C6 hsl

Solid standard substances C4-HSL, C6-HSL and 3-oxo-C12-HSL with certified purity of 97 % (Sigma-Aldrich, St. Louis, MO, USA) were used. Stock standard solutions of individual AHLs with the concentration of 1 mg/ml (C4-HSL, C6-HSL) and 0.91 mg/ml (3-oxo-C12-HSL) were prepared in methanol. Mixed working standard solutions for preparing the calibration curve containing all the three selected AHL signal molecules, each at the concentration from approx. 1 to 60 ng/ml, were prepared in a mixture of methanol and deionized water (35 + 65). Dichloromethane used for the extraction of AHLs and methanol used for the mobile phase were of analytical and chromatography grade purity, respectively (Merck, Darmstadt, Germany). As the mobile phase, a gradient mixing of methanol and deionized water containing 0.1 % formic acid (Merck) was used. For the rinsing of canine ear canals, saline solution (0.9 % NaCl) was employed.

The 827 strains tested in this study were previously isolated from the microbiota and faeces of marine invertebrates including anemones (A. sulcata and A. equina) and holothurians (H. tubulosa and H. forskali). The samples were collected and processed in a previous study at iMARE Natural S.L (http://www.imarenatural.com) aquaculture facilities located in Motril, Granada (36°44′33.4″N 3°31′12.1″W), in southern Spain [42 (link)]. All marine strains used were routinely grown at 28 °C in marine broth (MB, BD Difco^®, Franklin Lakes, NJ, USA). The human-related pathogen P. aeruginosa PAO1 was cultured in LB medium at 37 °C. C. violaceum ATCC 12472 [93 (link)], C. violaceum CV026 [94 (link)], and C. violaceum VIR07 [93 (link)] were grown at 28 °C in LB medium. In the case of strains CV026 and VIR07, the medium was supplemented with kanamycin (50 µg mL⁻¹). The synthetic AHLs used were C6-HSL (N-hexanoyl-dl-homoserine lactone) and C10-HSL (N-decanoyl-dl-homoserine lactone) (Sigma^®, Madrid, Spain).

Overnight cultures of QQ yeast cells were harvested by centrifugation. Cell pellets were washed twice and re-suspended in phosphate buffered saline (PBS) (100 mM, pH 6.5). Selected known concentrations of synthetic AHLs (C6-HSL, 3-oxo-C6-HSL and 3-hydroxy-C6-HSL, Sigma-Aldrich, St. Louis, MO, USA) were dispensed into sterile micro-centrifuge tubes and dried by evaporation. Yeast cell suspensions were then added to rehydrate the AHLs to final concentrations of 0.5 μM. The mixtures were then incubated at 28 °C with shaking (220 rpm) for 0 h and 24 h. All reactions were stopped by heat inactivation at 95 °C. For the detection of AHL degradation, 10 μL of reaction mixture was spotted onto sterile paper discs placed on a Chromobacterium violaceum CV026 lawn and incubated overnight at 28 °C. AHL inactivation assays involved incubation of E. coli TOP10 and PBS buffer as negative controls. Re-lactonisation with acidification using hydrochloric acid (HCl, 0.2 M) was performed as reported [11 (link)].

The ability of the tested Ochrobactrum sp. type strains to inactivate AHLs was determined in a modified assay described by Jafra and van der Wolf [44 (link)]. Briefly, bacterial cells from overnight cultures were harvested and re-suspended in the MOPS-buffered LB, pH 6.5. Turbidity was adjusted to 5 units in McFarland scale (McF) (~3.5×10⁸ cfu·mL^-1). 50 μl of cell suspension was added to 50 μl of 10 μM C6-HSL (Sigma-Aldrich, USA) in M63 0.12% agar in white/clear bottom 96-well plates [36 (link)]. The plates were incubated for 16 h at 28°C, without shaking. The remaining AHLs were detected using E. coli [pSB401] biosensor [45 (link)], emitting light in the presence of C6-HSL.

The following synthetic AHLs (Sigma-Aldrich) were used at a final concentration of 25 μM to evaluate the AHL degradation activity of PQQ-42 and PQQ-44: C4-HSL (N-butyryl-DL-homoserine lactone), C6-HSL (N-hexanoyl-DL-homoserine lactone), 3-O-C6-HSL (N-3-oxo-hexanoyl-DL-homoserine lactone), C8-HSL (N-octanoyl-DL-homoserine lactone), 3-O-C8-HSL (N-3-oxo-octanoyl-DL-homoserine lactone), C10-HSL (N-decanoyl-DL-homoserine lactone), 3-OH-C10-HSL (N-3-hydroxydecanoyl-DL-homoserine lactone), C12-HSL (N-dodecanoyl-DL-homoserine lactone), 3-O-C12-HSL (N-3-oxo-dodecanoyl-DL-homoserine lactone) and C14-HSL (N-tetradecanoyl-DL-homoserine lactone). Briefly, cultures of the two A. stellipolaris strains (OD₆₀₀ 1.5) were mixed with AHLs at the above mentioned final concentration. The mixtures (500 µl of culture supplemented with 0.5 µl of each synthetic AHL) were incubated at 25 °C for 16, 24 and 48 hours, and the remaining AHLs were detected using a well diffusion agar-plate assay technique described elsewhere^{24 (link)} with the aid of the biosensors C. violaceum CV026 and A. tumefaciens NTL4 (pZLR4). The diameters of the colored haloes were measured and compared to controls to determine the percentage of signal molecules remaining in each case. These assays were carried out in triplicate.

Three different AHL (Sigma-Aldrich, Spain) were used: C6-HSL (N-hexanoyl-dl-homoserine lactone), C8-HSL (N-octanoyl-dl-homoserine lactone), and C12-HSL (N-dodecanoyl-dl-homoserine lactone). To detect QQ activity of the isolated Gram-positive bacteria, both reporter strains mentioned previously and Aeromonas hydrophila (strain E2MB52) as negative control, were grown in 4 mL of LB broth at 28 °C and 120 rpm. After 24 h, the strains were diluted to an OD600 nm of 0.5 in the same medium. Subsequently, 5 µL of each AHL stock solution were added to Gram-positive cultures to achieve a final concentration of 0.5 µg mL⁻¹, and were incubated at 28 °C and 120 rpm. After 24 h, 50 µL of the supernatants were spotted in duplicate in wells that were made on LB agar plates overlaid with 5 mL of overnight cultures of C. violaceum CV026 (or A. tumefaciens NT1) in soft LB (0.8% agar). Fifty µL of A. hydrophila supernatant and 50 µL of sterile LB that was supplemented with C6-HSL, C8-HSL, or C12-HSL were used as negative controls in all of the plates. Fifty µL of sterile LB were used as positive control.

L-Carvone (≥99% purity) and AHL standards including C₄-HSL, C₆-HSL, C₈-HSL, C₁₀-HSL, C₁₂-HSL, and C₁₄-HSL were obtained from Sigma-Aldrich (United States). The molecular biology reagents were purchased from Thermo Fisher Scientific (Shanghai, China). Other chemical reagents used in this study were of analytical grade, except for methanol (Chromatographic grade). The bacterial strains used in this study were C. violaceum CV026 and H. alvei Ha-01, as an AHL-reporter organism and a test strain, respectively. C. violaceum CV026 was provided by Dr. Yang (Xinjiang Shihezi University, Xinjiang, China) and H. alvei (ATCC 13337) Ha-01 was originally isolated and identified from putrid turbot by our group. C. violaceum CV026 was a mini-Tn5 mutant derived from C. violaceum ATCC 31532; it was kanamycin-resistant. It could respond only when exogenous AHLs were present, after which it produced the characteristic violet pigment, violacein. Both the strains were overnight cultured in Luria-Bertani (LB) broth (Qingdao Hopebio Co., Ltd., China), at 28°C and 160 rpm; however, the LB broth culture medium for CV026 required 20 μg/mL kanamycin.