Upon acidification CUDC-907 mouse of the supernatant AHL biosensor activity could be restored thus confirming that AhlK has lactonase activity (data not shown). When Burkholderia strain GG4 was incubated with 3-oxo-C6-D-HSL for 3 h and examined by HPLC, we noted the appearance of a new peak (retention time 4.3 min) that was absent when either GG2 or Se14 was incubated with the same D-isomer (retention time 4.8 min) (Figure 2B).
Similar results were obtained following incubation of the natural L-isomer of 3-oxo-C6-HSL with GG4 and the new peak was found to co-migrate with the L-isomer of PRN1371 3-hydroxy-C6-HSL (data not shown) suggesting that GG4 has oxido-reductase activity towards 3-oxo-AHLs. To confirm the oxido-reductase activity of GG4, 3-oxo-C6-L-HSL
incubated with GG4 for 0 h and 24 h was analysed by mass spectrometry and the appearance of 3-hydroxy-C6-HSL was confirmed by detection of the precursor ion (m/z 216.2 ([M+H])) and fragment ions of m/z 198.0 ([M+H-H2O]) and 102.0 (Figure 2C) which are characteristic of 3-hydroxy-AHLs which readily lose a water molecule and the homoserine lactone moiety respectively [17, 18]. Similar results were obtained on incubation of GG4 with the L-isomers of 3-oxo-C4-HSL or 3-oxo-C8-HSL in that new HPLC peaks co-eluting with 3-hydroxy-C4-HSL and 3-hydroxy-C8-HSL synthetic standards, respectively, were observed after incubation for 6 h (data not shown). This oxido-reductase activity was only apparent when 3-oxo-AHLs were incubated with GG4 whole cells but not cell lysates (data not shown). Acinetobacter GG2 and Burkholderia GG4 produce AHLs Since some but not all Acinetobacter and Burkholderia strains have previously www.selleckchem.com/products/tideglusib.html been reported to produce AHLs, we wondered whether QQ and QS activities co-exist Y-27632 cell line in GG2, GG4 and Se14. To determine whether any of the three ginger rhizosphere strains produced AHLs, they were first cross-streaked against each of three different AHL biosensors namely C. violaceum CV026, E. coli [pSB401] and E. coli [pSB1075], and the plates examined over time for the induction of violacein or bioluminescence (data not shown). GG2 induced bioluminescence in E. coli [pSB1075] indicating
that it was producing long chain AHLs, GG4 activated both CV026 and E. coli [pSB401] indicative of short chain AHL production while Se14 failed to activate any of the three AHL biosensors. To identify unequivocally the AHLs produced by GG2, spent culture supernatant extracts were analysed by liquid chromatography (LC) coupled to hybrid quadruple-linear ion trap mass spectrometry (LC-MS/MS), which revealed the presence of 3-oxo-C12-HSL and 3-hydroxy-C12-HSL by comparison of their retention times, precursor and principal fragment ions with synthetic standards. Figure 3 shows the fragmentation patterns for 3-oxo-C12-HSL (precursor ion m/z 298.2 [M+H]; fragment ions m/z 197.2, 102.0 (Figure 3A and Figure 3C) and 3-hydroxy-C12-HSL (precursor ion m/z 282.2 [M-H2O]; fragment ions m/z 181.2, 102.