Specificity of Acyl-Homoserine Lactone Synthases Examined by Mass Spectrometry

Abstract

Many gram-negative bacteria produce a specific set of N-acyl-L-homoserine-lactone (AHL) signaling molecules for the purpose of quorum sensing, which is a means of regulating coordinated gene expression in a cell-density-dependent manner. AHLs are produced from acylated acyl-carrier protein (acyl-ACP) and Sadenosyl-L-methionine by the AHL synthase enzyme. The appearance of specific AHLs is due in large part to the intrinsic specificity of the enzyme for subsets of acyl-ACP substrates. Structural studies of the Pantoea stewartii enzyme EsaI and AHL-sensitive bioassays revealed that threonine 140 in the acyl chain binding pocket directs the enzyme toward production of 3-oxo-homoserine lactones. Mass spectrometry was used to examine the range of AHL molecular species produced by AHL synthases under a variety of conditions. An AHL selective normal-phase chromatographic purification with addition of a deuterated AHL internal standard was followed by reverse-phase liquid chromatography-tandem mass spectrometry in order to obtain estimates of the relative amounts of different AHLs from biological samples. The AHLs produced by wild-type and engineered EsaI and LasI AHL synthases show that intrinsic specificity and different cellular conditions influence the production of AHLs. The threonine at position 140 in EsaI is important for the preference for 3-oxo-acyl-ACPs, but the role of the equivalent threonine in LasI is less clear. In addition, LasI expressed in Escherichia coli produces a high proportion of unusual AHLs with acyl chains consisting of an odd number of carbons. Furthermore, these studies offer additional methods that will be useful for surveying and quantitating AHLs from different sources.

Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals

“…2F; also see Fig. S1) (18), whereas 3-oxo-C6-HSL and C6-HSL were produced (18,19) in E. coli by the T140A mutant of EsaI enzyme (referred to herein as EsaI). Over 90% (34/36) of the denatonium-responsive cells also showed robust responses to 60-to 120-μM concentrations of the LasI-or EsaI-produced AHLs as well as to the synthetic AHLs ( Fig.…”

“…AHL bacterial signaling molecules were produced in E. coli expressing AHL synthase genes (EsaI and LasI) and were purified using solid-phase extraction procedures as described previously (18,19). The purified extracts were analyzed (Fig.…”

A solo luxI-type gene directs acylhomoserine lactone synthesis and contributes to motility control in the marine sponge symbiont Ruegeria sp. KLH11

“…2a). Quantitative MS, as previously described (Gould et al, 2006;Zan et al, 2012), also failed to detect AHLs in this mutant (Fig. 2b).…”

“…KLH11 derivatives were grown in MB 2216 with appropriate antibiotics (and 0.5 mM IPTG to induce the P lac promoter) at 28 uC to stationary phase (OD 600~2 .0) in the presence of 5 g Amberlite XAD 16 resin l 21 for 36 h. Cells and resin were separated by centrifugation and extracted with 50 ml methanol and dried to 2 ml. Three nanomoles of D 3 -C 6 -HSL (D 3 indicates that there are three deuterium atoms at the terminal position of the acyl chain in the AHL molecule) was added to each sample as an internal standard and a volume of 0.2 ml of each extract was purified using solid phase extraction methods as described previously (Gould et al, 2006). Extracts were dried down and resuspended in 38 ml solvent A (8.3 mM ammonium acetate, pH 5.7) and 2 ml solvent B (methanol).…”

Bacterial Evolution by Intelligent Design

“…Curiously, overproduction of either protein also reduces its AHL specificity. Overproduction of AHL synthases is thought to deplete the cell of the favored acyl-carrier protein substrate, and the enzyme then uses less favored ones (17). Overproduction of several AHL receptors dramatically broadens their substrate specificities, though the reason for this is not clear (18).…”