A Novel Screening System to Characterize and Engineer Quorum Quenching Lactonases

Abstract

N-acyl l-homoserine lactones are signaling molecules used by numerous bacteria in quorum sensing. Some bacteria encode lactonases, which can inactivate these signals. Lactonases were reported to inhibit quorum sensing-dependent phenotypes, including virulence and biofilm. As bacterial signaling is dependent on the type of molecule used, lactonases with high substrate specificity are desirable for selectively targeting species in communities. Lactonases characterized from nature show limited diversity in substrate preference, making their engineering appealing but complicated by the lack of convenient assays for evaluating lactonase activity. We present a medium-throughput lactonase screening system compatible with lysates that couples the ring opening of N-acyl l-homocysteine thiolactones with 5,5-dithio-bis-(2-nitrobenzoic acid) to generate a chromogenic signal. We show that this system is applicable to lactonases from diverse protein families and demonstrate its utility by screening mutant libraries of GcL lactonase from Parageobacillus caldoxylosilyticus. Kinetic characterization corroborated the screening results with thiolactonase and homoserine lactonase activity levels. This system identified GcL variants with altered specificity: up to 1900-fold lower activity for long-chain N-acyl l-homoserine lactone substrates and ~38-fold increase in preference for short-chain substrates. Overall, this new system substantially improves the evaluation of lactonase activity and will facilitate the identification and engineering of quorum quenching enzymes.

Keywords: lactonase; medium throughput screening; quorum quenching; quorum sensing; thiolactone.

Figure 1

N‐acyl l‐homocysteine thiolactones hydrolysis assay. Chemical structures of (A) N‐acyl l‐homoserine lactones and (B) N‐acyl l‐homocysteine thiolactones. (C) Reaction scheme of the colorimetric assay system to measure lactonolysis of N‐acyl l‐homocysteine thiolactone using 5,5‐dithio‐bis‐(2‐nitrobenzoic acid) (DTNB).

Figure 2

Kinetics of N‐acyl l‐homocysteine thiolactone hydrolysis. (A) Real‐time increase in absorbance at 412 nm on hydrolysis of N‐acetyl homocysteine thiolactone at pH 8.0. (B) pH profile of the assay system with GcL and N‐butyryl l‐homocysteine thiolactone (red) or N‐octanoyl l‐homocysteine thiolactone (green). Shaded areas represent standard deviation (n = 3). (C–E) Kinetic curves for GcL against homocysteine thiolactone substrates: (C) N‐acetyl D‐/l‐homocysteine thiolactone, (D) N‐butyryl l‐homocysteine thiolactone, (E) N‐octanoyl l‐homocysteine thiolactone. Corresponding Michaelis–Menten curves for other lactonases can be found in the Supporting Information S1: Figures S1–S4. For substrates which did not fit the Michaelis–Menten equation, a linear regression was generated to determine catalytic efficiency. (F) N‐acetyl homocysteine thiolactonase activity in cell lysates overexpressing the lactonase GcL as reported by DTNB and recorded at 412 nm with lysates expressing active lactonase (GcL, blue), inactive lactonase (SsoPox 5A8, red). Shaded regions represent standard deviation (n = 8).

Figure 3

Substrate binding cleft of the GcL lactonase enzyme. Structure of GcL (PDB: 6N9R) bound to 3OC12‐HSL (yellow sticks) showing the active site metals (spheres; α: cobalt cation, β: iron cation) and the side chains of the amino acid residues lining the substrate binding cleft (sticks). Residues in the vicinity of the lactone ring are shown in green; residues interacting with the acyl chain are in magenta, and residues lining the cleft exposed to the solvent are in indigo. Residues mutated in this study are bolded, underlined, and highlighted in red circles. The flexible loop section between N152 and A157 that carries G156 is highlighted as an opaque cyan cartoon. The surrounding protein environment is shown as transparent cyan cartoon.

Figure 4

Heat maps showing normalized activity and substrate preference of GcL W26 (A) & F87 (B) saturation libraries. The color gradient of red–yellow–green reflects the normalized activity of each variant relative to the WT, with red representing the lowest activity and green representing WT‐like activity. Each row shows a different variant and each column represents a HTL substrate with a different length acyl‐chain. The color gradient of purple–white–orange shows changes in substrate preference relative to WT by calculating activity ratios between the different substrates. Purple indicates a shift in favor of longer‐chain HTLs, orange indicates a shift in favor of shorter‐chain HTLs, and white shows WT‐like substrate preference. Variants with extremely low activity (< 5% relative to WT) were omitted from substrate preference calculations and are shown in gray.

Figure 5

Heat maps representing changes in normalized activity level and substrate preferences for GcL M86 (A) and GcL G156 saturation libraries (B). The red‐green scale represents activity relative to the WT (green) while the purple‐orange scale represents preference changes for long (purple) or short (orange) HTL substrates based on activity ratios between substrates. Gray boxes denote values not calculated due to low activity (< 5% of the WT).