Many Gram-negative bacteria regulate gene expression in response to their population size by sensing the level of acyl-homoserine lactone signal molecules which they produce and liberate to the environment. We have developed an assay for these signals that couples separation by thin-layer chromatography with detection using Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. With the exception of N-butanoyl-L-homoserine lactone, the reporter detected acyl-homoserine lactones with 3-oxo-, 3-hydroxy-, and 3-unsubstituted side chains of all lengths tested. The intensity of the response was proportional to the amount of the signal molecule chromatographed. Each of the 3-oxo-and the 3-unsubstituted derivatives migrated with a unique mobility. Using the assay, we showed that some bacteria produce as many as five detectable signal molecules. Structures could be assigned tentatively on the basis of mobility and spot shape. The dominant species produced by Pseudomonas syringae pv. tabaci chromatographed with the properties of N-(3-oxohexanoyl)-L-homoserine lactone, a structure that was confirmed by mass spectrometry. An isolate of Pseudomonas fluorescens produced five detectable species, three of which had novel chromatographic properties. These were identified as the 3-hydroxy-forms of N-hexanoyl-, N-octanoyl-, and N-decanoyl-L-homoserine lactone. The assay can be used to screen cultures of bacteria for acyl-homoserine lactones, for quantifying the amounts of these molecules produced, and as an analytical and preparative aid in determining the structures of these signal molecules.

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...

The increase in drug-resistant pathogenic bacteria has created an urgent demand for new antibiotics. Among the more attractive targets for the development of new antibacterial compounds are the enzymes of fatty acid biosynthesis. Although a number of potent inhibitors of microbial fatty acid biosynthesis have been discovered, few of these are clinically useful drugs. Several of these fatty acid biosynthesis inhibitors have potential as lead compounds in the development of new antibacterials. This review encompasses the known inhibitors and prospective targets for new antibacterials.

No abstract