Materials
The TIANprep Midi Plasmid Kit was used for plasmid or genomic DNA extractions and the Efficom BL21 (DE3) Chemically Competent Cell was the host-expressing cell; the Uniclone One Step Seamless Cloning Kit recombinant plasmid construction kit and the total RNA Extraction Kit were purchased from Genesand company. T4 DNA Ligase, BamH I restriction endonuclease, and other reagents were used to construct the expression vectors. Kanamycin (30 mg/mL), ampicillin (10 mg/mL), 5-Bromo-4-chloro-3-indolyl β-D-galactoside (X-Gal), and Isopropyl β-D-Thiogalactoside (IPTG) solutions were purchased from TaKaRa and used as the recombinant strain selection medium. The Mag-Beads His-Tag protein purification beads were used for the purification of the His-Tag target protein, and the target protein concentration was determined by BCA Protein Assay Kit. Western blot analysis was performed on the target protein (PvdQ enzyme). Anti-6 His-Tag mouse monoclonal antibody was used as the primary antibody, HRP-conjugated rabbit anti-mouse IgG was the second antibody, and the EasyBlot ECL kit was used to observe the color reaction. The AHL standard reagents were purchased from Sigma (C4-HSL, C6-HSL, C8-HSL, 3-HSL, 3-oxo-C8-HSL, C10-HSL, 3-oxo-C10-HSL, C12-HSL, C14-HSL, 3-oxo-C14-HSL) and dissolved with methanol (HPLC grade, 99.9%), sealed, and stored at -20°C.
Strain Source And Culture
Agrobacterium tumefaciens A136 (NTL4) was purchased from Hongsai Biotech (Hangzhou, China) and was used for detecting the activity of AHL signaling molecules. Pseudomonas aeruginosa (QS009B) was purchased from the National Center for Medical Culture Collections (CMCC). Sludge bacteria samples were collected from six Membrane Bio-Reactors (MBR) in the laboratory (Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China). Six groups of samples were collected in different glass bottles, mixed, and 5 mL of sludge samples removed and mixed with 50 mL of Stroke-physiological saline solution. This was then sonicated at 20% power for 15 seconds, and subjected to centrifugation at 3,000 rpm for 1 minute to remove large particulate material. The supernatant was then subjected to centrifugation at 4,500 rpm for 5 min, and the precipitate was re-suspended in 15 mL of saline for culturing the biofilm.
Dna Extraction And Pcr Amplification
The PvdQ gene sequence (ID: NC002516.2) was obtained from NCBI GenBank as a reference, and the upstream and downstream homology arms (containing BamH I and Hind III restriction sites with His-tag) were selected in pET-28a plasmid polyclonal region (MCS). The primers were designed (Table 1, online Oligo) and synthesized by Sangon Biotech (Shanghai, China). The restriction sites are underlined in Table 1. Total genomic DNA from P. aeruginosa was extracted and utilized for PCR amplification. The PCR amplification conditions were: 95°C pre-denaturation 3 min; 35 cycles of 95°C denaturation 30 s, 57°C annealing 90s, 72°C extension 1 min; followed by a final extension of 72°C for 10 min. An adequate volume of the PCR amplicons was resolved using gel electrophoresis, viewed, and purified. The amplicons were stored at 4°C.
Table 1. Oligonucleotides used as primers.
Construction of pMD-PvdQ Cloning Vector
The PvdQ gene fragment was ligated with the pMD19-T cloning vector and transformed into E. coli JM109 competent cells using the heat shock method. The transformation steps were: incubation of 100 µL of JM109 competent cells on ice for dissolution, addition of 10 µL of connection system to JM109 and mixed gently. This was incubated on the ice for 30 min, then placed in a 42°C water bath for 90 s, and then immediately placed on ice for another 3 min. Then sterile SOC medium (800 µL) was added and incubated for 1 h at 37°C for oscillatory activation. The appropriate coating was applied to solid medium containing X-Gal, IPTG, and ampicillin and incubated 37°C for 24 h. Positive monoclones were selected for gene sequencing, and the correctly verified recombinant cloning vector was named pMD-PvdQ.
Construction of pET-PvdQ Expression Vector
The Uniclone One Step Seamless Cloning Kit was performed according to the manufacturer’s instructions. An appropriate amount of linearized pET-28a vector was combined with the target gene PvdQ to generate a 10 µL pET-PvdQ recombinant vector. The upstream and downstream of the experimental target gene contained approximately 15–20 base pair (bp) homology arms from the vector restriction site, which improved the recombination efficiency according to the principle of base complementary pairing. The recombinant vector (10 µL) was transformed into 100 µL BL21 competent cells, using the transformation conditions described above. An appropriate amount of bacterial broth was plated on LB solid medium containing X-gal, IPTG, and kanamycin, and cultured at 37°C for 24 h. The white colonies on the plates were selected. The positive clones were verified by colony PCR and samples requiring enzyme digestion were selected and then sequenced at Sangon Biotech (Shanghai, China). The sequencing was performed to ensure that the expression sequence constructed was not mutated and had no frameshift mutations. The correctly sequenced recombinant expression vector was named pET-PvdQ.
Sds-page Gel Electrophoresis
Ten microliters of recombinant bacteria containing pET-PvdQ and pET-28a empty plasmids were in inoculated into a conical flask containing 100 mL LB medium with 10 mM kanamycin, and cultured overnight at 37°C (OD600 was approximately 0.4–0.6). The temperature was reduced to 25°C, and 0.5 mM IPTG and 0.5 g α-lactose were added and cultured for an additional 8 to 16 h. The bacterial cells were collected by centrifugation at 5,000 rpm for 15 min and sonicated at 50% power. The sonicator was, operated for 4 s and then intermittently for 10 s, for a total of 60 min. After sonication, centrifugation was performed to collect the supernatant and an appropriate volume of the supernatant was used for SDS-PAGE protein gel electrophoresis.
Protein Purification And Western Blot Analysis
The PvdQ protein was purified according to the Mag-Beads His-Tag protein purification beads manufacturer’s instructions, and the protein concentration was determined by BCA Protein Assay Kit. Purified PvdQ protein was quantified by SDS-PAGE gel electrophoresis, followed by western blotting. Membrane transfer conditions were 150 V for 150 min. The primary antibody (Anti-6 His-Tag mouse monoclonal antibody) was incubated overnight at 4°C, and the secondary antibody (HRP-conjugated Rabbit anti-mouse IgG) was incubated at room temperature for 2 h. A color rendering reaction was performed by the EasyBlot ECL kit and observed in an imaging system to detect target protein expression.
Ahl Signal Degradation Testing
The QQ activity of the PvdQ engineered bacteria was detected by the chromogenic reaction of the A. tumefaciens A136, which was performed according to the method of Khalid et al. (2022) with slight modifications. Briefly, the A136 bacterial culture (OD600 = 0.8–1.0) containing X-Gal (10 mg/mL) was added to a 96-well microplate. The C12-HSL signal molecule solution and recombinant bacterial solution (OD600 = 0.8) were mixed in specific proportions (C12-HSL total concentration of 500 ng/L), incubated for 6 h at 28°C, and then added to the A136 indicator bacterial sample well. The C12-HSL pure dilution and E. coli transformed with plasmid blank were used as the controls. Each group was performed in triplicate with a final liquid volume of 200 µL per well, incubated at 28°C and the depth of color change (blue) in the wells was recorded.
Movability Measurement
The motility assays of P. aeruginosa were performed according to the method provided by Yin et al. (2021). Briefly, overnight cultures of P. aeruginosa were inoculated onto LB agar centers containing serial dilutions of PvdQ acylase (0, 0.1, 0.3, and 0.5 mg/mL), incubated at 37°C for 24 h, and the diffusion (coverage) radius of P. aeruginosa on agar plates was recorded.
Biofilm Microscopy Analysis
The biofilm formation of P. aeruginosa was analyzed using light microscopy according to Meena et al. (2021). Briefly, P. aeruginosa (OD600 = 0.8) was added to a 6-well plate at different concentrations of PvdQ acylase (0, 0.1, 0.3, and 0.5 mg/mL, 2 mL of the total volume per well), and a coverslip (20 × 20 mm area, 0.17 mm thick) was placed over each well. The mixture was incubated at 37°C for 24 h. The coverslips were removed, carefully washed with distilled water, and stained with 0.1% (m/v) crystal violet. The biofilm was observed at 600 (40×15) times using the high powered lens of a light microscope.
Biofilm Assay (Dup: Abstract ?)
The effect of PvdQ engineered bacteria on the extent of P. aeruginosa biofilm formation (Biofilm formation index, BFI) was determined using the crystal violet (CV) method (Zhang et al. 2020). The concentration of QQ engineered bacteria (PvdQ) and the QS bacterium P. aeruginosa in LB broth were adjusted to an OD600 = 0.5. The P. aeruginosa and QQ bacterial cells were then co-cultured at varying ratios of 1:0.3, 1:1, and 1:3 or inoculated with the diluted PvdQ enzyme (0.1, 0.3, and 0.5 mg/mL). In addition, the MBR sludge bacteria concentration was adjusted to OD600 = 0.5, and the sludge bacteria and the PvdQ engineered bacteria were co-cultured at a cell ratio of 1:1, 1:3, and 1:5.
After 24 h incubation at 37°C in the 96-well plates, CV was added and the cells were stained. The level of biofilm formed on the wells at 570 nm was quantified by a microplate reader (Noori et al. 2022). Cells containing E. coli with the transformed pET-28a empty plasmid, sludge bacteria, and P. aeruginosa was used as controls. All tests were performed six times.
Expression Assay Of Qs-related Genes
Genes related to QS-mediated biofilm formation, such as the AHL receptor protein genes lasR, pqsA, and cdrA associated with biofilm formation, were retrieved from GenBank, and the rpsl gene was selected as the internal control for normalizing gene expression (Yin et al. 2021). The PvdQ enzyme (0.1 mg/mL) was grown overnight, at 28°C and 150 rpm, in LB medium containing P. aeruginosa, with three parallel settings for each group. Total RNA was extracted from bacterial somatic cells using the Total RNA Extraction Kit. The mass concentration of RNA was then analyzed by a Ultramicro spectrophotometer (TIANGEN, Beijing, China). The cDNA was generated from the total RNA using the PrimeScriptTM-RT-reagent-Kit (TaKaRa, Dalian, China) and used as a template for the qRT-PCR (LightCycler® 96 SW 1.1). The mRNA expression levels of the QS-related genes were measured using the LightCycler® real-time PCR system and the TB Green® Fast qPCR Mix (2 Conc.) kit (TaKaRa, Dalian, China).
Statistical analysis
All the experiments performed were performed in at least three independent replicates. The data obtained in this experiment were analyzed by SPSS and Duncan software and expressed as percentage. The p < 0.05 was considered as having a significant level for all statistical tests.