Mining Marine Metagenomes Revealed a Quorum-Quenching Lactonase with Improved Biochemical Properties That Inhibits the Food Spoilage Bacterium Pseudomonas fluorescens

Abstract

The marine environment presents great potential as a source of microorganisms that possess novel enzymes with unique activities and biochemical properties. Examples of such are the quorum-quenching (QQ) enzymes that hydrolyze bacterial quorum-sensing (QS) signaling molecules, such as N-acyl-homoserine lactones (AHLs). QS is a form of cell-to-cell communication that enables bacteria to synchronize gene expression in correlation with population density. Searching marine metagenomes for sequences homologous to an AHL lactonase from the phosphotriesterase-like lactonase (PLL) family, we identified new putative AHL lactonases (sharing 30 to 40% amino acid identity to a thermostable PLL member). Phylogenetic analysis indicated that these putative AHL lactonases comprise a new clade of marine enzymes in the PLL family. Following recombinant expression and purification, we verified the AHL lactonase activity for one of these proteins, named moLRP (marine-originated lactonase-related protein). This enzyme presented greater activity and stability at a broad range of temperatures and pH, tolerance to high salinity levels (up to 5 M NaCl), and higher durability in bacterial culture, compared to another PLL member, parathion hydrolase (PPH). The addition of purified moLRP to cultures of Pseudomonas fluorescens inhibited its extracellular protease activity, expression of the protease encoding gene, biofilm formation, and the sedimentation process in milk-based medium. These findings suggest that moLRP is adapted to the marine environment and can potentially serve as an effective QQ enzyme, inhibiting the QS process in Gram-negative bacteria involved in food spoilage. IMPORTANCE Our results emphasize the potential of sequence and structure-based identification of new QQ enzymes from environmental metagenomes, such as from the ocean, with improved stability or activity. The findings also suggest that purified QQ enzymes can present new strategies against food spoilage, in addition to their recognized involvement in inhibiting bacterial pathogen virulence factors. Future studies on the delivery and safety of enzymatic QQ strategy against bacterial food spoilage should be performed.

Keywords: Pseudomonas fluorescens; bacterial food spoilage; biofilm; enzyme characterization; exoprotease activity; metagenomics; quorum-quenching lactonase; quorum-sensing.

FIG 1

Evolutionary relations of previously characterized phosphotriesterase-like lactonases and the newly identified homologs from metagenomics libraries. Phosphotriesterase (PTE) homologs were used as an outgroup. The evolutionary history was inferred using the neighbor-joining method. The optimal tree with the sum of branch length equal to 5.56804021 is shown. Percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches. The tree is drawn to scale; branch lengths are in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method; the units are the number of amino acid substitutions per site. The analysis involved 23 amino acid sequences. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA X. The newly identified marine-originated lactonase-related protein (moLRP) is indicated with a star, and marine-originated related protein (moRP) is indicated with an oval. PPH, putative parathion hydrolase; GKL, Geobacillus kaustophilus lactonase; and AhlA, acyl-homoserine lactonase A.

FIG 2

Sequence alignment of previously characterized phosphotriesterase-like lactonases (PLLs) and the newly identified homologs from marine metagenomes and the structural overlay of a model of moLRP and a thermostable quorum-quenching lactonase from Geobacillus kaustophilus GKL (PDB 3ojg). (A) The new marine PLLs (moLRP and moRP, first two sequences) aligned well with the PLL family using the MUSCLE algorithm. The previously characterized PLLs are SsoPox from Sulfolobus solfataricus (WP 009988477.1), SacPox from Sulfolobus acidocaldarius (WP 011278935.1), SisLac from Sulfolobus islandicus (WP 012710474.1), previously characterized parathion hydrolase (PPH) from Mycobacterium tuberculosis (ACF57854.1), acyl-homoserine lactonase A (AhlA) from Rhodococcus erythropolis (ACF57853.1), G. kaustophilus lactonase (GKL) from G. kaustophilus, and PTE from Pseudomonas diminuta (1HZY). (B) The backbone of the homology modeling of moLRP appears in blue, the solved structure of GKL is in pink, and N-butyryl-dl-homoserine lactone is in yellow (PDB 3ojg). The active site metal atoms (yellow spheres) and N-butyryl-dl-homoserine lactone are in the active site of lactonase from GKL. (C) Overlay of the binuclear catalytic center. Five of the zinc-ligating residues (His23, His25, His178, His266, and Asp301) align perfectly.

FIG 3

The newly identified moLRP is an N-acyl-homoserine lactonase. Michaelis-Menten analysis of the lactonase activity of moLRP with C4-HSL and C8-oxo-HSL was performed at E₀ = 0.3 μM, pH 8 at 27°C. AHL, N-acyl-homoserine lactone.

FIG 4

The biochemical properties of moLRP indicate that it is active and stable over a wide range of temperature, pH, and salinity levels. (A to C) Radar charts of the relative activity of moLRP and PPH at different temperature, pH, and salinity levels. (D to F) Radar charts of the residual activity of moLRP and PPH after 1 h of incubation of the enzymes in various conditions of temperature, pH, and salinity and then testing activity at optimal conditions. Enzyme concentrations are as follows: in panels B, C, and F, 0.3 μM moLRP were used. In panels A, D, and E, 0.025, 0.15, and 2.5 μM moLRP were used, respectively. In panels A and D, 1 μM PPH was used: in panels C and F, 2 μM PPH was used. In panels B and E, 1.75 and 1.4 μM PPH were used, respectively.

FIG 5

moLRP maintains its activity for 24 h in bacterial culture and presents higher inhibition than PPH of Pseudomonas fluorescens of extracellular proteolytic activity and of biofilm formation. (A) Enzyme activity (mOD/min) measured in supernatants of P. fluorescens culture (grown at 28°C, 170 rpm) following the exogenous addition of purified enzymes (1 μM) PPH or moLRP. Activity was measured with 0.05 mM TBBL. (B) Biofilm formation of P. fluorescens cultures in the presence of purified enzymes. Biofilm formation was quantified by crystal violet assay at an optical density at 590 nm (OD₅₉₀). The data are presented as means ± SD (control n = 5, PPH/moLRP n = 3). Statistical significance is according to one-way analysis of variance (ANOVA). ns, nonsignificant; ****, P < 0.001. P. fluorescens culture alone was used as a control. (C) Extracellular proteinase activity of P. fluorescens (skim-milk medium) in the presence of purified enzymes. Proteinase activity was quantified using azocasein assay at OD₄₄₀. The data are presented as means ± SD (n = 3). Statistical significance is according to one-way ANOVA. ****, P < 0.0001. P. fluorescens alone was used as a control. (D) Expression levels of aprX (proteinase gene) in cultures in the presence of purified moLRP and its activity buffer, as control. The transcription of QS-regulated gene aprX was normalized to the housekeeping gene 16S rRNA. Data points represent the means of three biological replicates ± standard error. Statistical significance is according to one-way ANOVA comparison (Tukey’s multiple-comparison test) (P value < 0.05*). (E) Growth curve measured for 69 h. Shown are the results of cultures of P. fluorescens alone, with enzyme activity buffer or with purified (1 μM) moLRP, grown in 200 μL LB media in 96-well plates to enable absorbance detection at OD₆₀₀.

FIG 6

moLRP inhibits sedimentation in P. fluorescens-treated milk-based cultures. (A) Skim-milk cultures of P. fluorescens at 28°C incubated with or without purified enzymes. Skim milk without bacteria as a negative control (sample a), P. fluorescens alone (sample b), P. fluorescens with 1 μM wtPPH (sample c), and P. fluorescens with 1 μM moLRP (sample d). The pictures were taken after 4 days. (B) Light transmission of bacterial cultures over time measured by LUMisizer analytical centrifuge. The graph presents the integrated light transmission of the upper 20% of each test tube with bacterial cultures against time. Bacterial cultures were incubated with and without purified moLRP. The data are presented as means ± SD (n = 3). Milk medium alone was used as a negative control. (C) Mean values of the integrated light transmission of the upper 20% of test tubes of bacterial cultures after 6 h. Statistical significance is according to one-way ANOVA. ****, P < 0.0001.