AidH, an alpha/beta-hydrolase fold family member from an Ochrobactrum sp. strain, is a novel N-acylhomoserine lactonase

Abstract

N-acylhomoserine lactones (AHLs) are signaling molecules in many quorum-sensing (QS) systems that regulate interactions between various pathogenic bacteria and their hosts. Quorum quenching by the enzymatic inactivation of AHLs holds great promise in preventing and treating infections, and several such enzymes have been reported. In this study, we report the characterization of a novel AHL-degrading protein from the soil bacterium Ochrobactrum sp. strain T63. This protein, termed AidH, shares no similarity with any of the known AHL degradases but is highly homologous with a hydrolytic enzyme from Ochrobactrum anthropi ATCC 49188 that contains the alpha/beta-hydrolase fold. By liquid chromatography-mass spectrometry (MS) analysis, we demonstrate that AidH functions as an AHL-lactonase that hydrolyzes the ester bond of the homoserine lactone ring of AHLs. Mutational analyses indicate that the G-X-Nuc-X-G motif or the histidine residue conserved among alpha/beta-hydrolases is critical for the activity of AidH. Furthermore, the AHL-inactivating activity of AidH requires Mn(2+) but not several other tested divalent cations. We also showed that AidH significantly reduces biofilm formation by Pseudomonas fluorescens 2P24 and the pathogenicity of Pectobacterium carotovorum, indicating that this enzyme is able to effectively quench QS-dependent functions in these bacteria by degrading AHLs.

FIG. 1.

Physical map of the aidH gene locus. The single-headed arrows represent the locations and orientations of the genes in the region of the Ochrobactrum sp. T63 chromosome that carries the aidH gene. A 2.3-kb HindIII fragment encoding the AHL inactivation function was inserted into pBluescript II SK(+) and pBBR1MCS-2 to create plasmids p8C-1 and pB8C-1, respectively. The construction of vector p47SΔAidH for deleting the aidH gene was described in Materials and Methods. Abbreviations: E, EcoRI; H, HindIII; EV, EcoRV; S, SalI.

FIG. 2.

Comparison of amino acid sequences of AidH and several alpha/beta-hydrolases. The alignment was generated by DNAMAN. Sections from left to right are the protein, species, and number of amino acids for the gene before the sequences shown. Identities are highlighted in white with a black background, and similarities are shaded gray. The catalytic triad residues are boxed with rectangles. The amino acid residues essential for AHL-degrading activity are indicated by asterisks. AidH_T63, AidH of Ochrobactrum sp. T63 (GenBank accession no. GQ849010); ab_OinLMG3301, alpha/beta-hydrolase fold of O. intermedium (accession no. EEQ96967); ab_OanATCC49188, alpha/beta-hydrolase fold of O. anthropi ATCC 49188 (accession no. YP001369382); ab_MopWSM2075, alpha/beta-hydrolase fold of Mesorhizobium opportunistum WSM2075 (accession no. EEW31886); ab_NwiNB255, alpha/beta-hydrolase fold of Nitrobacter winogradskyi Nb-255 (accession no. YP317111); MhpC_EcoW3110, MhpC from Escherichia coli W3110 (accession no. D86239); BphD_BceLB400, BphD from Burkholderia cepacia LB400 (accession no. X66123); ThnD_SmaTFA, ThnD from Sphingomonas macrogoltabidus TFA (accession no. AF204963); TodF_PpuF1, TodF from Pseudomonas putida F1 (accession no. Y18245).

FIG. 3.

aidH is the sole Ochrobactrum sp. T63 gene involved in AHL-degrading activity. (A) N-(3-Oxooctanoyl)-l-homoserine lactone (OOHL) was incubated with the wild-type, the aidH deletion mutant, or the complementation strain. Culture supernatants were extracted, and the AHLs were detected by the biosensor A. tumefaciens NTL4(pZLR4) as described in Materials and Methods. Error bars denoting standard deviations from three experiments are shown. (B) Plate assay of samples described above (A). The extracts were spotted onto an ABM minimal medium plate seeded with an A. tumefaciens NTL4(pZLR4) culture and X-Gal (40 μg/ml) and incubated at 28°C for 16 h. The control was an OOHL standard (10 pmol).

FIG. 4.

HPLC and ESI-MS spectrometry analysis of the AidH-catalyzed HHL product. (A) HPLC elution profiles of the reaction buffer (top), reaction buffer containing N-hexanoyl-l-homoserine lactone (HHL) (middle), or AidH-digested HHL products in the reaction buffer (bottom). The HHL peak eluted at 25.4 min (middle); the product peak eluted at 22.1 min (bottom). mAU, milli-absorbance unit. (B) HPLC elution profiles of lactonolysis solution (top), reaction solution containing undigested HHL (middle), and the lactonolysis reaction mixture (bottom). Both the lactonolysis product and AidH enzymatically digested HHL appeared with a retention time of 22.1 min. (C) ESI-MS analysis of the hydrolysis product of HHL. The fraction at 22.1 min from HPLC was collected and analyzed by ESI-MS as described in Materials and Methods.

FIG. 5.

The manganese(II) ion is important for the activity of AidH. (A) Effects of different divalent cations on the activity of AidH. E. coli BL21(DE3) cells carrying recombinant AidH were cultivated for 8 h after IPTG induction at 28°C. The indicated metal ions were added to the medium to 1 mM. E. coli BL21(DE3) carrying the vector pET-22b(+) or pET-AidH cultivated without exogenous metal ions was used as a control. Crude cell extracts were incubated with N-(3-oxooctanoyl)-l-homoserine lactone (OOHL) (final concentration, 100 nM) for 1 h at 37°C, extracted with ethyl acetate, and evaporated to dryness. The sample was dissolved in methanol, and the activity of AHLs was measured by using the biosensor A. tumefaciens strain NTL4(pZLR4). (B) EDTA abolishes AidH activity in the presence of Mn²⁺. Mn²⁺ and EDTA added to medium or in cell extracts were at 1 mM and 5 mM, respectively. Control indicates lysate from cells not expressing AidH. AHL activity was measured as described above (A). (C) Effects of different concentrations of Mn²⁺ and Zn²⁺ on the activity of AidH. Mn²⁺ (♦) or Zn²⁺ (□) at the indicated concentration was added to E. coli cultures expressing AidH, and the AHL-degrading activity of the cell extracts was evaluated with the A. tumefaciens biosensor. Error bars indicate standard deviations determined from three independent experiments.

FIG. 6.

Residues in the predicted catalytic triad are important for the enzymatic activity of AidH. (A) Mutations in G100, S102, G104, or H248 abolished AidH activity. The crude cell extracts of bacterial strains expressing the indicated AidH mutants were incubated with 100 nM N-(3-oxooctanoyl)-l-homoserine lactone (OOHL) for 1 h at 37°C and extracted with ethyl acetate, and AHL activity was detected by the A. tumefaciens biosensor. For samples, T63 indicates wild-type Ochrobactrum strain T63, pET-22b(+) indicates E. coli BL21(DE3) carrying pET-22b(+), and pET-AidH indicates E. coli BL21(pET-AidH). G100V, S102V, G104V, E219T, and H248S are substitution mutants of AidH in the indicated amino acids. The experiment was repeated three times, and data shown are means of three replicates. (B) Substitution mutants of AidH code for stable proteins. Total cellular proteins of IPTG-induced E. coli strains harboring each mutant on pET-22b(+) were analyzed by SDS-PAGE.

FIG. 7.

Effect of AidH on phenotypes of Pseudomonas fluorescens 2P24 and P. carotovorum subsp. carotovorum. Vectors or their derivatives expressing AidH were introduced into Pseudomonas fluorescens 2P24 and P. carotovorum subsp. carotovorum, and the resulting strains were tested for extracellular AHL accumulation (A and C), biofilm formation (B), and pathogenicity (D). AHLs produced by bacterial strains were evaluated for their abilities to activate traG on A. tumefaciens biosensor strain NTL4(pZLR4), measured by the expression of a TraR-dependent LacZ fusion. The activity of β-galactosidase is expressed in Miller units. The formation of biofilm in Eppendorf tubes was evaluated by crystal violet staining as described previously by Wei and Zhang (56). Error bars indicate standard deviations of data from three experiments. (D) Radish, potato, and Chinese cabbage were inoculated with 5 μl of bacterial culture (2 × 10⁹ CFU/ml) of Z3-3, Z3-3(pBBR1MCS-2) (vector), and Z3-3(pB8C-1), respectively. The development of disease symptoms was documented by photographing the inoculated plant tissues 48 h (radish and potato) or 5 days (Chinese cabbage) after inoculation. Similar results were obtained in multiple independent experiments, and images shown are representative of one experiment.