FIG. 3.

3-Oxo-acyl-HSLs but not C6-HSL are retained by A. tumefaciens cells expressing TraR. Cultures of A. tumefaciens NTL4(pZLQ), which does not express traR, and NTL4(pZLQR), which does express the activator from the trc promoter, were grown in ABM minimal medium to a density of about 10⁷ CFU/ml. The two cultures were each split into three subcultures, and 3-oxo-C8-HSL (50 nM) was added to one, 3-oxo-C6-HSL (150 nM) was added to a second, and C6-HSL (2.5 μM) was added to the third. At the same time IPTG was added to all cultures of NTL4(pZLQR) to induce expression of traR. The six cultures were grown to late exponential phase (OD₆₀₀ of about 1.2), at which time the cells were harvested, washed, and broken, and the cell extracts were assayed for intracellular quormone by thin-layer chromatography with A. tumefaciens NTL4(pZLR4) as the bioindicator, all as described in Materials and Methods. The thin-layer chromatography plates were spotted with the following samples: 1, pure 3-oxo-C8-HSL standard; 2, pure 3-oxo-C6-HSL standard; 3, pure C6-HSL standard; extracts of NTL4(pZLQR) grown with: 4, 3-oxo-C8-HSL; 5, C6-HSL; 6, 3-oxo-C6-HSL; and extracts of NTL4(pZLQ) grown with: 7, 3-oxo-C8-HSL; 8, 3-oxo-C6-HSL; and 9, C6-HSL.