incubation time might decrease the difference of the normal-
izedβ-galactosidase activities between candidate bacteria and
negative control.
- Possibleβ-galactosidases from candidate bacteria can be effec-
tively abolished by incubating the supernatant at 95C for
3 min without significant effect on the stability of C6-HSL.
- Many samples can cause significant reduction in the normalized
β-galactosidase activities if only Studentt-test was used to
compare the results. However, some of the obtained positive
samples did not show significant QQ activity in further tests. It
is inferred that the reduction of the normalizedβ-galactosidase
activity in some samples may be caused by unknown metabo-
lites of candidate bacteria. Therefore, we usually select samples
that caused more than 40% reduction in the normalizedβ-
galactosidase activities for further study.
- The cell suspension is clear if cells are completely lysed after
sonication. Most of Gram-negative bacteria can be completely
lysed after this sonication treatment. If cell suspension is not
clear, more times of sonication treatment are required.
- Cell contents are generally prepared in phosphate buffer in
order to avoid changes in pH caused by numerous enzymes
in the presence of high organic substrate (prepared in Marine
Broth). In this assay, PIPES is used to maintain a slightly acid
pH of mixture. Therefore, cell contents prepared in Marine
Broth is appropriate for subsequent tests. Just in case, pH of
each of the mixture should be checked after incubation to avoid
alkaline hydrolysis of AHL.
- It can be easily inferred that whether QQ activity is due to
enzymatic degradation of candidate bacteria from the results of
these steps (Fig.3). An identified AHL lactonase should be
included as a positive control.
- AHL lactonases hydrolyze the lactone ring of AHL, yielding
the correspondingN-acyl-homoserine. This hydrolysis may
also occur spontaneously at alkaline pH, and can be reversed
at lower pH [19]. Therefore, lactonase activity can be inferred
from the results of acidification.
In this step, enzymes in the samples can be abolished by
boiling treatment before acidification. The pH of mixture can
be monitored by pH-indicator strips to approximate pH 2. The
recovery of AHLs indicates AHL lactonase of candidate
bacteria.
It must be noticed that unrecovery of AHLs after acidifica-
tion does not mean no lactonase activity. As shown in Fig.3,
AHLs degraded by whole culture of Th120 are not recovered
even lactonase activity exists in the supernatant. This may be due
to further metabolization of ring-opened AHLs by unknown
Identifying Microbial Quorum Quenching Enzymes 307
