Acyl homoserine-lactone quorum-sensing signal generation

Abstract

Acyl homoserine lactones (acyl-HSLs) are important intercellular signaling molecules used by many bacteria to monitor their population density in quorum-sensing 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.

Figure 1

Purification of RhlI from clarified cell extracts. (A) HiTrap Q column chromatography. (B) HiTrap S column chromatography. (C) Superdex 75 column chromatography. ○, RhlI activity (nmol of butyryl-HSL produced min⁻¹ in each fraction); ●, protein levels. (D) SDS/PAGE of RhlI activity peaks. Lane 1, molecular mass standards (prestained low-range markers, Bio-Rad); lane 2, 30 μg of cell extract; lane 3, 35 μg of protein from pooled HiTrap Q fractions 3–8; lane 4, 30 μg of protein from pooled HiTrap S fractions 5–8; lane 5, 1.4 μg of protein from pooled Superdex 75 fractions 13–15. The numbers to the left indicate the molecular mass of the standard. The arrow indicates RhlI.

Figure 2

Substrate initial velocity patterns with SAM and butyryl-ACP. A double reciprocal plot with butyryl-ACP as the varied substrate at SAM concentrations of 61, 27, 10, and 4 μM.

Figure 3

HPLC analysis of acyl-HSLs synthesized by purified RhlI. Reaction mixtures contained saturating levels of SAM and 40 μM of both butyryl-ACP and hexanoyl-ACP. Synthetic butyryl-HSL was eluted in peak 7 and synthetic hexanoyl-HSL in peaks 21 and 22.

Figure 4

Analysis of inhibition kinetics. Double-reciprocal plots of product and dead-end inhibitors vs. the substrates butyryl-ACP and SAM. (A) Inhibition pattern with the product inhibitor MTA vs. varied concentrations of the substrate SAM with butyryl-ACP held at a nonsaturating concentration. (B) Inhibition pattern with the product inhibitor MTA vs. butyryl-ACP with SAM held at a nonsaturating concentration. (C) Inhibition pattern with the product inhibitor ACP vs. the substrate SAM with butyryl-ACP held at a nonsaturating concentration. (D) Inhibition pattern with the product inhibitor ACP vs. the substrate butyryl-ACP with SAM held at a nonsaturating concentration. (E) The inhibition pattern with differing amounts of the dead-end inhibitor S-adenosylhomocysteine vs. varied concentrations of the substrate SAM with butyryl-ACP held at nonsaturating levels.

Figure 5

The proposed enzymatic reaction mechanism for autoinducer synthesis by RhlI. Bound substrates and products are in parentheses. This is a bi ter sequential ordered reaction in which the substrates bind in a defined order and the products are released in a defined order.