PMC

Inhibiting effects of several compounds on E. coli JM109/pSB401, activated by 40 nM 3O-C₆-HSL (100%). The concentrations required for 50% inhibition are indicated in parentheses. The experiments were done in triplicate, and the standard deviations (not shown) did not exceed 9% of the means.

Degradation of lactone and thiolactone structures in the presence of the lactonase AiiA_soil. 3O-C₇-HSL (compound 4), 3O-C₇-HTL (compound 24), and 3O-C₇-ACH (compound 28) at a concentration of 25 μM were incubated in the absence (100% activity) (open bars) or presence (shaded bars) of AiiA_soil-producing E. coli DH5α/pMIR101. After 24 h, the cultures were centrifuged, and each cell-free supernatant was assayed to determine its ability to activate the bioreporter S. enterica serovar Typhimurium 14028/pJNS25. The data are the results of one experiment that was representative of two independent repeats, and the error bars indicate standard deviations for nine measurements.

Dose-response curves showing the activities with the reporter strain S. enterica serovar Typhimurium 14028/pJNS25. (A) ×, acetonitrile; ⧫, 3O-C₆-HSL (compound 3); ⋄, □, and ▵, chemically synthesized N-(3-oxo-acyl)-trans-2-aminocyclohexanols with chains that are six carbon atoms long (compound 19), seven carbon atoms long (compound 28), and eight carbon atoms long (compound 29), respectively. (B) ⧫, ○, and +, chemically synthesized 3O-AHLs (dotted lines) and 3O-AHTLs (solid lines) with chains that are 6 carbon atoms long (compounds 3 and 18), 8 carbon atoms long (compounds 5 and 25), and 12 carbon atoms long (compounds 7 and 26), respectively. The standard deviations are not shown for clarity but did not exceed 11% of the means. The data are the results of one experiment that was representative of five independent replicates. The gene expression was normalized by dividing the luminescence value by the A₆₂₀ of each sample. RLU, relative light units.

Degradation of lactone and thiolactone structures at alkaline pH values. Aqueous solutions (10 mM) of pure 3O-C₇-HSL (compound 4), 3O-C₇-HTL (compound 24), and 3O-C₇-ACH (compound 28) at different pH values were incubated for 1 h at 37°C, spotted on a hydrophilic TLC plate, and developed with 100% ethyl acetate. The spots were visualized as described in the supplemental material. The results are the results for 3O-C₇-HSL (compound 4) (lanes 2 and 3) and 3O-C₇-HTL (compound 24) (lanes 5 and 6) after incubation at pH 8 and 9, respectively, as well as the results for 3O-C₇-ACH (compound 28) after incubation at pH 8, 9, and 12 (lanes 8, 9, and 10). As positive controls, 10 mM stock solutions of the molecules in acetonitrile were run in parallel (lanes 1, 4, and 7). Since the opened lactone and thiolactone structures had higher polarity, they migrated more slowly than the intact compounds and remained at the bottom of the plate. The results show that 3O-C₇-HSL (compound 4) and 3O-C₇-HTL (compound 24) were both completely degraded at pH 9, while 3O-C₇-ACH (compound 28) remained active at pH 12. Similar results were obtained after 24 h of incubation. The results are representative of three independent repeats.

Analysis of the intracellular AHL remaining after extensive washing. S. enterica serovar Typhimurium strains 14028/pJNS25 and BA612/pJNS25 were incubated for 6 h in the presence of 100 μM 3O-C₆-HSL (compound 3). The cells were harvested and extensively washed five times with fresh LB medium. (A) Samples of the cell-free supernatant of 14028/pJNS25 obtained after each washing step were separated on a TLC plate and subsequently analyzed using an A. tumefaciens NT1/pJM749/pSVB33 AHL biosensor overlay. Lanes 1 to 5 show that the amount of signal decreased with extensive washing and almost disappeared after the fifth washing step. (B) After the final wash, the cells were lysed, and extracts of the lysates were assayed to determine the AHL activities using the bioreporter S. enterica serovar Typhimurium 14028/pJNS25 (0 h). A portion of the cells that were washed five times was resuspended in fresh LB medium and incubated for an additional 13 h. Samples were taken after 4 h and 13 h and lysed, and extracts were analyzed to determined their AHL activities. Lysates of unwashed 14028/pJNS25 cells were used as positive controls in all experiments. The data show the AHL activities of lysates of the sdiA mutant BA612/pJNS25 grown in the presence of 3O-C₆-HSL (open bars) and of wild-type strain 14028/pJNS25 grown in the presence (cross-hatched bars) or absence (negative control) (solid bars) of 3O-C₆-HSL. The activity is expressed as a percentage of the activity of the positive control (100%). We found that none of the extracts activated the reporter more strongly than the negative controls, indicating that SdiA is not able to retain detectable amounts of 3O,C₆-HSL after extensive washing. The data are the results of one experiment that was representative of three independent repeats, and the error bars indicate standard deviations for four measurements.