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.

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Acetyl-CoA carboxylase (ACC) catalyzes the first committed step of the fatty acid synthetic pathway. Although ACC has often been proposed to be a major ratecontrolling enzyme of this pathway, no direct tests of this proposal in vivo have been reported. We have tested this proposal in Escherichia coli. The genes encoding the four subunits of E. coli ACC were cloned in a single plasmid under the control of a bacteriophage T7 promoter. Upon induction of gene expression, the four ACC subunits were overproduced in equimolar amounts. Overproduction of the proteins resulted in greatly increased ACC activity with a concomitant increase in the intracellular level of malonyl-CoA. The effects of ACC overexpression on the rate of fatty acid synthesis were examined in the presence of a thioesterase, which provided a metabolic sink for fatty acid overproduction. Under these conditions ACC overproduction resulted in a 6-fold increase in the rate of fatty acid synthesis.Fatty acids are an essential component of the cellular membranes of all living organisms excepting the Archaea. AcetylCoA carboxylase (ACC) 1 catalyzes the first committed step of the fatty acid synthetic pathway, the formation of malonyl-CoA from acetyl-CoA plus bicarbonate, and ACC has often been postulated to be a rate-controlling step in fatty acid biosynthesis (see, e.g., Refs. 1 and 2). Consistent with this hypothesis, the activity of ACC, the rates of fatty acid synthesis, and the levels of malonyl-CoA are known to be well correlated during hormonal treatments of mammalian tissues (1, 2). However, interpretation of these data is greatly complicated by the recent discovery of a second ACC isoform present in mitochondria (3). Data obtained by use of ACC inhibitors in isolated chloroplasts are also consistent with a regulatory role for ACC in this fatty acid synthetic system (4), although no data on chloroplast malonyl-CoA concentrations were reported. The role of ACC in determining the rate of fatty acid synthesis in vivo seems to remain an open question. As first pointed out by Walsh and Koshland (5), a direct means to approach in vivo pathway regulation is to overproduce candidate enzyme(s) and measure the effect on the flux through the pathway. However, we know of no example in any organism where this approach has been utilized for ACC. A test of the rate-controlling nature of ACC in vivo requires significantly increased levels of ACC activity as well as a metabolic sink (6) for the overproduced fatty acid molecules. Provision of an appropriate sink precludes the possibility that complex lipid synthesis (or the capacity of cell membrane bilayers) could limit the rate of fatty acid synthesis. We have chosen the bacterium Escherichia coli to test if increased ACC activity results in increased rates of fatty acid synthesis. This organism has several experimental advantages. First, the E. coli ACC genes and proteins are well studied (7-13) and the enzyme does not appear to be regulated by small molecules (7). Second, in the presence of high levels of cytosol...

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Cyclopropane fatty acid (CFA) formation is a post‐synthetic modification of the lipid bilayer that occurs as cultures of Escherichia coli and many other bacteria enter stationary phase. We report the first distinct phenotype for this membrane modification; early stationary phase cultures of strains lacking CFA (as a result of a null mutation in the cfa gene) are abnormally sensitive to killing by a rapid shift from neutral pH to pH 3. This sensitivity to acid shock is dependent on CFA itself because resistance to acid shock is restored to cfa mutant strains by incorporation of CFAs from the growth medium or by introduction of a functional cfa gene on a plasmid. The synthesis of CFA depends in part on the RpoS sigma factor, but the role of RpoS in resistance to acid shock involves additional factors because strains with null mutations in both cfa and rpoS are more sensitive to acid shock than either single mutant strain. Exponential phase cultures of E. coli are much more sensitive to acid shock than stationary phase cultures, but survival is greatly increased if the exponential phase cultures are exposed to moderately acid conditions (pH 5) before shift to pH 3. We show that exposure to moderately acid conditions gives a marked increase in cfa transcription. The efficiency of the survival of acid shock is extremely strain dependent, even among putative wild‐type strains. Much, but not all, of this variability can be explained by the partially or totally defective RpoS alleles carried by many strains.

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