynthesis of these molecules. We used a radiolabeling protocol to quantify the relative amounts of AHLs synthesized over time by R. leguminosarum cells with and without the symbiosis plasmid pRLIJI. Cells containing pRLIJI were found to produce three predominant signals. In decreasing order of abundance,, these were N-(3-oxo)octanoyl homoserine lactone [(3-O)C8HSL], N-octanoyl homoserine lactone, and N-hexanoyl homoserine lactone. Cells without pRLIJI produced only two major signals, N-(3-hydroxy,7-cis)tetradecanoyl homoserine lactone [(3-OH)C1.4:1HSL] and (3-O)C8HSL. Each AHL exhibited a distinct temporal pattern of synthesis, suggesting that each AHL is subject to unique regulatory mechanisms. While (3-O)C8HSL was produced in both cultures, the patterns of synthesis were different in cells with and without pRLIJI, possibly as a result of redundant gene functions that are present on both the chromosome and the symbiosis plasmid. None of the AHLs appeared to regulate its own biosynthesis, although exogenous (3-OH)C1.4:1HSL did activate synthesis of the three NHLs made by cells containing pRLIJI. These results indicate that the synthesis of multiple AHLs in R. leguminosarum is regulated by complex mechanisms that operate independently of quorum sensing itself but that (3-OH)C14:1HSL can supersede these controls in pRLIJI-containing cells. This,work provides an important global perspective for AHL regulation that both complements and contrasts with the results of previous studies performed with isolated gene systems.