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Acyl homoserine lactones (acyl-HSLs) are important intercellular signaling molecules used by many bacteria to monitor their population density in quorumsensing control of gene expression. These signals are synthesized by members of the LuxI family of proteins. To understand the mechanism of acyl-HSL synthesis we have purified the Pseudomonas aeruginosa RhlI protein and analyzed the kinetics of acyl-HSL synthesis by this enzyme. Purified RhlI catalyzes the synthesis of acyl-HSLs from acyl-acyl carrier proteins and S-adenosylmethionine. An analysis of the patterns of product inhibition indicated that RhlI catalyzes signal synthesis by a sequential, ordered reaction mechanism in which S-adenosylmethionine binds to RhlI as the initial step in the enzymatic mechanism. Because pathogenic bacteria such as P. aeruginosa use acyl-HSL signals to regulate virulence genes, an understanding of the mechanism of signal synthesis and identification of inhibitors of signal synthesis has implications for development of quorum sensing-targeted antivirulence molecules.Many Gram-negative bacteria synthesize acyl-homoserine lactone (acyl-HSL) signal molecules that serve in a cell-to-cell communication system termed quorum sensing. Quorum sensing enables population density control of gene expression (for recent reviews of quorum sensing see refs. 1-4). Because quorum sensing has been implicated as an important factor in the expression of virulence genes in animal and plant pathogens (2, 5-7), understanding the mechanism of acyl-HSL synthesis is of importance. Although all acyl-HSLs possess an HSL ring, the length of the acyl side chain and the substitutions on the side chain differ and are specificity determinants for different quorum-sensing systems. In most systems, acyl-HSL signal synthesis requires a member of the LuxI family of proteins. LuxI family members occur in a number of different bacterial genera; all LuxI proteins direct the synthesis of specific acyl-HSLs and show sequence similarity (2-4, 8).There are three reports of in vitro catalysis of acyl-HSL synthesis by LuxI family members. The Vibrio fischeri LuxI protein was purified as a maltose-binding protein fusion (9) and the Agrobacterium tumefaciens TraI protein as a Histagged fusion (10). Both of these proteins functioned as acyl-HSL synthases when provided with S-adenosylmethionine (SAM) as the amino donor and an appropriate acyl-acyl carrier protein (acyl-ACP) as an acyl donor. Subsequently, the Pseudomonas aeruginosa RhlI protein was purified from recombinant Escherichia coli in the form of insoluble inclusion bodies. In vivo, RhlI directs the synthesis of N-butyryl-HSL and small amounts of N-hexanoyl-HSL (11). The purified protein was reported to catalyze the synthesis of butyryl-HSL when provided with butyryl-CoA, HSL, and NADPH (12). The activity of the RhlI preparation was substantially lower than the activity of the LuxI or TraI preparations (10 Ϫ6 ), thus raising concerns as to whether butyryl-CoA and HSL are relevant substrates for acyl-HSL synthesis...

Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxI gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding proteinLuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxI directs the synthesis of N-(3-oxohexanoyl)homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an autoinducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.Many Gram-negative bacteria synthesize diffusible acyl homoserine lactone molecules. These molecules serve as signals in quorum sensing, a system for cell density-dependent expression of specific sets of genes. As such, they have been termed autoinducers (for recent reviews of quorum sensing, see refs. 1-4). Acyl homoserine lactone signaling was first described in the luminous marine bacterium, Vibrio fischeri (5, 6), and V fischeri has since become a model for studies of quorum sensing (1, 3, 4). The luxI gene has been shown to direct V fischeri to synthesize N-(3-oxohexanoyl)homoserine lactone (5), and, more recently, it also has been shown to direct the synthesis of hexanoyl homoserine lactone (7). These acyl homoserine lactones bind to the product of luxR (8), which then serves as a transcriptional activator of the luminescence genes (1-4, 9). N-(3-oxohexanoyl)homoserine lactone shows more activity as an autoinducer than does hexanoyl homoserine lactone (10, 11). A variety of plant and animal pathogens use quorum sensing systems homologous to the luxR-luxI system to control expression of extracellular virulence factors (1-4).There is very little known about how luxI or any of its homologs direct the synthesis of acyl homoserine lactones. Crude cell extracts of V fischeri catalyze the synthesis of N...

The Vibrio fischeri luminescence genes are activated by the transcription factor LuxR in combination with a diffusible signal compound, N-(3-oxohexanoyl) homoserine lactone, termed the autoinducer. We have synthesized a set of autoinducer analogs. Many analogs with alterations in the acyl side chain showed evidence of binding to LuxR. Some appeared to bind with an affinity similar to that of the autoinducer, but none showed a higher affinity, and many did not bind as tightly as the autoinducer. For the most part, compounds with substitutions in the homoserine lactone ring did not show evidence of binding to LuxR. The exceptions were compounds with a homocysteine thiolactone ring in place of the homoserine lactone ring. Many but not all of the analogs showing evidence of LuxR binding had some ability to activate the luminescence genes. None were as active as the autoinducer. While most showed little ability to induce luminescence, a few analogs with rather conservative substitutions had appreciable activity. Under the conditions we employed, some of the analogs showing little or no ability to induce luminescence were inhibitors of the autoinducer.Quorum sensing is used by a number of gram-negative bacterial genera to regulate expression of specific sets of genes in a cell density-dependent fashion (10,20,21). Certain pathogenic bacteria use quorum sensing in the regulation of genes encoding extracellular virulence factors (17,19). The cell density control of luminescence in the symbiotic marine bacterium Vibrio fischeri is the best-studied quorum sensing system, and although each of the known systems has unique features, the V. fischeri luminescence system is considered the model (10, 20, 21). There are two regulatory genes involved in quorum sensing, the I and R genes. The I gene directs the synthesis of an N-acyl homoserine lactone (HSL) signal molecule termed the autoinducer. The R gene codes for a transcription factor that is responsive to the N-acyl HSL signal. In V. fischeri, the luxI gene directs the synthesis of 3-oxohexanoyl HSL, the autoinducer signal required for luminescence gene activation (6,8,9). Cells are permeable to this signal, and thus high cell densities are required to achieve a critical concentration of the autoinducer required to bind the luxR product, which in turn activates transcription of the luminescence genes (1,8,13,15).Little is known about the interaction of the V. fischeri autoinducer, 3-oxohexanoyl HSL, and the LuxR protein. The LuxR polypeptide consists of two domains. The available evidence indicates that 3-oxohexanoyl HSL binds to the N-terminal domain and that this binding allows a productive interaction of the LuxR C-terminal domain with the transcription-initiation complex of the luminescence genes (4, 13). There is one previous study of the influence of autoinducer analogs on induction of luminescence in V. fischeri (7). The analogs showed a spectrum of activities: some were capable of inducing luminescence, some inhibited activation by 3-oxohexanoyl HSL, and others showe...