weaken the selective pressure imposed on the pathogens and reduce
the potential for evolution of drug resistance [8]. To date, three
types of AHL-degrading enzymes have been found: AHL lacto-
nases (lactone hydrolysis), AHL acylases (amidohydrolysis), and
AHL oxidases and reductases (oxidoreduction).
Most of the identified AHL-degrading enzymes are derived
from nonmarine bacteria. However, the diversity of AHL-
degrading enzymes may be more than we expected. A novel
marine-derived AHL lactonase, MomL, was found inMuricauda
oleariain our previous work [9, 10]. Further study revealed that it
represents a novel type of secretory AHL lactonase widespread in
marine bacteria of the familyFlavobacteriaceae. Moreover, our
results showed that AHL acylases seem more common than lacto-
nases in the ocean. In other words, we may be only beginning to
understand the AHL-degrading enzyme diversity, especially in the
ocean.
A new high-throughput method was developed for identifying
QQ bacteria and 14 novel QQ bacterial species were found in our
previous work [9]. In this method, the biosensorAgrobacterium
tumefaciensA136 responses to a broad range of AHLs by expres-
singβ-galactosidase (encoded by the report genelacZ)[11]. By
using theβ-galactosidase substance 5-bromo-4-chloro-3-indolylβ-
D-galactopyranoside (X-gal), the β-galactosidase activity can be
quantified by spectrophotometric measurement of the final blue
products 5,5^0 -dibromo-4,4^0 -dichloro-indigo (indigo) at 630 nm
wavelength (OD 630 )[12]. However, only measurement of OD 630
is not accurate enough in a whole-cell assay because the absorbance
at 630 nm is actually a combination of absorption and light scatter-
ing by indigo andA. tumefaciensA136 biosensor cells. In the
mixture, according to the Beer-Lambert law [13], absorbance of
each component can be expressed as: OD630 cell¼aOD492 cell;
OD492 indigo¼bOD630 indigo;OD 630 ¼OD630 indigoþaOD 492
cell;OD 492 ¼bOD630 indigoþOD492 cell(wavelengths were
selected according to the absorbance spectra of indigo) [9]. The
correction factorsaandbare constants that can be easily obtained
by measuring the values of OD 492 and OD 630 of a dilution series of
biosensorA. tumefaciensA136 or indigo [9]. Therefore, theβ-
galactosidase activity is normalized to the cell amount of biosensor:OD630 indigo
OD492 cell¼0 : 653 OD 492 OD 630
0 : 267 OD 630 OD 492This improvement allows simultaneous measurement of large
numbers of samples with a minimum of hands-on time, requiring
merely standard equipment available in any research laboratory.
Herein, we describe the culture-dependent method for the
identification of microbial QQ enzymes, including high-through-
put screening, primary characterization of enzymatic AHL298 Kaihao Tang and Xiao-Hua Zhang