Bioluminescence in the microtitre plate

wells was visualized using Luminograph LB980 photon video camera (Berthold). To determine whether AHLs were being inactivated by lactonolysis, i.e. by the formation of the corresponding N -acylhomoserine compound, the method described by Yates et al [8] was used. This is based on AZ 628 manufacturer acidification of the reaction mixture to pH 2 with HCl (10 mM) to promote recyclization of the homoserine lactone ring. HPLC analysis of AHLs and AHL-degradation products HPLC analysis of AHLs and their degradation products was performed as described before [17, 20] on an analytical C8 reverse-phase preparative HPLC column (Kromasil C8; 250 × 4.6 mm) attached to a photodiode array (PDA) system (Waters 996 PDA system operating with a Millennium 2010 Chromatography Manager, Waters, England) and eluted with acetonitrile/water isocratic or gradient combinations SBI-0206965 as described before [17]. Identification of AHLs AHLs were unequivocally identified by LC-MS/MS as described before [17, Belnacasan chemical structure 42]

using enhanced product trap experiments (EPI) triggered by precursor ion scanning between the m/z range 150-500 and in particular for the fragment ion m/z 102 which is characteristic for the homoserine lactone ring moiety. The EPI spectra (m/z range 80-400) containing a fragment ion at m/z 102 were compared for the retention time and spectral properties to a series of corresponding synthetic AHL standards. The 3-hydroxy-AHLs were identified by comparison with a synthetic oxyclozanide standard based on the LC retention times, the MS-MS fragmentation product ions ([M+H-H2O] and m/z 102). 3-hydroxy-AHLs characteristically lose a water molecule during MS fragmentation generating a characteristic ion of [M-18] [17, 43]. P. aeruginosa QQ co-culture assays The ability of ginger rhizosphere isolates to attenuate P. aeruginosa QS-regulated virulence determinants (elastase and lectin A) were determined by growing cultures of P. aeruginosa PAO1, GG2, GG4 and Se14 separately at 28°C for 24 h with shaking (220 rpm), normalizing at an OD600 of 1.0 followed by co-culturing at a 1:1 ratio. Total viable cell counts were carried out to ensure that neither organism significantly reduced the growth of the other.

The elastolytic activity of P. aeruginosa was determined as described before using elastin-Congo red (ECR) as substrate. Briefly, 100 μl of cell free bacterial spent culture supernatants from both mono-culture and co-culture experiments were added separately to 900 μl ECR buffer (100 mM Tris [pH 7.5], 1 mM CaCl2) containing 20 mg of ECR and incubated with shaking at 37°C for 3 h. Insoluble ECR was removed by centrifugation at 7,000 × g for 5 min. The absorbance of the supernatant was determined at OD495. The expression of lecA was determined using a P. aeruginosa lecA :: lux reporter strain [35] in a 96-well microtitre plate using an automated combined luminometer/spectrometer (Anthos Labtech LUCYI). Briefly, 200 μl of a 1:500 dilution of an overnight culture of the P.