Environmental Microbiology

AidB, a Novel Thermostable N-Acylhomoserine Lactonase from the Bacterium Bosea sp.

Jun-Wei Zhang, Chen-Guang Xuan, Can-Hua Lu, Song Guo, Jin-Feng Yu, Muhammad Asif, Wen-Jun Jiang, Zhi-Gang Zhou, Zhao-Qing Luo, Li-Qun Zhang
Rebecca E. Parales, Editor
DOI: 10.1128/AEM.02065-19

ABSTRACT

Many Gram-negative bacteria employ N-acylhomoserine lactones (AHLs) as quorum-sensing (QS) signal molecules to regulate virulence expression in a density-dependent manner. Quorum quenching (QQ) via enzymatic inactivation of AHLs is a promising strategy to reduce bacterial infections and drug resistance. Herein, a thermostable AHL lactonase (AidB), which could degrade different AHLs, with or without a substitution of carbonyl or hydroxyl at the C-3 position, was identified from the soil bacterium Bosea sp. strain F3-2. Ultrahigh-performance liquid chromatography analysis demonstrated that AidB is an AHL lactonase that hydrolyzes the ester bond of the homoserine lactone (HSL) ring. AidB was thermostable in the range 30 to 80°C and showed maximum activity after preincubation at 60°C for 30 min. The optimum temperature of AidB was 60°C, and the enzyme could be stably stored in double-distilled water (ddH2O) at 4°C or room temperature. AidB homologs were found only in Rhizobiales and Rhodospirillales of the Alphaproteobacteria. AidB from Agrobacterium tumefaciens and AidB from Rhizobium multihospitium (with amino acid identities of 50.6% and 52.8% to AidB, respectively) also showed thermostable AHL degradation activity. When introduced into bacteria, plasmid-expressed AidB attenuated pyocyanin production by Pseudomonas aeruginosa PAO1 and the pathogenicity of Pectobacterium carotovorum subsp. carotovorum Z3-3, suggesting that AidB is a potential therapeutic agent by degrading AHLs.

IMPORTANCE A quorum-sensing system using AHLs as the signal in many bacterial pathogens is a critical virulence regulator and an attractive target for anti-infective drugs. In this work, we identified a novel AHL lactonase, AidB, from a soil bacterial strain, Bosea sp. F3-2. The expression of aidB reduced the production of AHL signals and QS-dependent virulence factors in Pseudomonas aeruginosa and Pectobacterium carotovorum. The homologs of AidB with AHL-degrading activities were found only in several genera belonging to the Alphaproteobacteria. Remarkably, AidB is a thermostable enzyme that retained its catalytic activity after treatment at 80°C for 30 min and exhibits reliable storage stability at both 4°C and room temperature. These properties might make it more suitable for practical application.

INTRODUCTION

Quorum sensing (QS) is a cell-cell communication mechanism that relies on the production, secretion, and detection of small-molecule signals to regulate gene expression in response to changes in population density (1). Many Gram-negative bacteria use one or more derivatives of N-acylhomoserine lactone (AHL) as QS signals, which share the homoserine lactone (HSL) ring but differ with respect to their lengths (C4 to C18) and the C-3 substitution of the acyl side chain (carbonyl and hydroxy moieties) (2, 3). AHL-dependent QS systems in bacteria regulate a variety of biological functions, such as bioluminescence in Vibrio fischeri (4), the production of virulence factors in Pseudomonas aeruginosa and Pectobacterium carotovorum (5), the CRISPR-Cas system-mediated adaptive immunity in Serratia sp. (6), biofilm formation and protease production in Aeromonas hydrophila (7), and conjugative transfer of the Ti plasmid in Agrobacterium tumefaciens (8). Some of these biological functions are essential for bacterial pathogenicity (2). Disruption or interference with the QS system, which is known as quorum quenching (QQ), is therefore a potential strategy to prevent bacterial infection. QQ enzymes have been found in different microorganisms, including bacteria, archaea, phages, and fungi (9, 10) and even in plants, mammals, and coelenterate cells, suggesting that convergent evolution events may have occurred between the host and the pathogen (11–13).

QQ enzymes are classified into three types according to their enzymatic mechanisms: AHL lactonases, AHL acylases, and AHL oxidoreductases. AHL lactonases cleave the ester bond of the homoserine lactone ring of AHLs, producing the corresponding N-acyl-homoserine. AHL acylases cleave the amide bond of AHLs, producing a fatty acid and homoserine lactone. Unlike lactonases and acylases, AHL oxidoreductases inactivate AHLs by oxidizing or reducing hydrogen atoms on their side chains (14). AHL lactonases are well studied in a wide variety of organisms and belong to different protein families (metallo-β-lactamase superfamily, alpha/beta hydrolase superfamily, phosphotriesterase family, and paraoxonase family) (14). AiiA from Bacillus sp. strain 240B1, the first identified AHL-degrading enzyme, is a member of the metallo-β-lactamase superfamily and is conserved in Bacillus species (15). The amino acid sequence of AiiA and its homologs contain a conserved HXHXDH-H zinc-binding motif, in which the conserved aspartate and histidine residues are required for AHL-degrading activity. Tobacco tissues expressing AiiA quenched bacterial QS signaling and showed significantly enhanced resistance to Erwinia carotovora infection (15, 16). To date, more than 20 metallo-β-lactamase superfamily AHL-degrading enzymes have been identified from different bacteria (14, 17). These lactonases exhibit a broad AHL degradation spectrum but differ in their enzymatic characteristics. For example, AiiT from Thermaerobacter marianensis JCM 11223 and AhlS from Solibacillus silvestris StLB098 share a high amino acid identity (59.4%), but AiiT exhibits a higher optimal temperature and thermostability than AhlS (18, 19).

Bosea thiooxidans TRT31, which was isolated from tobacco rhizospheres, has been identified as an AHL-degrading bacterium (20). However, the gene responsible for AHL degradation remains unknown. In the present study, we identified the aidB gene, which encodes a novel AHL lactonase, from Bosea sp. strain F3-2. The mechanism and biochemical properties of AidB were characterized systematically. The QQ activities of AidB in the plant-associated bacterium Pectobacterium carotovorum and human-associated bacterium Pseudomonas aeruginosa were investigated.

RESULTS

Isolation and identification of Bosea strains capable of degrading AHLs.In a large-scale screening of soil bacteria using Gavrish et al.’s in situ cultivation method (21), 68 bacterial strains with AHL-degrading activity were obtained from approximately 4,000 isolates. Among them, two strains, F3-2 and 12C, showed similar morphological characteristics. Sequence analysis of 16S rRNA genes indicated that F3-2 showed 97.9% to 99.5% nucleotide identity to type strains in the Bosea genus and that 12C showed 99.9% nucleotide identity to Bosea thiooxidans DSM 9653T (see Fig. S1 in the supplemental material). We therefore designated these two strains Bosea sp. strain F3-2 and Bosea sp. strain 12C, respectively. Strain F3-2, which was able to completely eliminate AHL activity at a low density (optical density at 600 nm [OD600] ≈ 0.5) within 2 h (Fig. 1), was chosen for further study.

FIG 1
FIG 1

Detection of the AHL-degrading activity of Bosea sp. F3-2. Individual components of F3-2 culture and LB medium were treated at 90°C or at room temperature (RT) for 30 min, and then 50 nM 3-oxo-C8-HSL was added as a substrate and the mixture was incubated for at 30°C for 2 h. The residual AHLs were qualitatively (A) and quantitatively (B) analyzed using AHL reporter strain A. tumefaciens NTL4(pZLR4). The AHL-degrading activity of Bosea sp. F3-2 culture incubated at RT for 30 min was defined as 100% relative activity. Statistical significance was calculated by a Student t test comparing the AHL-degrading activity of F3-2 culture at 90°C with that at RT (**, P < 0.01; ns, no significant difference).

To clarify the properties of the QQ determinant in F3-2, bacterial cells and the culture supernatant were separated by centrifugation. Resuspended cells exhibited high AHL degradation activity, whereas the filter-sterilized supernatant showed no AHL degradation activity. Heat treatment (90°C for 30 min) of the bacterial culture broth and cell pellet completely abolished their AHL degradation activities (Fig. 1). These results suggested that strain F3-2 is most likely to quench the QS signals via an enzymatic activity.

Cloning and identification of the QQ gene in Bosea sp. F3-2.We first tried a screening for QQ genes in a cosmid library of Bosea sp. F3-2 genome DNA constructed in Escherichia coli DH5α, but no positive clone was obtained from approximately 3,000 transformants, representing about 14× F3-2 genome coverage. Then, strain F3-2 was subjected to genome sequencing and gained the 7,043,791 bp of the whole-genome sequence, encoding 6,529 proteins. However, no gene was annotated as a homolog of AHL-degrading enzymes. We thus screened the genes encoding conserved motifs of known AHL lactonases, including 12 genes annotated as metallo-β-lactamase superfamily proteins with conserved zinc-binding motif HXHXDH-H and 19 genes annotated as alpha/beta hydrolase superfamily proteins with a conserved motif, G-X-Nuc-X-G, where “Nuc” is a nucleophile. Candidate genes were fused in-frame to a His6 tag under the control of the IPTG (isopropyl-β-D-thiogalactopyranoside)-inducible lac promoter. The recombinant strain E. coli BL21(pET-22b-05624) could completely degrade 50 nM 3-oxo-C8-HSL in 2 h (see Fig. S2 in the supplemental material). Gene 05624 (FQV39_28175), annotated as coding for a metallo-β-lactamase superfamily protein of 272 amino acids, was verified to encode an AHL-degrading enzyme and designated aidB (autoinducer-degrading gene from Bosea sp.).

The aidB gene encodes an AHL lactonase.The AidB-His6 fusion protein was expressed from the recombinant plasmid pET-22b-AidB. The purified His6-AidB protein had an apparent molecular mass of approximately 30 kDa upon 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 2A), which was consistent with its predicted molecular weight, and showed a degradation capacity toward a wide range of AHLs, including C6-HSL, 3-oxo-C6-HSL, 3-oxo-C8-HSL, 3-OH-C8-HSL, C10-HSL, C12-HSL, and 3-oxo-C12-HSL (Fig. 2B).

FIG 2
FIG 2

Mechanism of action of AidB. (A) Purified AidB-His6 was analyzed using SDS-PAGE and an 8% polyacrylamide gel. (B) Degradation spectrum of AidB. The reaction mixture containing 0.5 μg ml−1 AidB and individual AHLs (1,000 μM C6-HSL, 10 μM 3-oxo-C6-HSL, 0.25 μM 3-oxo-C8-HSL, 1 μM 3-OH-C8-HSL, 10 μM C10-HSL, 10 μM C12-HSL, and 1 μM 3-oxo-C12-HSL) in 50 mM Tris-HCl buffer (pH 8.0) were incubated at 30°C for 30 min. The residual AHLs were analyzed using A. tumefaciens NTL4(pZLR4). The activity of AidB toward C6-HSL was defined as 100% relative activity. (C) UPLC analysis of the enzymatic product of AidB. The substrate C6-HSL and its products hydrolyzed by 10 mM NaOH and by AidB were analyzed by UPLC as described in Materials and Methods. The retention times of C6-HSL and N-hexanoyl-homoserine (C6-HS) were 14.70 min and 12.37 min, respectively. The peaks corresponding to C6-HSL and C6-HS are indicated by arrows. mAu, arbitrary units (in thousands).

The mechanism of AidB was determined using C6-HSL as the substrate. Product analysis using reverse-phase ultrahigh-performance liquid chromatography (UPLC) identified a new peak with the same retention time (12.37 min) as the ring-opening product N-hexanoyl-homoserine generated by NaOH treatment (Fig. 2C) (22). When AidB was incubated with 2 mM C6-HSL at 30°C for 3 h or 4 h, 59.8% or 87.3% of the C6-HSL was hydrolyzed, respectively. However, C6-HSL treated with heat-denatured AidB protein shared the peak of the retention time (14.70 min) with the standard C6-HSL (Fig. 2C). The same results were obtained with different elution conditions (data not shown). These results suggested that aidB encodes an AHL lactonase that hydrolyzes the ester bond of the homoserine lactone ring of AHLs.

Characterization of AidB enzymatic activity.As a new member of the AHL lactonases, the environmental influences on AidB activity were carefully checked. A surprising finding was that AidB is a thermostable enzyme. Purified AidB (final concentration, 4 μg ml−1) retained high degradation activity after preincubation at temperatures from 30 to 80°C. The highest AHL degradation activity was observed after preincubation at 60°C for 30 min, and 93.0% of its maximal activity was retained after preincubation at 80°C for 30 min (Fig. 3A). In contrast, the degradation activity of the AHL lactonase from Bacillus sp. strain 240B1 (AiiA240B1) continuously declined with the increase of preincubation temperature, and only 19.2% of its maximal activity was found after preincubation at 70°C for 30 min (Fig. 3A). Similarly, high temperature was also required under the optimal conditions for the enzymatic reaction. The optimal temperature of AidB to degrade 3-oxo-C8-HSL was 60°C. AidB maintained a high degradation activity at temperatures from 0 to 80°C and then markedly decreased from 80 to 90°C. In contrast, the activity of AiiA240B1 was maximal at 40°C and decreased from 40 to 90°C (Fig. 3B).

FIG 3
FIG 3

Characteristics of AidB. (A) Thermostability. AidB or AiiA240B1 protein was preincubated at temperatures from 30 to 100°C for 30 min before the assay. After the proteins cooled, the AHL degradation activities were determined by incubating the heat-treated proteins with 3-oxo-C8-HSL at 30°C for 30 min. (B) Optimal temperatures. The optimal temperatures for AHL degradation were determined by incubating the proteins with 3-oxo-C8-HSL at temperatures from 0 to 90°C for 30 min. (C) Storage stability. AidB or AiiA240B1 protein was stored in ddH2O at 4°C or room temperature (RT), respectively. The AHL degradation activities were determined every 24 h by incubating the proteins with 3-oxo-C8-HSL at 30°C for 30 min. The residual AHLs were analyzed with A. tumefaciens NTL4(pZLR4), and the activity of AidB toward 3-oxo-C8-HSL at 30°C for 30 min was defined as 100% relative activity.

AidB also showed promising storage stability. Compared with AiiA240B1, AidB retained full AHL degradation activity on the 15th day of storage at 4°C and room temperature. However, the degradation activity of AiiA240B1 continuously declined from the 1st day under the same conditions (Fig. 3C).

AidB disrupts QS-mediated functions in Pseudomonas aeruginosa and Pectobacterium carotovorum subsp. carotovorum.The effect of AidB on virulence factor production in Pseudomonas aeruginosa PAO1 was evaluated. The growth of Pseudomonas aeruginosa PAO1 was not detectably affected by the expression of the plasmid-borne aidB gene (Fig. 4A), whereas the C4-HSL and 3-oxo-C12-HSL productions of Pseudomonas aeruginosa PAO1(pBBR1MCS2-AidB) were significantly decreased compared with those of the control strain, Pseudomonas aeruginosa PAO1(pBBR1MCS-2) (Fig. 4B and C), indicating the degradation of AHLs by AidB. The pyocyanin production and swarming motility in strain PAO1, containing the aidB gene, were all decreased significantly (Fig. 4D to F), suggesting a reduction in virulence factor production caused by AHL degradation.

FIG 4
FIG 4

Effects of AidB on QS-dependent functions in Pseudomonas aeruginosa PAO1 and Pectobacterium carotovorum subsp. carotovorum Z3-3. Strains PAO1 and Z3-3 containing pBBR1MCS2-AidB (with the empty plasmid pBBR1MCS-2 as a control) were used to test growth and extracellular AHL accumulation (A, B, C, and G), production of pyocyanin (D and E), swarming (F), and pathogenicity on Chinese cabbage and potato tissue (H). (A, B, C, and G) The growth and AHL production of PAO1 or Z3-3 derivative strains were tested by the methods described in Materials and Methods. Statistical significance was calculated by the Student t test in a comparison of the C4-HSL (B) and 3-oxo-C12-HSL (C) production of strain PAO1(pBBR1MCS2-AidB) with that of the control strain, PAO1(pBBR1MCS-2) (**, P < 0.01). (D) Pyocyanin productions of PAO1 derivative strains were tested every 3 h with the methods described in Materials and Methods. Statistical significance was calculated by the Student t test in a comparison of the pyocyanin production of strain PAO1(pBBR1MCS2-AidB) with that of the control strain, PAO1(pBBR1MCS-2) (*, P < 0.05; **, P < 0.01). (E) Image of pyocyanin production of PAO1 derivative strains, taken after inoculation at 37°C for 24 h. (F) For swarming assays, 2 μl of fresh bacterial suspension was inoculated in the central portion of the swarming plates and then statically incubated at 37°C for 24 h. (H) For pathogenicity assays, 5 μl fresh bacterial suspension was inoculated on the wound sites of potato or Chinese cabbage leaves and incubated at 28°C for 48 h (for potato) or 72 h (for Chinese cabbage) in crispers moisturized with wet filter papers.

The pathogenicity factors of the plant-pathogenic bacterium Pectobacterium carotovorum are expressed in a QS-dependent way, mediated by C6-HSL and 3-oxo-C6-HSL (23). Expression of the plasmid-borne aidB gene in Pectobacterium carotovorum subsp. carotovorum Z3-3 did not affect bacterial growth (Fig. 4G) but significantly reduced the AHL concentration in the culture supernatant compared with that in the control strain containing the empty plasmid (Fig. 4G). Accordantly, AidB significantly reduced the pathogenicity of strain Z3-3 on tissues of Chinese cabbage and potato (Fig. 4H and Table S1). These results indicated that AidB functions as an AHL-degrading enzyme in both Pseudomonas and Pectobacterium and could quench QS-dependent functions in these bacteria.

AidB is a new subclass of the AHL lactonase.The amino acid sequence of AidB showed very low identity to all known AHL lactonases. The most similar is that of AhlS (GenBank accession no. BAK14506) from Solibacillus silvestris StLB046 (18), which has 22.3% identity to AidB. The well-studied AHL lactonase AiiA (GenBank accession no. AAF62398) from Bacillus sp. 240B1 (15) has 21.7% identity (Fig. S3). Phylogenetic analysis of all known QQ enzymes indicted that AidB belongs to a new clade of the metallo-β-lactamase superfamily of AHL lactonases, which is distant from the other known lactonases (Fig. 5A).

FIG 5
FIG 5

Phylogenetic analysis of AidB. (A) Phylogenetic tree based on the amino acid sequences of metallo-β-lactamase (MBL) superfamily members, including AidB, AidB12C, AidBRm, AidBAt, AaL (30), GcL (31), AhlS (18), AiiT (19), AiiB (49), AhlD (50), AiiA (15), QlcA (24), PONX_OCCAL (12), SsoPox (33), MCP (51), QsdA (52), AiiM (53), AidH (54), and Aii810 (55). (B) Phylogenetic analysis of AidB and its highly homologous MBL fold metallo-hydrolases. All the amino acid sequences used were obtained from the NCBI database. The dendrograms were constructed after ClustalW alignment using the neighbor-joining and Kimura two-parameter methods and were subjected to 1,000 bootstrap trials with MEGA 5.05. The proteins in bold were confirmed to have AHL-degrading activities.

A BLASTp search of the NCBI nonredundant protein sequences database revealed a group of metallo-β-lactamase fold hydrolase proteins with high amino acid identities (76.9 to 87.9%) with AidBs from Bosea and Methylobacterium genera, and a number of metallo-β-lactamase superfamily proteins in Rhizobiales and Rhodospirillales of Alphaproteobacteria have significant identities (49.6 to 56.8%) with AidB (Fig. 5B). Phylogenetic analysis further supported the above finding and suggested that AidB homologs are conserved across the Bosea, Rhizobium, Agrobacterium, Roseomonas, and Methylobacterium genera of the Alphaproteobacteria. To identify the AHL-degrading activities of these AidB homologs, two genes encoding proteins with lower identities to AidB were cloned. AidBs from Agrobacterium tumefaciens C58 (AidBAt) and from Rhizobium multihospitium CCBAU 83401T (AidBRm) show amino acid identities of 50.6% and 52.8% to AidB, respectively, and the identity between AidBAt and AidBRm is 50.9%. Another aidB gene (whose product shows 87.5% amino acid identity to AidB) from Bosea sp. strain 12C was also cloned to test its AHL-degrading activity.

The purified AidB homologs (Fig. S4) showed different degrading activities of 3-oxo-C8-HSL under the same conditions. When the purified proteins were applied at concentrations of 3.125 μg ml−1, 6.25 μg ml−1, 12.5 μg ml−1, and 25 μg ml−1, AidB-His6 and AidB12C-His6 completely degraded 50 nM 3-oxo-C8-HSL at 28°C in 2 h, while AidBAt-His6 and AidBRm-His6 degraded only part of 3-oxo-C8-HSL. The AHL-degrading activity of AidBAt-His6 was higher than that of AidBRm-His6 at the same concentrations (Fig. 6A and B).

FIG 6
FIG 6

Characterization of AidB homologs, namely, AidB12C from Bosea sp. strain 12C, AidBAt from Agrobacterium tumefaciens C58, and AidBRm from Rhizobium multihospitium CCBAU 83401T. Purified AidB-His6 and its homologs at different concentrations (0, 3.125, 6.25, and 25 μg ml−1) were mixed with 50 nM 3-oxo-C8-HSL in 50 mM Tris-HCl buffer (pH 8.0) and incubated at 30°C for 2 h. The residual AHLs were qualitatively (A) and quantitatively (B) analyzed using A. tumefaciens NTL4(pZLR4). The activity of purified AidB-His6 (25 μg ml−1) toward 3-oxo-C8-HSL was defined as 100% relative activity. (C) Thermal stability of the AidB homologs. Proteins at 10 μg ml−1 were preincubated at temperatures from 30 to 90°C for 30 min. After they cooled, their AHL degradation activities were determined at 30°C for 1.5 h using the method described above. The activity of preincubated AidB protein toward 3-oxo-C8-HSL at 30°C was defined as 100% relative activity. Statistical significance was calculated by the Student t test in a comparison of the AHL-degrading activities of AidB and its homologs (*, P < 0.05; **, P < 0.01; ns, no significant difference).

Thermal-stability assays showed that all homologs of AidB are thermostable. Proteins at a high concentration of 10 μg ml−1 (compared with 4 μg ml−1 in the above-described tests) were used to compare the thermal stabilities of AidB and its homologs due to their differences in AHL-degrading activity (Fig. 6A and B). Purified AidBAt-His6 retained high AHL-degrading activity after preincubation at 30 to 70°C for 30 min, and 54.1% of its maximal activity was retained after preincubation at 80°C. In contrast, purified AidB12C-His6 and AidBRm-His6 retained high activity from 30 to 60°C and 40 to 60°C, respectively, and 34.5% of the maximal AidBRm-His6 activity was retained at 70°C. However, the activity of purified AidB12C-His6 was almost completely lost at 70°C (Fig. 6C).

DISCUSSION

In the present study, two QQ strains, Bosea sp. F3-2 and 12C, were successfully isolated using in situ trap cultivation, a method for isolating rare actinomycetes (21). A number of QQ bacteria belonging to different genera were obtained in our screening, and most of them, including Pseudomonas, Arthrobacter, Bacillus, Variovorax, Ralstonia, Ochrobactrum, Microbacterium, Agrobacterium, Delftia, Enterobacter, and Flavobacterium, have been described in previous publications (data not shown). Bosea spp. did not appear in our previous screening, possibly because of the different cultivation method used in this study. Compared with conventional petri dish cultivation, in situ trap cultivation produced more bacterial colonies with higher variety from the same soil sample. Identification of AidB from Bosea spp. indicated the potential to discover new QQ enzymes using different cultivation approaches.

Almost all reported QQ genes have been cloned from culturable QQ bacteria and metagenomic DNA (17). The former is cultivation dependent and thus excluded QQ genes in unculturable bacteria, which may account for the vast majority of the bacterial population. The cloning of metagenomic DNA overcomes the problem of noncultivable microorganisms by screening QQ genes directly from the total DNA sample of a biological niche, and more than eight QQ genes have been successfully cloned from metagenomic libraries. However, the frequency of positive clones was low (varying from 1 × 10−4 to 1.89 × 10−2 per Mbp) (24–27), which makes metagenomic library screening labor-intensive. In addition, the restriction-modification system of the host bacterium may affect the efficiency of the metagenomic library by destroying foreign DNA, and some foreign DNA fragments may not normally be transcribed in the host bacteria because of a lack of suitable sigma factors (28, 29). At the beginning of this study, we failed to obtain positive clones in the screening of the F3-2 cosmid library. After identification of the aidB gene with the assistance of genomic sequence analysis, we rechecked the cosmid library and found two different cosmids containing complete aidB genes, but neither was expressed in E. coli (data not shown). Further complementation experiments identified a special sigma factor from Bosea sp. F3-2 required for AidB expression in E. coli (data not shown). This finding described the impacts of special sigma factors on the screening of functional genes in heterologous hosts, like E. coli.

The metallo-β-lactamase superfamily QQ enzymes share the Zn2+ binding motif HXHXDH-H at the active center that is essential for protein folding and enzyme activity. According to the conserved Zn2+ binding motif and low amino acid sequence identities (≤22.3%) with other members, AidB was assigned to a new subclass in the AHL lactonase family. Phylogenetic analysis revealed a restricted distribution of aidB homologs in several genera of Alphaproteobacteria, including Bosea, Rhizobium, Agrobacterium, Roseomonas, and Methylobacterium (Fig. 5B). Three homologs of AidB from the genera Bosea, Rhizobium, and Agrobacterium were obtained, and all exhibited AHL-degrading activity. The AHL-degrading activity of AidB homologs might be beneficial to bacterial fitness under specific environments, allowing them to be retained in some Alphaproteobacteria organisms during the process of evolution (17).

A surprising finding in this study was that AidB is a thermostable enzyme. Actually, a number of thermostable AHL lactonases have been identified in the metallo-β-lactamase superfamily, including AaL from Alicyclobacillus acidoterrestris ATCC 49025 (30), GcL from Geobacillus caldoxylosilyticus CIC9 (31), and AiiT from Thermaerobacter marianensis JCM 11223 (19), and in the phosphotriesterase-like lactonase superfamily, including GKL from Geobacillus kaustophilus HTA426 (32) and SsoPox from Sulfolobus solfataricus P2 (33). All above-mentioned thermostable AHL lactonases were isolated from thermophilic bacteria. AidB, however, is from a mesophilic bacterium, Bosea sp. F3-2, which could not grow even at 37°C (data not shown). Although the thermostability mechanism of AidB is unknown, it has been reported that an increase in arginines in the amino acid sequence contributes to thermostability, possibly because the guanidium group in arginine forms salt bridges to create stabilizing hydrogen bonds (34). This is very effective both in short, local and in long-range interactions and promotes the stability of the enzyme (19). Although the amino acid sequence of AidB did not show high similarities to thermostable AHL lactonases in the metallo-β-lactamase superfamily (Fig. S3), the frequency of arginine in AidB and other thermostable AHL lactonases is higher than that in nonthermostable AHL lactonases (Table S2). On the other hand, ionic bonds formed on the protein surface were supposed to contribute to the protein thermostability (35). For example, comparative study of the protein structures of thermostable organophosphorus hydrolases MPH_OCH (GenBank accession no. ACC63894, melting temperature of 66.5°C) from Ochrobactrum sp. strain M231 and OPHC2 (GenBank accession no. AJ605330, melting temperature of 97.8°C) from Pseudomonas pseudoalcaligenes C2-1 revealed an ionic bond formed by the charged residues Asp66 and Lys68 on the surface of OPHC2, but not on the surface of MPH_OCH, where the corresponding residues were Pro76 and Pro78 (Fig. S5A). Double point mutations (P76D and P78K) generated an ionic bond on the protein surface and, as expected, improved the thermal stability of MPH_OCH (35, 36). The predicted three-dimensional structures of AidB by Phyre2 (37) were highly similar to the reported crystal structure of OPHC2 (100% confidence and 35.0% amino acid identity), and an ionic bond formed by the charged residues Arg32 and Glu34 was predicted in AidB at the corresponding position of the ionic bond formed by Asp66 and Lys68 in OPHC2, indicating the possible involvement of the surface ionic bonds in the thermostability of AidB (Fig. S5B). The thermostable characteristics provide the enzymes with additional benefits, such as ease of purification, higher stability, better productivity, and higher resistance to organic solvents, acidic and alkaline conditions, and detergents (38). These advantages might make AidB suitable for the development of quorum-quenching products to prevent infection by animal and plant pathogens.

MATERIALS AND METHODS

Bacterial strains, plasmids, medium, growth conditions, and chemicals.The bacterial strains and plasmids used in this study are listed in Table 1. Pectobacterium carotovorum subsp. carotovorum Z3-3, Bosea sp. F3-2, Bosea sp. 12C, and Agrobacterium tumefaciens NTL4(pZLR4) (39) were grown in Luria-Bertani (LB) medium or AB minimal medium (40) at 28°C. Without special instructions, Pseudomonas aeruginosa PAO1 and Escherichia coli strains were grown in LB medium at 37°C. Swarming medium (5 g liter−1 Difco Bacto agar, 8 g liter−1 Difco nutrient broth, 5 g liter−1 glucose) were used for motility assays with Pseudomonas aeruginosa (41). Appropriate antibiotics were added when necessary at the following concentrations: ampicillin, 50 μg ml−1, gentamicin, 30 μg ml−1, and kanamycin, 50 μg ml−1. 5-Bromo-4-chloro-3-indolyl-l-d-galactopyranoside (X-Gal) was purchased from Sangon Biotech (Shanghai, China). The AHLs used in this study, N-butyryl-dl-homoserine lactone (C4-HSL; catalog no. 09945), N-hexanoyl-l-homoserine lactone (C6-HSL; catalog no. 56395), N-(β-ketocaproyl)-dl-homoserine lactone (3-oxo-C6-HSL; catalog no. K3255), N-(3-hydroxyoctanoyl)-dl-homoserine lactone (3-OH-C8-HSL; catalog no. 61698), N-(3-oxooctanoyl)-dl-homoserine lactone (3-oxo-C8-HSL; catalog no. O1639), N-decanoyl-dl-homoserine lactone (C10-HSL; catalog no. 17248), N-dodecanoyl-dl-homoserine lactone (C12-HSL; catalog no. 17247), and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL; catalog no. O9139) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the AHLs were dissolved in methanol and stored at −20°C.

View this table:
TABLE 1

Strains and plasmids used in this study

Isolation and screening of AHL-degrading bacteria.Soil samples collected from different areas of China were used for bacterial isolation. The in situ trap cultivation method was used to isolate bacteria as previously described, with some modifications (21). Briefly, a 0.45-μm-pore-size membrane (diameter, 5 cm) was glued to the bottom of a stainless steel washer (6.5 cm by 3.7 cm by 0.44 cm); the washer was then placed on top of a soil sample with proper humidity in the plate. Three milliliters of sterile 0.5% agar with 1% vitamin supplement (ATCC MD-VS) was added to the center of the washer. After the medium solidified, a 0.2-μm-pore-size membrane (diameter, 5 cm) was glued to the upside of the washer to seal the trap. Another washer was placed on the top of a 0.2-μm-pore-size membrane in case the membrane loosened. The plates were sealed with Parafilm to prevent evaporation and incubated at room temperature for 14 days in the dark. After incubation, the semisolid agar was transferred into a sterile petri dish, ground with a grinding rod, diluted with sterile water, and plated on CN medium (0.1% Casamino Acids, 0.1% nutrient broth, 1% Bacto agar). The plates were incubated at 28°C for 3 to 4 days until single colonies appeared.

The AHL degradation activities of the isolated bacteria were determined using the biosensor strain A. tumefaciens NTL4(pZLR4) (39). Simply, single colonies were inoculated into 200 μl of LB broth in 96-well plates using sterile toothpicks and cultivated at 28°C for 48 to 72 h. 3-Oxo-C8-HSL was added to the bacterial culture at a final concentration of 50 nM. After 4 h of incubation, the residual 3-oxo-C8-HSL was evaluated by transferring 5 μl of bacterial culture onto a reporter plate (13 cm by 13 cm), which comprised 50 ml of AB minimal medium seeded with 1 ml of A. tumefaciens NTL4(pZLR4) (OD600 ≈ 1.0), gentamicin (30 μg ml−1), and X-Gal (40 μg ml−1). The reporter plates were incubated at 28°C for about 12 h, and the size of the blue zone was checked. The bacterial isolates showing smaller blue zones than those of the control possibly inactivated 3-oxo-C8-HSL.

Extraction and detection of AHLs.The AHLs were extracted using an equal volume of ethyl acetate, the organic phase was then evaporated to dryness, and the pellet was resuspended in the proper volume of methanol. The AHLs were relatively quantified using A. tumefaciens strain NTL4(pZLR4) or absolutely quantified by UPLC. For relative quantification, 2 μl of the AHL samples was mixed with 300 μl of A. tumefaciens strain NTL4(pZLR4) (OD600 ≈ 0.8) in 2-ml tubes and incubated at 30°C for 3 h. The samples were then centrifuged at 13,000 × g for 5 min to collect the bacteria. Then, 100 μl of ddH2O was added to resuspend the bacteria, and 900 μl Z buffer (60 mM Na2HPO4·12H2O, 40 mM NaH2PO4·2H2O, 1 mM MgSO4·7H2O, 10 mM KCl, 50 mM β-mercaptoethanol, pH 7.0), 40 μl of chloroform, and 20 μl of 0.1% sodium dodecyl sulfate (SDS) were added in sequence and mixed thoroughly. After incubation at 30°C for 10 min, 200 μl of 4 mg ml−1 O-nitrophenyl-β-d-galactopyranoside (ONPG) was added to the samples as a substrate, and 200 μl of 1 M Na2CO3 was added to terminate the reaction when the color of the reaction mixture became yellow. The reaction time was recorded to calculate the β-galactosidase activity. The OD420 value was measured using a spectrophotometer after centrifugation at 13,000 × g for 15 min. The β-galactosidase activity was calculated using the following formula: 1 U = (1,000 × OD420)/(t × V × OD600), where t is the reaction time (in minutes), V is the volume of the biosensor (in milliliters), and OD600 is the absorbance value of A. tumefaciens NTL4(pZLR4) at 600 nm (42).

For absolute quantification of AHLs produced by Pseudomonas aeruginosa PAO1, 5 μl of the sample was analyzed on an Agilent 1290 system with a symmetry C18 reverse-phase column (4.6 by 150 mm, Agilent TC-18; Agilent Technologies, Santa Clara, CA, USA). C4-HSL and 3-oxo-C12-HSL were detected at 210 nm. C4-HSL was eluted isocratically with 30:70 methanol-water (vol/vol) at a flow rate of 0.5 ml min−1, and 3-oxo-C12-HSL was eluted with 80:20 methanol-water (vol/vol) at a flow rate of 0.6 ml min−1. The concentrations of C4-HSL and 3-oxo-C12-HSL were quantified by comparison with a set of standards.

Whole-genome sequencing and candidate QQ gene screening.Genomic DNA of Bosea sp. F3-2 was extracted using the standard method (43). DNA quality was confirmed using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and gel electrophoresis. Whole-genome sequencing was performed by Sinobiocore Biotechnology Co., Ltd. (Beijing, China) using the Pacific Biosciences (PacBio, Menlo Park, CA, USA) RSII-supplemented Illumina (Illumina, Inc., CA, USA) sequencing platform. The reads were assembled de novo using the hierarchical genome assembly process (HGAP) algorithm, version 3.0, into a circular contig, and genome annotation was performed using Prodigal version 2.6 (44, 45). Sequences that encoded amino acids sharing the conserved zinc-binding motif HXHXDH-H of the metallo-β-lactamase superfamily or the G-X-Nuc-X-G motif of the alpha/beta hydrolase superfamily were PCR amplified using primers listed in Table 2 and fused in frame to a His6 tag in pET-22b(+). The recombinant plasmids were transformed into Escherichia coli BL21(DE3) and cultured in 2 ml LB medium. When the OD600 of the transformants reached 0.6 to 0.8, IPTG (final concentration, 100 μM) was added to induce protein expression. After induction for 2 h, 3-oxo-C8-HSL was added to the bacterial cultures at a final concentration of 50 nM and incubated at 30°C for 2 h. The residual 3-oxo-C8-HSL was evaluated using A. tumefaciens NTL4(pZLR4) reporter plates as described above.

View this table:
TABLE 2

Primers used in this study

Protein expression and purification.To express the aidB gene and its homologous genes (aidB12C, aidBAt, and aidBRm), the coding regions were amplified using PCR, digested with NdeI and HindIII, and inserted into pET-22b(+) to give pET-22b-AidB, pET-22b-AidB12C, pET-22b-AidBAt, and pET-22b-AidBRm, respectively. Primers used are listed in Table 2. The aiiA240B1 gene (GenBank accession no. AF196486) was synthesized to construct pET-22b-AiiA240B1 by Genewiz (Suzhou, China) for the expression of the AiiA240B1 protein. The recombinant plasmids were transformed into E. coli BL21. IPTG at a final concentration of 100 μM was added to the transformant culture (OD600 = 0.6 to 0.8) to induce protein expression. The induction was allowed to proceed for 10 h at 18°C. Proteins were purified using Ni2+-nitrilotriacetic acid (NTA) Superflow according to the manufacturer’s instructions (Thermo Scientific, USA). Finally, the proteins were eluted using 250 mM imidazole in Tris-HCl buffer (10% glycerin, 20 mM Tris-HCl, and 150 mM NaCl, pH 8.0) and dialyzed in buffer (20% glycerin, 20 mM Tris-HCl, and 150 mM NaCl, pH 8.0) at 4°C for 24 h. The purified proteins were assessed using 8% SDS-PAGE.

The mechanism of AidB catalysis.The catalytic mechanism of AidB for degrading C6-HSL was analyzed using UPLC. Briefly, 80 μg purified AidB-His6 was added to 500 μl of the reaction buffer (50 mM Tris-HCl, pH 8.0) with 2 mM C6-HSL. After incubation at 30°C for 3 or 4 h, the mixtures were extracted three times with ethyl acetate and evaporated. The samples were redissolved in 100 μl of methanol and analyzed using the Agilent 1290 system. Fractions were eluted isocratically with 40:60 methanol-water (vol/vol) at a flow rate of 0.5 ml min−1 and detected at 210 nm. Two millimolar C6-HSL incubated with 10 mM NaOH and 2 mM C6-HSL incubated with heat-inactivated AidB were used as the positive control and negative control, respectively (22).

Characterization of AidB enzymatic activity.To determine the physical parameters that affect AidB activity, purified AidB (final concentration, 4 μg ml−1) and 3-oxo-C8-HSL (final concentration, 50 nM) were used in all subsequent tests. In the thermostability test, purified AidB was preincubated at temperatures ranging from 30 to 100°C (with an interval of 10°C) for 30 min; after cooling, 3-oxo-C8-HSL was added into the reaction buffer and incubated at 30°C for 30 min. The storage stability of AidB was determined in ddH2O at 4°C or room temperature; the activity of AidB was evaluated every 24 h during the 15-day storage by incubating the samples with 3-oxo-C8-HSL at 30°C for 30 min. The optimal reaction temperature was determined by evaluating AidB activity at temperatures ranging from 0 to 90°C (with an interval of 10°C) for 30 min. The residual AHL in the above-described experiments was extracted using ethyl acetate and evaporated. The samples were dissolved in 50 μl methanol and were relatively quantified using A. tumefaciens strain NTL4(pZLR4). Purified AiiA240B1 was used as a control.

Pyocyanin produced in Pseudomonas aeruginosa PAO1 and the virulence of Pectobacterium carotovorum subsp. carotovorum Z3-3.The ability of AidB to interfere with QS-regulated functions was tested in the human-pathogenic bacterium Pseudomonas aeruginosa PAO1 and plant-pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum Z3-3. The aidB gene was amplified using PCR with primers AidB-F/AidB-R (Table 2) and inserted into the broad-host-range vector pBBR1MCS-2 (46), which had been digested with BamHI and XbaI. The recombinant plasmid pBBR1MCS2-AidB was transferred into Pseudomonas aeruginosa PAO1 by electroporation and into Pectobacterium carotovorum subsp. carotovorum Z3-3 by mating. The empty vector pBBR1MCS-2 was used as the control. To detect growth, AHL production, and pyocyanin production, the derivative strains of PAO1 and Z3-3 were cultured at 180 rpm for 18 h before centrifugation at 13,000 × g for 5 min. The pellet was resuspended in fresh LB broth to an OD600 of 1.0 (2.1 × 109 CFU ml−1 for PAO1 and 2.3 × 109 CFU ml−1 for Z3-3), diluted 1:1,000 (vol/vol) in 30 ml LB medium with the appropriate antibiotics, and cultured at 180 rpm for 33 h. The cell density was measured at 600 nm, and the AHLs were extracted and analyzed using the method described above. To extract pyocyanin, 1 ml of culture supernatant was collected by centrifugation at 13,000 × g for 5 min and mixed with 600 μl of chloroform; the organic phase was reextracted by adding 200 μl of 200 mM HCl. Subsequently, the absorbance was determined at 520 nm. The pyocyanin concentration was calculated by multiplying the values of the samples at OD520 by 17.072 to obtain the concentrations in micrograms per milliliter (47). The motility assays with PAO1 were conducted as described previously (41). Two microliters of resuspended fresh strains was inoculated in the center of a swarming plate for static incubation at 37°C for 24 to 28 h. For the pathogenicity assay of Pectobacterium carotovorum subsp. carotovorum Z3-3 and its derivatives, 5 μl of bacterial suspension was inoculated at the center of a potato slice or wound site of Chinese cabbage after treatment with 70% ethanol. Fresh LB medium was inoculated as the negative control. The inoculated samples were incubated at 28°C for 48 h or 72 h in a crisper moisturized with wet filter papers before the maceration areas were recorded and analyzed. Three replicates were used for each condition.

Molecular identification and phylogenetic analysis.The 16S rRNA gene of strain Bosea sp. 12C was amplified by PCR with primers 12C-16S-F/12C-16S-R (Table 2). PCR was performed in 20-μl reaction mixtures containing 10 μl PrimeSTAR Max DNA polymerase (TaKaRa; R045Q), 1 μl 10 μM 12C-16S-F/12C-16S-R primers, and 50 ng purified genomic DNA of strain 12C with the following procedure: 30 cycles of denaturation at 98°C for 10 s, annealing at 59°C for 10 s, and extension at 72°C for 1 min. The PCR products were sequenced at Genewiz (Suzhou, China). Homologous sequences used in this study were obtained from the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/) by using BLASTn or BLASTp. Sequences were aligned using the program DNAMAN version 7.0. Phylogenetic and molecular evolutionary relationships were analyzed using Molecular Evolutionary Genetics Analysis (MEGA) version 5.05 (48).

Statistical analysis.The data were analyzed using the Student t test in DPS v9.50. Error bars represent the means ± standard deviations (SD). P values of less than 0.05 were considered statistically significant.

Accession numbers.The sequences of the 16S rRNA gene and the aidB12C gene from strain Bosea sp. 12C have been deposited in the GenBank database under accession numbers MN240442 and MN240477, respectively. The whole-genome sequence of strain Bosea sp. F3-2 described here has been deposited in the NCBI nucleotide sequence database under the accession numbers CP042331 (chromosome sequence), CP042332 (plasmid pB32-1), and CP042333 (plasmid pB32-2).

ACKNOWLEDGMENTS

This work was funded by the Key Project of Chinese National Programs for Fundamental Research and Development (973 Program) (grant 2015CB150605), the National Key Research and Development Program (grant 2017YFD0201106), the 111 Project (grant B13006), and the National Natural Science Foundation of China (grants 31572045 and 31872020).

FOOTNOTES

    • Received 18 September 2019.
    • Accepted 4 October 2019.
    • Accepted manuscript posted online 11 October 2019.

  • Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02065-19.

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KEYWORDS

quorum quenching
AHL lactonase
thermostability

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