4 mM IPTG induced and uninduced cultures, and then reacted with various AHLs to perform whole cell bioassays. Cytoskeletal Signaling inhibitor Identical to the result in Fig. 1, the absence of a violet lawn indicates a positive AHL-degrading ability and is defined as’+'; a violet lawn indicates no AHL-degrading ability and is defined as’-’ Figure 1 The Aac acylase degrades C7-HSL in C. violaceum CV026 cultures and inhibits violacein production. The E. coli DH10B (pS3aac) overnight culture was centrifuged, and the harvested cells were
suspended into 100 mM Tris buffer (pH 7.0). The cell suspensions and cell free supernatants were mixed with 25 μM C7-HSL each and then incubated at 30°C for 1 h. The mixtures were assayed by the in vitro whole cell bioassay. Well 1, C7-HSL (AHL-non-degrading Napabucasin cost control); well 2, Tris buffer (AHL-degrading control); well 3, the mixture of cell suspensions with C7-HSL; well 4, the mixture of supernatants with
C7-HSL. Figure 2 SDS-PAGE analysis Aac expressed by E. coli BL21(DE3). The crude proteins were prepared from the recombinant E. coli BL21 (pET21aac) and analysed by 6% SDS-PAGE. The arrow indicates the Aac. Lane 1, pre-stained protein ladder marker; lane 2, IPTG-induced crude proteins; lane 3, IPTG-non-induced crude proteins. Aac is an AHL-acylase and not an aculeacin A acylase To demonstrate whether the Aac protein is an AHL-acylase, we performed Suplatast tosilate the ESI-MS analysis. E. coli DH10B (pS3aac) cells were first reacted with C7-HSL at 30°C for 60 CFTRinh-172 in vivo min. If the enzyme encoded by the aac gene is an AHL-acylase, we predicted that two free digested products, HSL and heptanoic acid, would be detected. Since ESI+-MS could not detect the carboxylic group (COO-), only HSL was detectable. The fatty acids containing the carboxylic group would have to be detected by ESI–MS. The analytic results showed that C7-HSL (M+H m/z = 214) could be digested into HSL (M+H m/z = 102) and heptanoic acid (M-H m/z = 129) (Fig. 3).
We also observed that the amount of the heptanoic acid gradually increased, starting from the 15th min until the 60th min of reaction times (data not shown). Thus, our results indicate that the aac gene encodes an AHL-acylase. Figure 3 ESI-MS spectrometry analysis of C7-HSL degradation by AHL-acylase Aac. The E. coli DH10B (pS3aac) cells were suspended in 0.1 mM sodium phosphate and 0.01 mM ammonia acetate, respectively, and then mixed with 25 μM C7-HSL for the degradation reaction described in Materials and Methods. (a) To detect HSL, the ESI+-MS spectra of undigested C7-HSL (top) and degraded C7-HSL products (bottom) were shown. (b) To detect heptanoic acid, the ESI–MS spectra of undigested C7-HSL (top) and degraded C7-HSL products (bottom) were shown. (c) Mechanism of C7-AHL degradation by Aac. The white arrow indicates the Aac catalytic site.