tube. Add 2μl of C6-HSL and 20μl of PIPES stock solutions
to the tube. Mix gently by pipetting and incubate the mixtures
at 28C for 24 h under static condition. Detect the QQ activity
according to the method described in Subheading3.1,steps
4 – 8 (seeNote 10).- Determine whether QQ activity is due to AHL lactonase. After
transferring 10μl of untreated whole culture, supernatant, and
cell content of Th120 to a 96-well plate in the previous step,
boil the rest of the reaction mixtures (190μl) for 5 min. Add
2–3μl of 6 M HCl. Mix gently by pipetting and incubate the
mixtures at 28C for 24 h under static condition. Detect the
QQ activity according to the method described in Subheading
3.1,steps 4– 8 (seeNote 11). The QQ activity of each compo-
nent of Th120 is shown in Fig.3.
3.3 Identification of
QQ Enzymes from
Selected Bacteria
Many methods can be used to identify the responsible QQ enzymes
in bacteria, such as purification of QQ enzyme [14] and construc-
tion of a genomic library [15]. Here, we only describe searching
putative QQ enzymes through local BLASTP in a computer with
Windows operating system. Detailed methods for heterologous
overexpression, purification, and in vitro characterization of puta-
tive QQ enzymes are described elsewhere [16].- Inoculate Th120 in MB, and incubate the cultures at 28C,
170 rpm for 24 h. - Collect cell pellets by centrifuging the culture at 13,000gfor
10 min at 4C. - Extract genomic DNA of Th120 by using a commercial kit for
genomic DNA extraction.
Fig. 3Characterization of the AHL degradative activity ofM. oleariaTh120. AHL degradative activity of Th120
was analyzed by the A136 liquid X-gal assay. AiiA and Marine Broth medium were used as the positive and
negative control, respectively.WCwhole culture,Ssupernatant,CCcell content,NCnegative control. In
consideration of incomplete degradation of proteins, no proteinase K-treated WC was included. Four Arabic
numbers represent different pretreatments of each sample: ( 1 ) untreated, ( 2 ) recovery of AHLs by acidification,
( 3 ) heat-treated, and ( 4 ) proteinase K-treated. Reproduced with permission from Copyright©2015, American
Society for Microbiology, Appl Environ Microbiol, 81, 2015:774–782. doi:10.1128/AEM.02805-14 [10]
Identifying Microbial Quorum Quenching Enzymes 303