QsdH, a Novel AHL Lactonase in the RND-Type Inner
Membrane of Marine Pseudoalteromonas byunsanensis
Wei Huang1, Yongjun Lin3, Shuyuan Yi1, Pengfu Liu1, Jie Shen1, Zongze Shao2, Ziduo Liu1*
1State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, P. R. China, 2The State Oceanic
Administration, The Third Marine Research Institute, Xiamen, P. R. China, 3National Key Laboratory of Crop Genetic Improvement, College of Life Science and Technology,
Huazhong Agricultural University, Wuhan, P. R. China
N-acyl-homoserine lactones (AHLs) are the main quorum-sensing (QS) signals in gram-negative bacteria. AHLs trigger the
expression of genes for particular biological functions when their density reaches a threshold. In this study, we identified
and cloned the qsdH gene by screening a genomic library of Pseudoalteromonas byunsanensis strain 1A01261, which has
AHL-degrading activity. The qsdH gene encoded a GDSL hydrolase found to be located in the N-terminus of a multidrug
efflux transporter protein of the resistance-nodulation-cell division (RND) family. We further confirmed that the GDSL
hydrolase, QsdH, exhibited similar AHL-degrading activity to the full-length ORF protein. QsdH was expressed and purified
to process the N-terminal signal peptide yielding a 27-kDa mature protein. QsdH was capable of inactivating AHLs with an
acyl chain ranging from C4to C14with or without 3-oxo substitution. High-performance liquid chromatography (HPLC) and
electrospray ionization-mass spectrometry (ESI-MS) analyses showed that QsdH functioned as an AHL lactonase to
hydrolyze the ester bond of the homoserine lactone ring of AHLs. In addition, site-directed mutagenesis demonstrated that
QsdH contained oxyanion holes (Ser-Gly-Asn) in conserved blocks (I, II, and III), which had important roles in its AHL-
degrading activity. Furthermore, the lactonase activity of QsdH was slightly promoted by several divalent ions. Using in silico
prediction, we concluded that QsdH was located at the first periplasmic loop of the multidrug efflux transporter protein,
which is essential to substrate selectivity for these efflux pumps. These findings led us to assume that the QsdH lactonase
and C-terminal efflux pump might be effective in quenching QS of the P. byunsanensis strain 1A01261. Moreover, it was
observed that recombinant Escherichia coli producing QsdH proteins attenuated the plant pathogenicity of Erwinia
carotovora, which might have potential to control of gram-negative pathogenic bacteria.
Citation: Huang W, Lin Y, Yi S, Liu P, Shen J, et al. (2012) QsdH, a Novel AHL Lactonase in the RND-Type Inner Membrane of Marine Pseudoalteromonas
byunsanensis Strain 1A01261. PLoS ONE 7(10): e46587. doi:10.1371/journal.pone.0046587
Editor: Eric Cascales, Centre National de la Recherche Scientifique, Aix-Marseille Universite ´, France
Received April 12, 2012; Accepted September 1, 2012; Published October 8, 2012
Copyright: ? 2012 Huang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant from the National Natural Sciences Foundation of China (NO. u1170303) and the Genetically Modified Organisms
Breeding Major Projects of China (2011zx08001-001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Numerous bacteria monitor their own population densities by
sensing the concentration of small signaling molecules called
autoinducers, which further regulate the expression of specific
genes, thus resulting in better adaptation to a novel environment
. The cell-to-cell communication phenomenon of quorum
sensing (QS) depends on the production, secretion and response to
diffusible autoinducers . Many gram-negative bacteria commu-
nicate with each other through AHLs. Once these chemicals reach
a threshold concentration, they activate QS-dependent gene
expression and produce phenotypic effects, including biolumines-
cence, Ti plasmid conjugal transfer and swarming motility [2,3].
Many pathogens rely on quorum sensing to synchronize microbial
activities essential for infection by triggering expression of
particular virulence genes [4,5]. Recently, many quorum-quench-
ing phenomena have been observed, and strategies have been
developed to disturb quorum sensing. The discovery of quorum-
quenching mechanisms has demonstrated that these mechanisms
are widely conserved in many prokaryotic and eukaryotic
organisms and have important roles in microbe/microbe and
To date, several groups of potent quorum-quenching enzymes
have been identified, including AHL lactonase, AHL amidohy-
drolase (AHL acylase), paraoxonases (PONs), and AHL oxidore-
ductase [4,6]. AHL lactonases catalyze the lactone ring opening by
hydrolyzing AHL. The first cloned AHL lactonase was aiiA from
Bacillus spp. 240B1 (hereafter referred to as AiiA240B1)  followed
by aiiB and attM from Agrobacterium [8,9], ahlD from Arthrobacter spp.
, qsdA from Rhodococcus erythropolis strain W2 , aiiM from
Microbacterium testaceum StLB037 , and aidH from Oclrobacterium
spp. . These AHL lactonases come from several different
hydrolase families, such as the metallo-beta-lactamase superfamily,
a/b hydrolase superfamily, and PTE superfamily. Site-directed
mutagenesis based on a sequence alignment of AiiA homologues
has elucidated that AiiA contains a HXHXDH zinc-binding motif,
which is widely conserved in several groups of metallohydrolases
and is essential for the enzymatic activity of AHL lactonase . In
PLOS ONE | www.plosone.org1 October 2012 | Volume 7 | Issue 10 | e46587
contrast with previous studied AHL lactonases, AiiM and AidH
show similarity to predicted a/b hydrolase fold family proteins but
exhibit variable substrate specificity of AHLs [12,13]. Similarly,
several bacteria have been reported to encode an AHL acylase for
hydrolyzing the amide bond of AHL and releasing the fatty acid.
For example, AHL acylases have been discovered in Ralstonia spp.
XJ12B, Streptomyces spp. M664, Shewanella spp., Pseudomonas
aeruginosa, Ralstonia solanacearum GM1000, and Ochrobactrum sp.
A44 [3,14,15,16,17,18,19,20]. Interestingly, strong AHL inactiva-
tion activity of paraoxonases has been observed in mammalian
species. The human paraoxonase gene family has three members
(PON1, PON2 and PON3) that exhibit wide physiological effects,
including quorum-quenching enzymatic activity. Enzymatic char-
acterization of purified PONs has revealed them to be lactonases
with some overlapping substrates (e.g., aromatic lactones) [21,22].
In vitro assays have shown that PON1 from mouse serum is
required and sufficient to degrade 3OC12-HSL and that PON2
and PON3 also effectively degrade 3OC12HSL .
All AHLs are amphipathic molecules that have been assumed to
be freely diffuse or to be transported outside of bacterial cells .
Recently, studies have found that short-chain AHL signals can
freely diffuse across bacterial membrane but that long-chain AHLs
are not freely permeable. In Pseudomonas aeruginosa, cell-to-cell
signaling controls the expression of extracellular virulence factors
and biofilm differentiation . To date, several multidrug efflux
systems have been described in P. aeruginosa that contribute to the
active efflux of AHLs, N-butanoyl-L-homoserine lactone (C4HSL)
and N-(3-oxododecanoyl) homoserine lactone (3OC12HSL) and
then regulate the intracellular signal molecule levels. For example,
3OC12HSL efflux in P. aeruginosa cells relies on active transpor-
tation by the MexAB-OprM multidrug efflux pump .
Overexpression of MexEF-OprN is correlated with a decrease in
the production of extracellular virulence factors, particularly those
controlled by the rhl system, thus affecting the intracellular C4HSL
and Pseudomonas quinolone signal (PQS) . Similarly, the
MexGHI-OpmD pump in P. aeruginosa cells involves an inability
to produce 3OC12HSL and PQS as well as a drastic reduction of
N-butanoyl-L-homoserine lactone levels, thus indicating its essen-
tial function in facilitating cell-to-cell communication and antibi-
otic susceptibility in addition to promoting virulence and growth
. In Burkholderia pseudomallei, the extracellular secretion of AHL
relies on the BpeAB-OprB efflux pump [27,28], which is required
for efflux but not for influx of acyl-HSLs during quorum sensing
. These active transportation mechanisms reduced the
production of AHL signals and virulence factors.
In this study, we reported the isolation of the AHL-inactivating
enzyme, QsdH, which is a GDSL hydrolase from P. byunsanensis
strain 1A01261. QsdH was found to be located in the N-terminus
of a multidrug efflux transporter protein functioning as a novel
broad spectrum AHL lactonase. In addition, QsdH was found to
be integrated into the C-terminal cytoplasmic RND-type mem-
brane protein, which is the first known example of a multidrug
efflux pump transporter protein with an N-terminal periplasmic
extension in the genus Pseudoalteromonas. Additionally, the recom-
binant protein may regulate the expression of certain phenotypic
genes by controlling intercellular AHL levels in P. byunsanensis
1A01261. The result showed that over-producing QsdH in E. coli
inhibits virulence of E. carotovora, a pathogenic bacterium of plants.
Materials and Methods
Chemicals, strains, plasmids and culture conditions
N-butanoyl-L-homoserine lactone (C4HSL), N-nexanoyl-L-ho-
moserine lactone (C6HSL), 3-oxohexanoyl-L-homoserine lactone
(3OC6HSL), N-octanoyl-L-homoserine lactone (C8HSL), 3-ox-
ooctanoyl-L-homoserine lactone (3OC8HSL), N-decanoyl-L-ho-
moserine (C10HSL), N-dodecanoyl-L-homoserine (C12HSL) and
N-tetradecanoyl-L-homoserine lactone (C14HSL) were purchased
from the Cayman Chemical Company. All strains and plasmids
used in this study are listed in Table 1. E. coli were cultured in
Luria-Bertani medium (the pH was buffered to 6.8 using HCl) at
37uC. The marine bacteria were supplied by the Marine Culture
Collection of China, and were cultured in modified LB medium
(1% tryptone, 3% NaCl, and 0.5% yeast extract; pH 7.0) at 25uC.
The Agrobacterium tumefaciens strain NT1 (traR; tra::lacZ749) and E.
carotovora SCG1 was cultured in a minimal medium as previously
described (Zhang et al, 2000). For a bioassay plate to measure
AHL degradation, 5-bromo-4-chloro-3-indolyl-b-D-galactopyra-
noside (X-gal) was added to minimal solid medium to a final
concentration of 40 mg/ml. In addition, ampicillin was added to a
final concentration of 100 mg/ml.
Screening of AHL-degrading marine bacteria
The biosensor A. tumefaciens strain NT1 was applied to detect
residual AHLs on agar plates as previously described [7,29].
Strains from the Marine Culture Collection of China (MCCC)
were cultured at 28uC with gentle shaking to an OD600 of
approximately 1.5. The cells were collected by centrifugation,
washed and suspended in 200 ml of PBS buffer (140 mM NaCl,
2.7 mM KCl, 10 mM Na2HPO4and 1.8 mM KH2PO4; pH 7.4)
to an approximate OD600of 0.6. 3OC8HSL was then added into
the suspensions to a final concentration of 1 mM and reacted at
28uC for 2 h. The supernatants from the cultures were also
isolated and reacted with 3OC8HSL for 2 h. After incubation,
these mixtures were spotted onto the bottoms of solidified agar
medium plates, which were cut into separated slices. The A.
tumefaciens strain NT1 (approximate OD600of 0.6) was then spotted
at distances progressively further from the loaded samples. After
incubation at 28uC for 24 h, the results were analyzed by
monitoring the precipitate color of the A. tumefaciens strain NT1.
b-galactosidase expression was induced by AHL, which digested
X-gal in the medium to give blue colonies. Therefore, the residual
AHL in the samples was determined by the distance of the blue
colonies on the agar plate.
To further analyze the characteristic of AHL-degrading strains,
the supernatant from bacterial cultures was incubated at 60uC for
30 min, and then reacted with 1 mM 3OC8HSL at 28uC for 2 h.
The reaction was stopped and residual 3OC8HSL was detected
Cloning and sequencing of an AHL-degrading gene from
P. byunsanensis strain 1A01261
A common protocol for genetic manipulation was exploited to
construct the genomic library. The DNA fragments, partially
digested by Sau3A I from the total genomic DNA of strain
1A01261, were ligated into the pUC118 cloning vector (TaKaRa),
which was digested by BamH I and dephosphorylated. The
genomic library was transformed into E. coli DH5a. Subsequently,
transformants were grown overnight on LB agar plates containing
ampicillin. The colonies were inoculated into fresh LB medium
and incubated at 37uC overnight. The colony cultures were then
reacted with 250 nM 3OC8HSL for 4 h. The reactions were
halted by heating, and residual 3OC8HSL was detected with A.
tumefaciens NT1. A total of 6,000 clones were screened, and several
different transformants manifested AHL-degrading activity. The
E. coli DH5a harboring positive plasmids were rescreened by
inoculation with 2 mM 3OC8HSL. Finally, one positive plasmid,
pUC118-sm20, was obtained and sequenced. The positive DNA
A Novel Lactonase in RND-Type Transporter Protein
PLOS ONE | www.plosone.org2October 2012 | Volume 7 | Issue 10 | e46587
fragment was analyzed with DNASTAR software (DNASTAR,
Inc.) and by BLAST (http://www.ncbi.nlm.nih.gov/), BPROM
(http://linux1.softberry.com/berry.phtml) and SIGNALP (http://
Given that the cloned positive gene encoded a two-domain
protein, including the N-terminal GDSL hydrolase domain and C-
terminal RND-type transporter domain, we further explored
which domain was responsible for the AHL-degrading activity.
Thus, we cloned a DNA fragment (named qsdH, the abbreviation
of quorum sensing degrading hydrolase) encoding the GDSL
hydrolase and the open reading frame (ORF) gene, orf, into the
pUC118 vector. The orf gene was amplified from the 1A01261
genome withthe following
ATGCGCCGACGTCGCCGCGC-39 and 59-CGCGGATCCT-
TAGGATCCCTTTACTATGC-39 (EcoR I and BamH I sites are
underlined). The orf’ gene fragment was amplified with the
following primers to process the N-terminal signal sequence to
yield the mature
CGCGGATCCTTAGGATCCCTTTACTATGC-39. The qsdH
gene was clonedwith the
these DNA fragments were cloned into pUC118 and transformed
into E. coli DH5a. The AHL-degrading activity of these
transformants was then detected by the biosensor A. tumefaciens
Expression and purification of QsdH protein
As the qsdH GDSL hydrolase gene exhibits AHL activity, the
gene fragment was amplified without the N-terminal signal
sequence using the
and Xho I sites are underlined). The PCR products were inserted
into the EcoR I/Xho I site of the pGEX-6p-1 vector and
transformed into the E. coli BL21 (DE3) expression host.
The E. coli BL21 (DE3) strain harboring the recombined
plasmid was inoculated into fresh LB medium at 37uC with gentle
shaking. After the OD600of the culture reached 0.6, the QsdH-
GST fusion protein was induced by the addition of isopropyl-D-
thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM
followed by further incubation at 22uC for 6 h. The cells were then
harvested by centrifugation, washed with PBS buffer, resuspended
in PBS buffer, lysed under high pressure and finally centrifuged at
4,0006g for 30 min at 4uC to obtain the supernatant. The QsdH-
GST protein was bound to glutathione affinity resin with the GST
fusion protein purification kit (GE Healthcare). Then, 10 ml of the
3C protease stock solution (10 U/l; GE) diluted with PBS buffer
was added into the purification column to digest the GST tag
linker overnight after the unbound bacterial proteins completely
washed off the column. Finally, the purified protein was stored
with an equivalent amount of pure glycerol at 280uC and
analyzed with 12% SDS-PAGE.
Construction of the qsdH mutant
The mutants were constructed by mutating specific amino acids
according to the protocol of the TransGen Easy Mutagenesis
System. Site-directed mutagenesis was carried out with the pGEX-
6p-qsdH plasmid and pairs of complementary oligonucleotides
containing the desired mutation (Table 2). The PCR products
(10 ml) were digested with 1 ml of Dpn I (TaKaRa) for 2 h at 37uC,
which removed the wild-type templates. The digested PCR
samples were then transformed into DMT competent cells for
further digestion of the parental plasmid. Subsequently, the
transformants were screened with ampicillin. Finally, all mutations
were verified by double-strand DNA sequencing. Plasmids
harboring the desired mutation were transformed into E. coli
BL21 (DE3) for further protein expression and purification.
Analysis of the AHL-degrading activity of QsdH
For in vivo assays, E. coli DH5a containing constructs of interest
were inoculated into LB medium containing 100 mg/ml ampicillin
and grown overnight followed by incubation with 3OC8HSL to a
final concentration of 2 mM for 4 h at 37uC. Subsequently, the
mixtures were heated in a 95uC water bath and detected by A.
tumefaciens strain NT1. For the purified protein assay, a standard
reaction mixture containing 90 ml of 200 mM NaH2PO4-
Table 1. Bacterial strains and plasmids used in the study.
Strain or plasmid Characteristics source and reference
E. coli strain DH5a
a host for DNA cloning Lab collection
E. coli BL21 (DE3) a cloning and expression hostLab collection
E. carotovora SCG1 plant-pathogenic bacteriaLab collection
A. tumefaciens NT1 AHL-detecting strain; traR; tra::lacZ749Dr. Lianhui Zhang
P. byunsanensis 1A01261AHL-degrading strain; marine bacteriaMCCC
pUC118-sm20 Apr; screened target gene inserted into pUC118In this study
pUC118-orforf amplified and cloned into pUC118In this study
pUC118-orf’orf (process signal peptide) amplified and cloned into pUC118In this study
pUC118-qsdHGDSL hydrolase gene qsdH amplified and cloned into pUC118 In this study
pGEX-6p-qsdHGDSL hydrolase gene qsdH amplified and cloned into pGEX-6p-1In this study
MCCC, Marine Culture Collection of China.
A Novel Lactonase in RND-Type Transporter Protein
PLOS ONE | www.plosone.org3October 2012 | Volume 7 | Issue 10 | e46587
Na2HPO4buffer (pH 7.3), 5 ml of 100 mM 3OC8HSL and 5 ml of
the purified protein (2.5 mg) was used. A mixture composed of
95 ml of the same buffer and 5 ml of 100 mM 3OC8HSL was used
as a control. After incubation at 40uC for 30 min, the mixture was
heated in boiling water for 5 min and detected with A. tumefaciens
HPLC was performed according to a previously described
method . The amount of AHL was estimated by comparing
the reduction in peak areas with an AHL solution of known
concentration and was further calibrated with pure AHLs in the
concentration range of 100 nM to 2, 000 nM. The optimal
temperature of activiy was measured at 20uC to 60uC, under
enzymatic reaction system of 5 ml of purified QsdH in 490 ml of
NaH2PO4- Na2HPO4 buffer (pH 7.3) and 5 ml of 100 mM
3OC8HSL; the non-enzymatic degradation of 3OC8HSL in
reaction system at different temperatures were detected as
controls. After incubation of 30 min, the reactions were stopped,
and the AHLs were extracted three times with an equal volume of
ethyl acetate. The combined organic phase in sample was
evaporated with nitrogen to dryness, and then the residual
3OC8HSL were detected by HPLC. For HPLC analysis, the
samples were dissolved in 500 ml of column buffer (methanol:
water at 4:6; v/v), which was introduced into a symmetry C18
reverse-phase column (4.6 mm6250 mm) (Kromasil C18-5u) and
detected with an UV/visible light (Waters) detector set at 200 nm.
Fractions were eluted with a mobile phase of methanol: water
(60:40; v/v) at a flow rate of 1 ml/min. To detect the substrate
specificity of purified QsdH for AHL-degrading activity, 5 ml of
purified QsdH reacted with different AHLs (C4HSL, C6HSL,
C8HSL, C10HSL, C12HSL,
3OC8HSL) respectively. After incubation for 30 min at 40uC,
the reaction was halted with a 95uC water bath. AHLs were
extracted three times with an equal volume of ethyl acetate, and
the combined organic phase was then evaporated. AHLs (1 mM)
were incubated in the same enzyme reaction buffer at 40uC for
30 min and extracted as a control. Samples were dissolved in
500 ml of column buffer and detected by HPLC. To investigate the
effects of divalent ions on enzymatic activity, several divalent ions
at final concentrations of 0.2 and 10 mM were added into the
enzymatic reaction system. After incubating 1 mM 3OC8HSL
(final concentration) with 5 ml of purified QsdH in a reaction
buffer containing different divalent ions for 30 min at 40uC, the
residual 3OC8HSL were extracted three times with ethyl acetate
respectively. The combined organic phase was then evaporated to
dryness. The samples were redissolved in methanol, and
3OC8HSL was detected and quantified by HPLC.
Determining the mechanism of AHL degradation of
To determine the chemical structure of products of the AHLs
and QsdH, 3OC8HSL was digested by QsdH, and the reaction
products were analyzed using HPLC and ESI-MS. A 5 ml of the
purified QsdH (2.5 mg) enzyme solution with 1 mM 3OC8HSL
(final concentration) was added to the Na2HPO4-KH2PO4
reaction buffer (pH 7.3). After incubation for 30 min at 40uC,
the reaction was halted with a 95uC water bath, and the reacted
products were extracted three times with ethyl acetate. The
combined organic phase was then evaporated to dryness. The
samples were dissolved in methanol and introduced into a
symmetry C18 reverse-phase column for HPLC analysis. The
lactonolysis product of 3OC8HSL, 3-oxooctanoyl-L-homoserine,
was prepared by hydrolysis of the lactone ring in alkaline buffer as
previously described . 3OC8HSL (1 mM) was digested in a
solution containing 200 ml of dimethyl sulphoxide (DMSO) and
300 ml of 1 M NaOH for 6 h at 37uC, and the mixture was then
adjusted with H3PO4 to a pH of 5.0. Next, 3-oxooctanoyl-L-
homoserine was extracted three times with ethyl acetate and
evaporated to dryness. The sample was redissolved in a methanol:
water solution and purified by HPLC using a C18reverse-phase
column. ESI-MS was performed with an API5000 triple-quadru-
pole instrument from Applied Biosystems (USA). Samples were
dissolved in methanol and ionized by negative-ion electrospray.
The production of AHLs and the expression of qsdH in P.
byunsanensis strain 1A01261
To determine whether the production of AHLs was reliable to
the cell density of P. byunsanensis strain 1A01261, we extracted
AHLs from culture of strain 1A01261. The amount of AHLs was
detected by HPLC with standard AHLs as controls. Strain
1A01261 was cultured in 2 L modified LB media (LB medium
with 3% (w/v) NaCl) at 25uC. At 24-h intervals a 50-ml aliquot of
the culture was centrifuged, and the supernatant was extracted 3
times with 50 ml of chloroform. The extract was evaporated,
dried, and dissolved in 1 ml of methanol for HPLC analysis. The
growth of the bacteria was calculated by measuring the
absorbance at 600 nm; bacteria was inoculated into medium
and cultured for 0 h as a control.
Table 2. Primers for site-directed mutagenesis.
Amino acid SubstitutionSequence2
1F, forward primer; R, reverse primer.
2The bases changed are shown in boldface type in each primer.
A Novel Lactonase in RND-Type Transporter Protein
PLOS ONE | www.plosone.org4 October 2012 | Volume 7 | Issue 10 | e46587
To detect the expression of QsdH in strain 1A01261, we
identified the expression of qsdH in RNA level. At 24-h intervals
2 ml culture of bacteria incubated in modified LB medium was
centrifuged with a control of uncultured bacteria, and RNA in cell
was extracted by Trizol reagent as the method on Operating
instruction in kit (Invitrogen, USA). After removal of DNA
template in samples, cDNA was amplified from RNA with random
9 mers by RT-PCR as the method annexed in RT-PCR kit
(Ferments, USA). Finally, the gene qsdH was amplified with
specific primers of 59-GCGCCCCCTTCGCCGCAGTA-39 and
E. carotovora as a plant pathogen produces and secretes
exoenzymes that act as virulence determinants for soft rot diseases
of various plants. To test the effect of AHL-lactonase on bacterial
infection, we detect the prevention of bacterial infection of E. coli
BL21 containing over-producing QsdH. After E. carotovora strain
SCG1 was cultivated until the OD600was 1.0, 200 ml of culture
was centrifuged and then diluted with saline solution (0.15 M
NaCl) to a final OD600 of 0.1. Recombinant E. coli carrying
pGEX-6p-qsdH and E. coli carrying pGEX-6p-1 were harvested
6 h after IPTG induction at 22uC, and resuspended in saline
solution to a final OD600of 3.0. After bacteria were diluted, equal
volumes of E. carotovora SCG1 and recombinant E. coli were mixed
respectively. Equal volumes of 0.15 M NaCl solution incubated
with E. carotovora SCG1 as a negative control. 20 ml of the mixtures
were loaded onto potato slices and incubated at 30uC for 24 h. In
addition, 20 ml of E. coli contaning pGEX-6p-qsdH or pGEX-6p-1,
and NaCl were loaded onto potato slices respectively as positive
controls. Watery rotten lesions around inoculation sites were
observed as evidence of the activation of virulence, the area of
rotten lesions in potato representing amount of virulence.
Nucleotide sequence accession number
The nucleotide sequences of orf gene, encoding the RND-type
efflux transporter protein from P. byunsanensis strain 1A01261 have
been deposited in the DDBJ/EMBL/GenBank databases under
accession number JX392407.
Identification of AHL-degrading activity of marine
bacteria P. byunsanensis strain 1A01261
One thousand independent isolates from the Marine Culture
Collection of China (MCCC) were incubated with 3OC8HSL,
and the AHL-degrading activities were detected on agar plates
with A. tumefaciens NT1. Among these isolates, ten strains displayed
different AHL-degrading activity, including P. byunsanensis, Alcani-
vorax dieselolei, Bacillus cereus, Alcanivorax venusti and Marinobacter
hydrocarbonoclasticus, Halomonas sp. and so on. In which P. byunsanensis
(MCCC1A01261) displayed high AHL-degrading activity. More-
over, the main AHL-degrading activity of strain 1A01261 was
exhibited in supernatant of the marine strain. In addition, the
AHL-degrading activity of P. byunsanensis strain 1A01261 was
thermostable because it still harbored high activity after the
supernatant of bacterial culture incubated at 60uC for 30 min.
Finally, P. byunsanensis spp. represents a novel species within the
Pseudoalteromonas genus and was first isolated in 2009 , study
about which was little. Therefore, as a novel material, P.
byunsanensis had the potential value for study.
Cloning and characterization of the AHL-degrading gene
from P. byunsanensis strain 1A01261
After screening approximately 6, 000 transformants from a
genomic library of P. byunsanensis 1A01261, several positive clones
were obtained and then rescreened with the biosensor A. tumefaciens
NT1. The positive transformant-containing plasmid, pUC118-
sm20, displayed AHL- degrading activity with complete inactiva-
tion of 2 mM 3OC8HSL in 4 h (Fig. 1A). Sequencing analysis
demonstrated that pUC118-sm20 contained a cloned genomic
fragment of 3, 116 bp, which encompassed one complete open
reading frame (ORF) (Fig. 1B). The orf gene was predicted to
encode a protein of 968 amino acids with a predicted isoelectric
point at 4.84. BLAST analysis suggested that this gene encom-
passed the following two catalytic domains: an N-terminal GDSL
hydrolase fold family domain (the highly conserved residues ‘Gly-
Asp-Ser-X’ around the catalytic site) and a C-terminal RND-type
multidrug efflux transporter domain.
The measurement of the AHL-degrading activities of E. coli
DH5a harboring different recombinant plasmids, including
pUC118-orf, pUC118-orf’ and pUC118-qsdH, elucidated that all
three of the transformants completely digested 2 mM 3OC8HSL
within 4 h (Fig. 1A). This result suggested that the catalytic region
containing a GDSL hydrolase possessed similar enzymatic activity
to the full-length ORF. Among enzymes with demonstrated
functions, QsdH was most similar to the secreted hydrolase from
Streptomyces spp. AA4 (GI 302529957), sharing 47% identity at the
amino acid level. QsdH also shared 45% identity with the
triacylglycerol lipase from Kribbella flavida DSM17836 (GI
284031490) and 34% identity with EstA from Pseudoalteromonas
spp. 643A (GI 194369063). However, the tertiary fold of the
GDSL hydrolase family is substantially different from that of the
alpha/beta hydrolase family, which is unique among all known
hydrolases. The active site of GDSL hydrolase proteins contains
two of the three components of a typical Ser-His-Asp (Glu) triad
found in other serine hydrolases but may lack the carboxylic acid
[32,33]. QsdH was found to contain three conserved residues in I,
II and III blocks, which formed the oxyanion holes (Ser42-Gly83-
Asn183) of the GDSL hydrolase fold according to the analysis of a
multiple protein sequence alignment (Fig. 2). However, QsdH was
found to lack the catalytic His and Asp in block V.
Figure 1. Screening an AHL-degrading activity gene qsdH and
the physical map of qsdH locus. (A) The AHL-degrading activity of E.
coli DH5a harboring different plasmids detected with biosensor A.
tumefaciens strain NT1. CK, pUC118, used as a control; 1, pUC118-sm20,
which was the screened positive transformant; 2, pUC118-orf (encodes
the full ORF protein); 3, pUC118-orf’ (encodes the ORF protein removing
the N-terminal signal peptide); and 4, pUC118-qsdH. (B) A schematic
representation of the complete ORF, which contains an N-terminal
GDSL hydrolase domain and a RND-type multidrug efflux protein
domain with the GDSL-lipolytic enzyme named QsdH.
A Novel Lactonase in RND-Type Transporter Protein
PLOS ONE | www.plosone.org5 October 2012 | Volume 7 | Issue 10 | e46587
Enzymatic characterization of the AHL-degrading activity
Because the qsdH gene lacking the signal peptide still manifested
AHL-degrading activity, the mature QsdH was purified using
GST affinity chromatography. The calculated molecular weight of
27.0 kDa was consistent with the molecular mass determined by
12% SDS-PAGE (Fig. 3A). An examination of the AHL-degrading
activity showed that purified QsdH was able to completely
degrade 5 mM 3OC8HSL at 40uC for 30 min (Fig. 3B). To
determine if QsdH degraded AHL by acting as a lactonase or as
an acylase, 3OC8HSL was digested with purified QsdH, and the
reaction products were then analyzed via HPLC and ESI-MS.
Fractionation of pure 3OC8HSL revealed one major HPLC peak
with a retention time of 17.3 min and a minor peak with a
retention time of 12.7 min, which was the sodium salt of
3OC8HSL based on MS analysis (data not shown). Meanwhile,
the ring-opened product of 3OC8HSL, which was prepared by
hydrolyzing 3OC8HSL with 1 M NaOH, displayed a HPLC peak
with a retention time of approximately 4.3 min (Fig. 4A). The
enzyme digestion products contained a similar peak with a
retention time of 4.3 min and a peak of undigested 3OC8HSL
with a retention time of 17.3 min. To confirm product identity,
enzyme-digested product fragments were collected for ESI-MS
analysis. The results demonstrated that the 4.3 min HPLC
fragment was a substrate of a quasimolecular (M-H) ion at m/z
of 258.1 (Fig. 4B), which indicated that an enzymatic digestion of
3OC8HSL led to a mass increase of 18 in its products. In addition,
MS2 analysis of the parent ion at m/z of 258 by tandem mass
spectrometry showed a daughter ion of 118.1 (Fig. 4B; down
column), which was consistent with the formula of C4H9NO3
(homoserine; M-H ion m/z of 118.1). Together, these data
demonstrated that qsdH encodes an AHL lactonase that hydrolyzes
the homoserine lactone ring of 3OC8HSL, thus releasing N-acyl-
QsdH exhibited activity at temperature ranging from 20uC to
60uC, reaching its optimum activity at 40uC after incubation of
30 min at pH 7.3 using 3OC8HSL as a substrate (Fig. 5A). The
substrate specificity of QsdH was determined by detecting the
enzymatic activity against a range of AHLs with or without
substitution of carbon 3 by HPLC. Figure 5B shows that QsdH
exhibited high relative activities to all tested AHLs but worked
better with short- and medium-chain acyl homoserine lactones
(such as C4HSL, C6HSL, C8HSL and C10HSL) than long-chain
acyl homoserine lactones (C12HSL and C14HSL). A 3-oxo
substitution of AHLs (3OC8HSL and 3OC6HSL) was detected
and efficiently degraded by QsdH.
To determine the effect of divalent ions on the AHL-degrading
activity of QsdH, several divalent ions (Zn2+, Cu2+, Ca2+, Mg2+,
Ni2+, Ba2+, Sr2+and Mn2+) were added into the enzyme reaction
system, and the AHL-inactivating function of QsdH was evaluated
by HPLC. The effects of two concentrations (0.2 and 10 mM) of
various metal ions on the enzymatic activity of QsdH are shown in
Table 3. The presence of all assayed metal ions at 0.2 mM showed
a slight enhancement of the AHL-degrading activity of QsdH,
which was approximately 5–15% greater than the control.
However, most of the tested metal ions at 10 mM inhibited the
AHL-degrading activity of QsdH.
QsdH is a member of the AHL lactonase family
Sequence alignment was performed with QsdH and previously
studied AHL lactonases. QsdH shared a low similarity to known
lactonases. For example, the sequence similarity of QsdH to
several AHL lactonases was as follows: 26% similarity to AiiA
Figure 2. Multiple alignment of the deduced amino acid sequence of qsdH and GDSL-like lipase/esterase. 1, GDSL family lipase from
Amycolatopsis mediterranei U32 (GI: 300788557); 2, a putative secreted hydrolases from Streptomyces sp. Tu6071 (GI: 333028773); 3, a GDSL family
lipase from Pseudonocardia sp. P1 (GI: 324999532); 4, a GDSL family lipase from Segniliparus rotundus DSM 44985(GI: 296394487); 5, a secreted
hydrolases from Streptomyces sp. AA4 (GI: 302529957); 6, a triacylglycerol lipase from Kribbella flavida DSM 17836 (GI: 284031490) and 8, a esterase
from Coccidioides posadasii str. Silveira (GI: 320039410). Conserved residues are shaded in gray, and the catalytic amino acid residues (Ser, Gly and
Asn) in the consensus sequences are marked.
Figure 3. Purification of QsdH and identification of AHL-
degrading activity of QsdH. (A) Analysis of the expression and
purification of QsdH protein by 12% SDS-PAGE. M is the standard
molecular weight markers (TaKaRa); Lanes 1 and 2 are uninduced and
induced cell lysates of E. coli BL21 (DE3) harboring pGEX-6p-qsdH,
respectively. Lane 3 is the purified QsdH from P. byunsanensis 1A01261.
The protein bands of GST-QsdH and QsdH are marked respectively. (B)
AHL-degrading activity of purified QsdH. The solution of purified QsdH
was mixed into the reaction buffer containing 5 mM 3OC8HSL (final
concentration) and incubated at 40uC for 30 min. The residual 3OC8HSL
was detected by A. tumefaciens strain NT1. The control consisted of
5 mM 3OC8HSL in reaction buffer incubated at 40uC for 30 min.
A Novel Lactonase in RND-Type Transporter Protein
PLOS ONE | www.plosone.org6 October 2012 | Volume 7 | Issue 10 | e46587
(Bacillus spp.), 24% similarity to QsdA (R. erythropolis W2), 25%
similarity to AhlD (Arthrobacter spp.), 32% similarity to AiiM (M.
testaceum), and no similarity to AidH (Oclrobacterum spp.), AiiB and
AttM (A. tumefaciens). The main zinc-binding motif of the conserved
HXHXDH sequence is found in many AHL lactonases. For
example, AidH and AiiM have a a/b hydrolase fold in which a
conserved G-X-Nuc-X-G or histidine is critical to the AHL-
degrading activity. However, these conserved motifs were not
found in QsdH, which suggested that QsdH was quite different
from the abovementioned AHL lactonases. EstA, a homologous
gene of QsdH from Pseudoalteromonas spp. 643A, has been shown to
have activity for esters of short- to medium-chain (C4and C10)
fatty acids and to not have activity for long-chain fatty acid esters
or any lactones . Meanwhile, the EstA of Serratia liquefaciens
MG1, which is a GDSL-lipolytic enzyme, has been shown to
hydrolyze only short-chain naphthol esters with a maximum of six
carbons . Together, these data suggest that the functional
QsdH may not be present in the two strains.
The conserved serine, glycine and asparagine residues
are required for QsdH activity
Site-directed mutagenesis was used for exploring the roles of
these conserved residues (Ser42, Gly83 and Asn183) in three
conserved blocks. We replaced Ser42, Gly83 and Asn183 with a
Val residue and replaced Asn183 with a Ser residue. These
mutants were then purified in a manner indistinguishable from
that of wild-type QsdH. The activities for AHL inactivation of
these mutants are shown in Figure 6A. The enzymatic activity of
the S42V, G83V, N183V and N183S mutants was drastically
reduced. These results suggested that the Ser, Gly and Asn
oxyanion residues in the conserved blocks have important roles in
QsdH activity and that the loss of activity is due to changes in the
biochemical properties of the mutant proteins but not in the
expression conditions (Fig. 6B).
In silico prediction of QsdH localization within the cell
A BLAST search revealed that the amino acid sequences
deduced for the ORF protein showed a high identity with the
CzcA family metal efflux proteins from Alteromonas spp. SN2 and
Glaciecola spp. HTCC2999, as well as the RND divalent metal
cation efflux transporters from Collimonas fungivorans Ter331 and
Alcanivorax borkumensis SK2 (Fig. 7). These results demonstrated that
the ORF protein belongs to a RND-type transporter protein
family, which is one part of a three-component multidrug efflux
pump. Subsequent determination of the cellular location of the
RND-type transporter protein by the SPORT computer program
revealed that this protein is located at the inner membrane. These
data suggested that the N-terminal GDSL hydrolase, QsdH, is
located on the cytoplasmic membrane of the multidrug efflux
protein. As predicted by the SIGNALP program, SOPM program
and secondary structure prediction method (http://pbil.ibcp.fr/
htm/index.php), the N-terminal 29 amino acids of QsdH are a
signal sequence. The secondary structure of the signal peptide
domain was found to have the following features: 1) N-domain
with positively charged amino acids (MRRR); 2) hydrophobic
region (H-domain) of neutral amino acids (RRALSIATALAA-
LAAGVG); and 3) signal peptidase recognition sites (AGA) with
cleavage occurring after the second Ala. This type of signal peptide
is common in previously studied autotransporter proteins in gram-
negative bacteria . In addition, domain analysis via SMART
(http://smart.embl-heidelberg.de/) predicted that the full length
ORF protein has 12 transmembrane domains (a-helix) leaving
both the amino and carboxyl termini on the cytoplasmic side of
the inner membrane, and that two periplasmic loops of a long
hydrophilic domain containing 300 amino acids are formed
between TM1 (transmembrane 1) and TM2 and between TM7
and TM8. According to sequence alignment among RND-type
transporter proteins, the GDSL hydrolase, QsdH, is located in the
first periplasmic loop. Based on these predictions and analyses, we
concluded that QsdH may be inserted into a RND-type inner
membrane, which is exposed to the exterior periplasm.
Figure 4. HPLC and ESI-MS spectrometry analysis of the QsdH-catalyzed OHHL product. (A) HPLC analysis of the AHL lactonase digestion
product of 3OC8HSL. (Upper) HPLC profile of 3OC8HSL (retention time of 17.3 min). The minor peak at 12.7 min was the sodium salt of 3OC8HSL
based on MS analysis (data not shown). (Middle) HPLC profile of 3OC8HSL hydrolyzed by NaOH, which released the ring-opened product of 3OC8HSL
with a retention time of 4.3 min. (Lower) HPLC profile showed that 3OC8HSL digestion by AHL lactonase resulted in products with a retention time of
4.3 min. (B) ESI-MS and MS2 analysis of the hydrolysis product of 3OC8HSL by QsdH. (Upper) ESI-MS analysis of the 4.3 min HPLC fragment of
enzymatic-digested products showed a quasimolecular (M–H) ion substrate at m/z of 258.1. (Lower) MS2 analysis of the parent ion at m/z of 258 by
tandem mass spectrometry showed a daughter ion of 118.1.
A Novel Lactonase in RND-Type Transporter Protein
PLOS ONE | www.plosone.org7 October 2012 | Volume 7 | Issue 10 | e46587
The production of AHLs and the expression of qsdH in P.
byunsanensis strain 1A01261
The time course for the growth of bacteria and production of
AHLs was shown in Fig. 8A. In this study, bacterial growth of P.
byunsanensis strain 1A01261 reached the high point at 72 h, and
decreased thereafter. At 24-h intervals 50 ml culture of strain was
centrifuged and detected the production of AHL by HPLC. The
result demonstrated that the production of 3OC8HSL (sodium salt
of 3OC8HSL was detected in bacterial culture of modified LB
media which contain high concentration of NaCl) was increased at
48 h, and decreased thereafter till to disappear at 144 h.
We also detected the qsdH expression at 24-intervel culture of
strain 1A01261. The result demonstrated that qsdH expression was
appeared at 24 h to 72 h, on which the cell grew in log phrase,
(Fig. 8B). After 72 h, the qsdH expression was not detected, and
now the signal molecule, 3OC8HSL, was accumulated.
QsdH-overproducing E. coli for the attenuation of plant-
pathogenic E. carotovora
The QsdH-overproducing E. coli mixed with E. carotovora SCG1,
and displayed a decrease of watery rotten lesions in the potato
slices. In contrast, attenuation was not observed in recombinant E.
coli containing plasmid pGEX-6p-1 (Figure 9). This result showed
that the potato virulence of E. carotovora whose virulence was
regulated by AHLs was attenuated by AHL-degrading enzyme-
overproducing E. coli. This suggested that the enzymatic quench-
ing of AHL quorum-sensing signals by QsdH is a feasible
approach for prevention of bacterial infection, which might has
potential to use for the control of gram-negative plant-pathogenic
Figure 5. Enzymatic characteristics of QsdH for AHL-degrading
activity. (A) Effect of temperature on enzyme activity. The activity of
QsdH to 3OC8HSL was measured at temperatures ranging from 20uC to
60uC. The residual 3OC8HSL in reaction system without enzyme at
different temperatures was detected as controls. The highest activity at
40uC was defined as 100%. (B) Detect the substrate specificity of AHL-
lactonase QsdH. 1 mM different substrate incubated with purified
QsdH, and then the residual substrate was quantified by HPLC. Data are
the means of 3 measurements. The activity of QsdH toward 3OC8HSL
was defined as 100%. The 3OC8HSL-degrading activity without QsdH is
represented as the control.
Table 3. Effects of metal ions on QsdH activity for 3OC8HSL.
Relative activity (%)
10 mM0.2 mM
Figure 6. Conserved residues are important for the enzymatic
activity of QsdH. (A) Site-directed mutagenesis replacing Ser42,
Gly83, and Asn183 with Val and replacing Asn183 with Ser reduced
QsdH activity. The purified mutants were incubated with 1 mM
3OC8HSL, and the residual 3OC8HSL was detected by HPLC. Data are
the mean values of three measurements. The activity of wild-type QsdH
toward 3OC8HSL was defined as 100%. (B) Substitution mutants of
QsdH made stable proteins. The mutants were expressed and purified
in the same manner as wild-type QsdH, and purified mutants were
analyzed by SDS-PAGE. 1, standard molecular weight markers (TaKaRa);
2–6, purified proteins of wild-type QsdH and mutants S42V, Gly83V,
N183V and N183S, respectively.
A Novel Lactonase in RND-Type Transporter Protein
PLOS ONE | www.plosone.org8 October 2012 | Volume 7 | Issue 10 | e46587
bacteria in the production of crops.
The P. byunsanensis strain 1A01261 produces an enzyme that can
inactivate a broad range of AHLs with or without 3-oxo
substitutions. In this study, the orf gene encoding an AHL
inactivation protein was cloned and fully sequenced. Sequence
analyses with BLAST suggested that this protein contained the
following two distinct domains: an N-terminal catalytic domain
that harbored a GDSL hydrolase domain and a C-terminal RND-
type efflux pump domain. Moreover, removal of the C-terminal
RND-type transporter membrane domain rendered a stand-alone
GDSL hydrolase domain, which still retained full AHL-degrading
activity. The purified QsdH exhibited broad spectrum substrate
specificity to AHLs. The HPLC and ESI-MS analysis demon-
strated that QsdH was an AHL lactonase able to hydrolyze the
homoserine lactone ring to release N-acyl-homoserine. Moreover,
QsdH shared both low similarity to known AHL lactonases and
low identity to reported GDSL-like hydrolases.
These enzymes of the GDSL hydrolase family have two obvious
features as follows: 1) the enzymes share little sequence homology
with true lipases; and 2) unlike other lipases where the GXSXG
motif is near the center, the serine-containing motif of the GDSL
subfamily (Gly-Asp-Ser-X) is closer to the N-terminus [32,37].
GDSL-like hydrolases are useful to many branches of industry
because of their multifunctional properties, such as board substrate
specificity, regiospecificity, flexibility of active site and capability of
changing the conformation of the substrate after binding
[32,37,38]. Riedel et al. reported that the GDSL esterase, EstA,
which is located proximal to the swr quorum-sensing system of
Serratia liquefaciens MG1, is required for AHL biosynthesis when
cells are grown on certain lipidic substrates to provide fatty acids
. In the present study, however, the GDSL hydrolase, QsdH,
conferred strong enzymatic activity to all tested AHLs, which
suggested that N-acyl-homoserine lactones are the first reported
substrates of the GDSL hydrolase family. In addition, mutagenesis
of the conserved catalytic residues (Ser, Gly and Asn) in the three
conserved regions (blocks I, II, III) led to greatly reduced lactonase
activity of QsdH, which indicated that QsdH was clearly a
Figure 7. QsdH is localized in the inner membrane of a RND-type multidrug efflux transporter. The alignment of the ORF protein with
two multidrug efflux pumps was showed. 1, ORF protein; 2, CzcA family heavy metal efflux protein from Alteromonas spp. SN2 (GI: 333892934); and 3,
CzcA family heavy metal efflux protein from Pseudoalteromonas atlantica T6c (GI: 109898348). Structural predictions illustrate 12 transmembrane
helices with two perisplasmic loops located between TM1 and TM2 and between TM7 and TM8 in the ORF protein (RND-type efflux transporter). The
12 transmembrane helices are shaded with a green box, and the typical signal peptide is marked in the ORF protein with a red box. Using in silico
prediction, the GDSL hyrolase, QsdH, was predicted to form the first perisplasmic loop of the RND-type multidrug efflux transporter.
A Novel Lactonase in RND-Type Transporter Protein
PLOS ONE | www.plosone.org9 October 2012 | Volume 7 | Issue 10 | e46587
member of the GDSL hydrolase family. In particular, these
conserved active site residues had pivotal roles in the enzymatic
activity of AHL inactivation. GDSL hydrolases are not known to
require cofactors for their activity , which was consistent with
our observations that the lactonase activity of QsdH did not rely
on any divalent ion.
Intriguingly, the GDSL hydrolase, QsdH, was found to be
integrated into the inner membrane protein, RND-type efflux
transporter, which is involved in multidrug transportation.
Recently, several GDSL family lipolytic enzymes covalently
attached to outer membranes and secreted by gram-negative
bacteria have been designated to be members of an autotran-
sporter family [35,39,40,41]. To our knowledge, this is the first
report of a GDSL hydrolase anchored with a C-terminal
cytoplasmic membrane of the multidrug efflux transporter protein
in the Pseudoalteromonas genus. The following three distinct domains
have been suggested to be present in nearly all autotransporter
proteins: amino-terminal leader peptide; surface-localized mature
protein; and carboxy-terminal domain that mediate secretion
through the outer membrane. Moreover, prior investigations have
identified the key structural features of the amino-terminal leader
peptide of autotransporter proteins involved in exporting precur-
sors through the inner membrane in a Sec-independent manner
. In the present study, a typical signal peptide of inner
membrane proteins was predicted in the N-terminus of QsdH and
was predicted to constitute the first transmembrane structure of
the multidrug protein. These data revealed that the signal peptide
may lead to the translocation of QsdH to the inner membrane
without being cleaved.
The RND-type transporter is one part of the three-component
efflux pump, which has a significant role in multidrug efflux. These
tripartite pumps are composed of an integral inner membrane
drug-proton antiporter of the RND family of exporters, a channel-
forming outer membrane efflux protein (or outer membrane
factor; OMF) and a periplasmic membrane fusion protein (MFP)
. Studies over the past few years have documented that these
multiprotein complexes transport a wide variety of substrates,
including antibiotics, dyes, detergents and host-derived molecules,
from the periplasm to the extracellular space . However,
previous investigations have outlined that the efflux of long-chain
Figure 8. Production of OOHL and QsdH expression in P. byunsanensis. (A) Time Courses for the Growth of P. byunsanensis Strain 1A01261,
and the Production of N-Acylhomoserine Lactones (AHLs). The growth of the bacteria was calculated by measuring the absorbance at 600 nm. Strain
1A01261 was cultured in modified LB media (LB medium with 3% (w/v) NaCl) at 25uC. At 24-h intervals a 50-ml aliquot of the culture was centrifuged,
and AHLs were extracted from supernatants of cultures, which were measured by HPLC. The extracted AHL was identified as sodium salt of 3OC8HSL
compared with control. Maximum abundance of sodium salt of 3OC8HSL was measured at 72 h, which was defined as 100%. (B) The qsdH expression
was detected with RT-PCR. M, DNA marker (TaKaRa); 0–7, qsdH amplified from total RNA from cultures of P. byunsanensis strain 1A01261, with strain
incubating for 7 days; the culture for 24-h intervals as a sample, the PCR products amplified from samples was detected by agar gel electrophoresis.
A Novel Lactonase in RND-Type Transporter Protein
PLOS ONE | www.plosone.org 10October 2012 | Volume 7 | Issue 10 | e46587
AHLs used to synchronize quorum sensing in many gram-negative
bacteria dependson active
[24,25,26,27,28]. Furthermore, previous studies have illustrated
that the integral inner membrane RND component of three-
component multidrug efflux systems defines the substrate selectiv-
ity of these efflux systems [43,44]. The inner membrane RND-
type transporter contains 12 membrane-spanning helices and 2
periplasmic loops of approximately 300 amino acids between
helixes 1 and 2 and between helixes 7 and 8 . More recently,
the importance of the periplasmic loops for substrate recognition
and transport in P. aeruginosa has been demonstrated [43,44,46]. In
this study, the GDSL hydrolase, QsdH, lies in the first periplasmic
loop. Thus, the possibility that AHLs are digested by QsdH when
these signal molecules influx or efflux through the inner
membrane should not be excluded.
The Pseudoalteromonas genus is a marine group of bacteria known
to influence boil formation in various marine econiches . P.
byunsanensis spp. represents a novel species within the Pseudoalter-
omonas genus and was first isolated from tidal sediment in Korea.
The phenotypic features of P. byunsanensis spp. are similar to those
of Pseudoalteromonas phenolica and Pseudoalteromonas luteoviolacea
exhibiting alkaline phosphatase, esterase (C4), esterase lipase (C8)
and leucine arylamidase activities . Several phenotypic
characterizations of the Pseudoalteromonas spp. marine bacterium
associated this species with quorum sensing. For example, the
antibacterial activity of Pseudoalteromonas spp. NJ6-3-1 is regulated
by quorum sensing . Additionally, N-(3-oxooctanoyl)-homo-
serine lactone has been shown to be a signaling molecule involved
in the production of violacein of Pseudoalteromonas spp. 520P1 .
Furthermore, the expression of five enzymes (VioA-VioE) is
responsible for synthesizing violacein of Pseudoalteromonas spp.
520P1, which is regulated by a quorum-sensing mechanism .
In this study, bacterial growth of P. byunsanensis strain 1A01261
reached the high point at 72 h, and decreased thereafter.
Moreover, the production of 3OC8HSL was increased at 48 h,
and decreased thereafter till to disappear at 144 h (Fig. 8A),
suggesting that AHL was accumulated after the cell growth on log
phrase. Detect qsdH expression in strain 1A01261, and the result
showed that qsdH expression was appeared at 24 h to 72 h, on
which the cell grew in log phrase, suggesting the expression was
dependent on the cell growth phrase (Fig. 8B). After 72 h, the qsdH
expression was not detected in this condition, and at the same time
the signal molecule, 3OC8HSL, was accumulated. Therefore, the
AHLs accumulated in P. byunsanensis strain 1A01261 until the
AHL-lactonse QsdH was disappeared in culture. The phenome-
non indicated that the QsdH regulated the production of AHL in
strain on log phrase of cell growth when strain was cultured in this
condition. After 6 days, the AHLs might be degraded as pH
changed in culture or utilized by cell lacking of nutrition in this
phrase. The QsdH lactonase located in the RND-type inner
membrane with the catalytic domain exposed to the surface may
be a quorum-quenching mechanism of P. byunsanensis 1A01261
used to control the intracellular concentration of AHLs, and
further to regulate the expression of relevant phenotypic genes.
These findings illustrate the important roles of QsdH in microbe/
microbe and pathogen/host interactions of P. byunsanensis
In conclusion, it was demonstrated that QsdH possesses
interesting features with respect to both biological and biocatalyst
functions. The localization of QsdH in the inner membrane with
the catalytic domain oriented into the periplasm and its ability to
hydrolyze AHLs suggest an important role for this lactonase in vivo
and in vitro. The AHL-degrading activity of QsdH may perform
useful functions that affect the quorum sensing-associated pheno-
typic characterization of genus Pseudoalteromonas. The attenuation
of plant pathogenicity shows the possibility of biocontrol of gram-
negative bacteria by the use of recombinant QsdH-overproducing
microbes, indicating that P. byunsanensis might have an additional
function, including the regulation of gram-negative pathogenic
The authors would like to thank Prof. Lianhui Zhang (Institute of
Molecular & Cell Biology, Singapore) for providing the sensor strain of A.
tumefaciens, strain NT1 (traR; tra::lacZ749).
Conceived and designed the experiments: WH ZDL. Performed the
experiments: WH SYY PFL. Analyzed the data: WH JS. Contributed
reagents/materials/analysis tools: ZZS YJL ZDL. Wrote the paper: WH.
Supplied the funding: ZDL YJL.
1. Fuqua WC, Winans SC, Greenberg EP (1994) Quorum sensing in bacteria: the
LuxR-LuxI family of cell density-responsive transcriptional regulators. Journal of
Bacteriology 176: 269.
2. Miller MB, Bassler BL (2001) Quorum sensing in bacteria. Annual Reviews in
Microbiology 55: 165–199.
3. Morohoshi T, Nakazawa S, Ebata A, Kato N, Ikeda T (2008) Identification and
characterization of N-acylhomoserine lactone-acylase from the fish intestinal
Shewanella sp. strain MIB015. Bioscience, Biotechnology, and Biochemistry 72
4. Dong YH, Wang LH, Zhang LH (2007) Quorum-quenching microbial
infections: mechanisms and implications. Philosophical transactions of the
Royal Society B: Biological Sciences 362: 1201–1211.
5. Finch R, Pritchard D, Bycroft B, Williams P, Stewart G (1998) Quorum sensing:
a novel target for anti-infective therapy. The Journal of Antimicrobial
Chemotherapy 42: 569.
6. Czajkowski R, Jafra S (2009) Quenching of acyl-homoserine lactone-dependent
quorum sensing by enzymatic disruption of signal molecules. Acta Biochimica
Polonica 56: 1–16.
Figure 9. Attenuation of potato pathogenicity of Erwinia
carotovora by recombinant QsdH- producing E. coli. 1, saline
solution; 2, E. coli carrying pGEX-6p-1; 3, E. coli carrying pGEX-6p-qsdH;
4, E. carotovora; 5, mixture of E. carotovora and E. coli carrying pGEX-6p-
1; 6, mixture of E. carotovora and E. coli carrying pGEX-6p-qsdH.
A Novel Lactonase in RND-Type Transporter Protein
PLOS ONE | www.plosone.org11 October 2012 | Volume 7 | Issue 10 | e46587
7. Dong YH, Xu JL, Li XZ, Zhang LH (2000) AiiA, an enzyme that inactivates the Download full-text
acylhomoserine lactone quorum-sensing signal and attenuates the virulence of
Erwinia carotovora. Proceedings of the National Academy of Sciences 97: 3526.
8. Carlier A, Uroz S, Smadja B, Fray R, Latour X, et al. (2003) The Ti plasmid of
Agrobacterium tumefaciens harbors an attM-paralogous gene, aiiB, also encoding N-
acyl homoserine lactonase activity. Applied and Environmental Microbiology
9. Zhang HB, Wang LH, Zhang LH (2002) Genetic control of quorum-sensing
signal turnover in Agrobacterium tumefaciens. Proceedings of the National Academy
of Sciences 99: 4638.
10. Park SY, Lee SJ, Oh TK, Oh JW, Koo BT, et al. (2003) AhlD, an N-
acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other
bacteria. Microbiology 149: 1541.
11. Uroz S, Oger PM, Chapelle E, Adeline MT, Faure D, et al. (2008) A Rhodococcus
qsdA-encoded enzyme defines a novel class of large-spectrum quorum-
quenching lactonases. Applied and Environmental Microbiology 74: 1357–1366.
12. Wang WZ, Morohoshi T, Ikenoya M, Someya N, Ikeda T (2010) AiiM, a novel
class of N-acylhomoserine lactonase from the leaf-associated bacterium
Microbacterium testaceum. Applied and Environmental Microbiology 76: 2524–
13. Mei GY, Yan XX, Turak A, Luo ZQ, Zhang LQ (2010) AidH, an alpha/beta-
hydrolase fold family member from an Ochrobactrum sp. strain, is a novel N-
acylhomoserine lactonase. Applied and Environmental Microbiology 76: 4933–
14. Czajkowski R, Krzyz _anowska D, Karczewska J, Atkinson S, Przysowa J, et al.
(2011) Inactivation of AHLs by Ochrobactrum sp. A44 depends on the activity of a
novel class of AHL acylase. Environmental Microbiology Reports 3: 59–68.
15. Huang JJ, Han JI, Zhang LH, Leadbetter JR (2003) Utilization of acyl-
homoserine lactone quorum signals for growth by a soil pseudomonad and
Pseudomonas aeruginosa PAO1. Applied and Environmental Microbiology 69:
16. Huang JJ, Petersen A, Whiteley M, Leadbetter JR (2006) Identification of QuiP,
the product of gene PA1032, as the second acyl-homoserine lactone acylase of
Pseudomonas aeruginosa PAO1. Applied and Environmental Microbiology 72:
17. Lin YH, Xu JL, Hu J, Wang LH, Ong SL, et al. (2003) Acyl-homoserine lactone
acylase from Ralstonia strain XJ12B represents a novel and potent class of
quorum-quenching enzymes. Molecular Microbiology 47: 849–860.
18. Park SY, Kang HO, Jang HS, Lee JK, Koo BT, et al. (2005) Identification of
extracellular N-acylhomoserine lactone acylase from a Streptomyces sp. and its
application to quorum quenching. Applied and Environmental Microbiology 71:
19. Sio CF, Otten LG, Cool RH, Diggle SP, Braun PG, et al. (2006) Quorum
quenching by an N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa
PAO1. Infection and Immunity 74: 1673.
20. Uroz S, Chhabra SR, Camara M, Williams P, Oger P, et al. (2005) N-
Acylhomoserine lactone quorum-sensing molecules are modified and degraded
by Rhodococcus erythropolis W2 by both amidolytic and novel oxidoreductase
activities. Microbiology 151: 3313.
21. Draganov DI, Teiber JF, Speelman A, Osawa Y, Sunahara R, et al. (2005)
Human paraoxonases (PON1, PON2, and PON3) are lactonases with
overlapping and distinct substrate specificities. Journal of Lipid Research 46:
22. Ozer EA, Pezzulo A, Shih DM, Chun C, Furlong C, et al. (2005) Human and
murine paraoxonase 1 are host modulators of Pseudomonas aeruginosa quorum-
sensing. FEMS Microbiology Letters 253: 29–37.
23. Poole K, Srikumar R (2001) Multidrug Efflux in Pseudomonas aeruginosa
Components, Mechanisms and Clinical Significance. Current Topics in
Medicinal Chemistry 1: 59–71.
24. Pearson JP, Van Delden C, Iglewski BH (1999) Active efflux and diffusion are
involved in transport of Pseudomonas aeruginosa cell-to-cell signals. Journal of
Bacteriology 181: 1203.
25. Kohler T, Van Delden C, Curty LK, Hamzehpour MM, Pechere JC (2001)
Overexpression of the MexEF-OprN multidrug efflux system affects cell-to-cell
signaling in Pseudomonas aeruginosa. Journal of Bacteriology 183: 5213.
26. Aendekerk S, Diggle SP, Song Z, Hoiby N, Cornelis P, et al. (2005) The
MexGHI-OpmD multidrug efflux pump controls growth, antibiotic susceptibil-
ity and virulence in Pseudomonas aeruginosa via 4-quinolone-dependent cell-to-cell
communication. Microbiology 151: 1113.
27. Chan YY, Bian HS, Tan TMC, Mattmann ME, Geske GD, et al. (2007)
Control of quorum sensing by a Burkholderia pseudomallei multidrug efflux pump.
Journal of Bacteriology 189: 4320–4324.
28. Chan YY, Chua KL (2005) The Burkholderia pseudomallei BpeAB-OprB efflux
pump: expression and impact on quorum sensing and virulence. Journal of
Bacteriology 187: 4707–4719.
29. Piper KR, von Bodman SB, Farrand SK (1993) Conjugation factor of
Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature
30. Dong YH, Wang LH, Xu JL, Zhang HB, Zhang XF, et al. (2001) Quenching
quorum-sensing-dependent bacterial infection by an N-acyl homoserine
lactonase. Nature 411: 813–817.
31. Park YD, Baik KS, Yi H, Bae KS, Chun J (2005) Pseudoalteromonas byunsanensis sp.
nov., isolated from tidal flat sediment in Korea. International journal of
systematic and Evolutionary Microbiology 55: 2519–2523.
32. Akoh CC, Lee GC, Liaw YC, Huang TH, Shaw JF (2004) GDSL family of
serine esterases/lipases. Progress in Lipid Research 43: 534–552.
33. Molgaard A, Kauppinen S, Larsen S (2000) Rhamnogalacturonan acetylesterase
elucidates the structure and function of a new family of hydrolases. Structure 8:
34. Cies ´lin ´ski H, Białkowska AM, Długołe ˛cka A, Daroch M, Tkaczuk KL, et al.
(2007) A cold-adapted esterase from psychrotrophic Pseudoalteromas sp. strain
643A. Archives of Microbiology 188: 27–36.
35. Riedel K, Talker-Huiber D, Givskov M, Schwab H, Eberl L (2003)
Identification and characterization of a GDSL esterase gene located proximal
to the swr quorum-sensing system of Serratia liquefaciens MG1. Applied and
Environmental Microbiology 69: 3901.
36. Henderson IR, Navarro-Garcia F, Nataro JP (1998) The great escape: structure
and function of the autotransporter proteins. Trends in Microbiology 6: 370–
37. Upton C, Buckley JT (1995) A new family of lipolytic enzymes? Trends in
Biochemical Sciences 20: 178.
38. Suzuki T, Nakayama T, Choo DW, Hirano Y, Kurihara T, et al. (2003)
Cloning, heterologous expression, renaturation, and characterization of a cold-
adapted esterase with unique primary structure from a psychrotroph Pseudomonas
sp. strain B11-1. Protein Expression and Purification 30: 171–178.
39. Carinato ME, Collin-Osdoby P, Yang X, Knox TM, Conlin CA, et al. (1998)
The apeE gene of Salmonella typhimurium encodes an outer membrane esterase not
present in Escherichia coli. Journal of Bacteriology 180: 3517.
40. Talker-Huiber D, Jose J, Glieder A, Pressnig M, Stubenrauch G, et al. (2003)
Esterase EstE from Xanthomonas vesicatoria (Xv_EstE) is an outer membrane
protein capable of hydrolyzing long-chain polar esters. Applied Microbiology
and Biotechnology 61: 479–487.
41. Wilhelm S, Tommassen J, Jaeger KE (1999) A novel lipolytic enzyme located in
the outer membrane of Pseudomonas aeruginosa. Journal of Bacteriology 181: 6977.
42. Blair J, Piddock LJV (2009) Structure, function and inhibition of RND efflux
pumps in Gram-negative bacteria: an update. Current Opinion in Microbiology
43. Eda S, Maseda H, Nakae T (2003) An elegant means of self-protection in gram-
negative bacteria by recognizing and extruding xenobiotics from the periplasmic
space. Journal of Biological Chemistry 278: 2085–2088.
44. Middlemiss JK, Poole K (2004) Differential impact of MexB mutations on
substrate selectivity of the MexAB-OprM multidrug efflux pump of Pseudomonas
aeruginosa. Journal of Bacteriology 186: 1258–1269.
45. Guan L, Ehrmann M, Yoneyama H, Nakae T (1999) Membrane topology of the
xenobiotic-exporting subunit, MexB, of the MexA, B-OprM extrusion pump in
Pseudomonas aeruginosa. Journal of Biological Chemistry 274: 10517.
46. Sennhauser G, Bukowska MA, Briand C, Gru ¨tter MG (2009) Crystal structure
of the multidrug exporter MexB from Pseudomonas aeruginosa. Journal of
Molecular Biology 389: 134–145.
47. Bowman JP (2007) Bioactive compound synthetic capacity and ecological
significance of marine bacterial genus Pseudoalteromonas. Marine Drugs 5: 220–
48. Guo X, Zheng L, Cui Z, Han P, Tian L, et al. (2008) Antibacterial activity of
sponge associated marine bacterium Pseudoalteromonas sp. NJ6-3-1 regulated by
quorum sensing. Acta microbiologica Sinica 48: 545–550.
49. Wang Y, Ikawa A, Okaue S, Taniguchi S, Osaka I, et al. (2008) Quorum sensing
signaling molecules involved in the production of violacein by Pseudoalteromonas.
Bioscience, Biotechnology, and Biochemistry 72 (7): 1958–1961.
50. Zhang X, Enomoto K (2011) Characterization of a gene cluster and its putative
promoter region for violacein biosynthesis in Pseudoalteromonas sp. 520P1. Applied
Microbiology and Biotechnology 90: 1963–1971.
A Novel Lactonase in RND-Type Transporter Protein
PLOS ONE | www.plosone.org12 October 2012 | Volume 7 | Issue 10 | e46587