N -acyl-homoserine lactones from enterobacter sakazakii (Cronobacter spp.) and their degradation by bacillus cereus enzymes

Article abstract of DOI:10.1021/jf203846f

A chemical study of acyl-homoserine lactones (acyl-HSLs) produced by Enterobacter sakazakii resulted in the identification of three molecules: (S)-N-heptanoyl-HSL, (S)-N-dodecanoyl-HSL and (S)-N-tetradecanoyl-HSL. Mixed cultures of E. sakazakii and Bacillus cereus depleted E. sakazakii acyl-HSLs, suggesting acyl-HSL degradation by B. cereus hydrolases (hydrolysis of the lactone or amide moiety). The expression of B. cereus acyl-HSL lactonase and acyl-homoserine acylase was confirmed by monitoring the biotransformation of (S)-N-dodecanoyl-HSL into (S)-N-dodecanoyl-homoserine, dodecanoic acid and homoserine in the presence of B. cereus whole cells, using electrospray-mass spectrometry (ESI-MS).

Full text of DOI:10.1021/jf203846f

Article
pubs.acs.org/JAFC
N-Acyl-homoserine Lactones from Enterobacter sakazakii
Cronobacter spp.) and Their Degradation by Bacillus cereus Enzymes
(
†
‡,§
‡
́
Francisca Diana da Silva Araujo, Luciana Maria Ramires Esper, Arnaldo Yoshiteru Kuaye,
Marcelo Palma Sircili, and Anita Jocelyne Marsaioli*
∥
,†
†
Chemistry Institute, University of Campinas, POB 6154, 13084-971, Campinas, SP, Brazil
‡
Department of Food Technology, Faculty of Food Engineering, University of Campinas, POB 6121, Campinas, SP, Brazil
∥
̃
Laboratory of Bacteriology, Butantan Institute, 05503-900, Sao Paulo, SP, Brazil
*
S Supporting Information
ABSTRACT: A chemical study of acyl-homoserine lactones (acyl-HSLs) produced by Enterobacter sakazakii resulted in the
identification of three molecules: (S)-N-heptanoyl-HSL, (S)-N-dodecanoyl-HSL and (S)-N-tetradecanoyl-HSL. Mixed cultures
of E. sakazakii and Bacillus cereus depleted E. sakazakii acyl-HSLs, suggesting acyl-HSL degradation by B. cereus hydrolases
(
hydrolysis of the lactone or amide moiety). The expression of B. cereus acyl-HSL lactonase and acyl-homoserine acylase was
confirmed by monitoring the biotransformation of (S)-N-dodecanoyl-HSL into (S)-N-dodecanoyl-homoserine, dodecanoic acid
and homoserine in the presence of B. cereus whole cells, using electrospray-mass spectrometry (ESI-MS).
KEYWORDS: Enterobacter sakazakii (Cronobacter spp.), Bacillus cereus, N-acyl-homoserine lactones, foodborne diseases, enzymes
INTRODUCTION
Enterobacter sakazakii (Cronobacter spp.), a Gram-negative
bacteria of the Enterobacteriaceae family, is an opportunistic
pathogen associated with meningitis, necrotizing enterocolitis
and sepsis in neonates and immunocompromised patients, con-
sequently gaining the attention of public health authorities and
several bacterial species (e.g., Erwinia carotovora, Chromobacte-
■
7
−9
rium violaceum and Ralstonia solacearum).
4
1
,2
Lehner et al. investigated the ability of E. sakazakii to
produce acyl-HSL using thin layer chromatography and
detected two molecules: 3-oxo-hexanoyl-HSL and 3-oxo-
10
octanoyl-HSL. Pinto et al. also detected short chain acyl-
HSL in E. sakazakii, through bioassays with C. violaceum
CV026. However, isolation and full chemical characterization of
these molecules are missing.
2
−5
researchers in different countries.
Research concerning outbreaks of E. sakazakii infections in
hospitals indicated powdered infant formula as a common
contamination source, both in closed bottles or as reconstituted
Interference in quorum sensing mechanisms, known as
quorum quenching, involves, among other strategies, degrada-
tion of acyl-HSL molecules, decreasing the concentration of
these molecules in the immediate vicinity of the bacterial cells,
and has been proposed as a promising alternative to control
2
−5
milk.
The presence of E. sakazakii in milk processing
equipment and utensils may be due to its ability to adhere and
form biofilms on many surfaces, including silicone, latex,
polycarbonate (used in the bottle manufacture) and stainless
11−13
bacterial virulence.
2
,4
steel.
Quorum quenching by acyl-HSL enzymatic degradation can
be promoted by acyl-HSL acylases that cleave the acyl chain to
produce homoserine lactone and fatty acids and by acyl-HSL
Bacterial processes involved in biofilm formation, virulence
and bioluminescence, among other phenotypic expressions, are
mediated by quorum sensing, a mechanism based on the
production, release and detection of signaling molecules of low
molar mass, the autoinducers (AI). Extracellular concentrations
of AI are related to the microorganism population density, and,
upon reaching a concentration threshold, these signaling
molecules are detected by the cells initiating a coordinated
1
1−13
lactonases that cleave the lactone ring.
Acyl-HSL degrada-
14
tion activity was initially discovered in species of Bacillus sp.
and Variovorax paradoxus but is relatively widespread among
diverse bacteria.
15
Bacillus cereus, a Gram-positive bacterium and member of the
Bacillaceae family, is a microorganism that produces acyl-HSL
6
−8
16−19
action.
lactonase and peptidases.
This bacterium is responsible for
Quorum sensing mechanisms are at the basis of bacterial
contamination in the food industry and in hospitals. Among
others, these include acyl-homoserine lactones (acyl-HSL)
two types of food poisoning, the emetic and the diarrheal
syndromes, and a variety of local and systemic infections, such
2
0,21
as meningitis.
Similar to E. sakazakii, B. cereus also affects
22
(AI-1) in Gram-negative bacteria, oligopeptides in Gram-
milk quality and its derivatives, mainly due to its ability to
positive bacteria, autoinductor-2 (AI-2) in Gram-positive and
Gram-negative interspecies communication, butyrolactones in
Streptomyces, fatty acids, usually classified as diffusible signal
factors, in Xanthomonas fastidiosa, diketopiperazines in Proteus
mirabilis, and AI-3/epinefrin/norepinefrin, which is present in
Received: September 22, 2011
Revised: November 29, 2011
Accepted: December 1, 2011
Published: December 1, 2011
©
2011 American Chemical Society
585
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Journal of Agricultural and Food Chemistry
Article
form biofilms and spores that are heat-resistant, surviving food
processing and remaining for long periods in dried foods.
peptone (Oxoid), 0.5% NaCl (Sigma) and 0.5% yeast extract (Oxoid)
to 100 mL of sterile water.
23
Agrobacterium tumefaciens NTL4(pZLR4) was kept in LB medium
plus gentamicine (50 μg/mL). C. violaceum CV026 strain also was
grown in LB medium supplemented with 20 μL/mL kanamycin at
25 °C for 24 h. Solid media were prepared with 2% bacteriological agar
(Sigma).
Bioassays with Agrobacterium tumef aciens NTL4(pZLR4). E.
sakazakii, B. cereus ATCC 14579 and mixed cultures of E. sakazakii
and B. cereus ATCC 14579 were grown in slants and inoculated in
Both the genus Bacillus and the genus Enterobacter occur in
dairy environments and in pasteurized liquid milk products and
are responsible for food spoilage together with other micro-
2
4,25
organisms coexisting in biofilms.
The existence of multiple
species biofilms has motivated the present investigation on cell-
to-cell communication via the acyl-HSL of a Gram-negative
pathogenic bacterium (E. sakazakii) in the presence of a
cooccurring Gram-positive bacterium (B. cereus).
2
mL of LB liquid medium in a BOD (biochemical oxygen demand)
incubator at 28 °C for 24 h. Cultivation media with the pure strains of
microorganisms and in mixed cultures were maintained at pH 7.0 in
all experiments. Assays were performed in test tubes. Test tube
MATERIALS AND METHODS
General Experimental Procedures. All solvents were treated
■
1
(negative control): 20 μL of A. tumefaciens NTL4(pZLR4), 20 μL of
X-Gal (50 mg/mL in DMF), 2 mL of LB. Test tube 2: 20 μL of A.
tumefaciens NTL4(pZLR4), 20 μL of E. sakazakii, 20 μL of X-Gal,
with anhydrous Na SO and distilled. The reagents were from Aldrich,
2
4
Sigma and Merck. Column chromatography purifications were carried
out with silica gel (Acros, 0.035−0.070 mm) and thin layer chro-
matographic analyses with silica gel 60 F254 plates (Merck), visualized
2
mL of LB medium. Test tube 3: 20 μL of A. tumefaciens
NTL4(pZLR4), 20 μL of B. cereus ATCC 14597, 20 μL of X-Gal,
mL of LB medium. Test tube 4: 20 μL of A. tumefaciens
NTL4(pZLR4), 20 μL of E. sakazakii + B. cereus ATCC 14597,
0 μL of X-Gal, 2 mL of LB medium. All experiments were performed
in duplicate (two different batches). The test tubes were incubated in
2
with UV light (254 nm) or sprayed with a solution of KMnO and
4
p-anisaldehyde (5%), acetic acid (50 mL), and concentrated sulfuric
acid (1 mL) and heated to ∼100 °C with a heat gun.
GC−MS analyses. GC−MS analyses (70 eV) were carried out
with an Agilent 6890 chromatograph coupled to a Hewlett-Packard
2
2
6,27
BOD at 30 °C, and were visually evaluated after 24 h.
General Procedures for Extract Preparations. E. sakazakii, B.
cereus ATCC 14579 and a mixed culture (E. sakazakii and B. cereus
ATCC 14579) in 10 mL of LB were kept in a BOD at 30 °C for 24 h.
These were then transferred to separate Erlenmeyers (2 L) containing
liquid LB (1 L) and incubated at 30 °C for 24 h under stirring. After
this time the mixtures were centrifuged at 5000 rpm for 20 min and
NaCl was added to the aqueous solution that was then extracted with
ethyl acetate (3 × 300 mL). The combined organic phases were dried
over anhydrous magnesium sulfate and filtered, and the solvent was
evaporated under reduced pressure, producing crude extracts of
E. sakazakii (87.52 mg), B. cereus ATCC 14579 and mixed cultures of
E. sakazakii and B. cereus ATCC 14579 (89.15 mg). A control experi-
ment was set with sterile LB culture medium (1 L) which was extracted
with ethyl acetate as described above, producing 80.50 mg. The micro-
organism culture extracts were dissolved in ethanol (2 mg/mL), and
20 μL of each solution was used in the bioassays with A. tumefaciens
NTL4(pZLR4) to detect the expression of β-galactosidase.
Purification of the Acyl-HSLs of E. sakazakii and Mixed
Culture Extracts. Cultivation and extraction with ethyl acetate
was repeated eight times yielding 0.70 g (E. sakazakii) and 0.71 g
(E. sakazakii + B. cereus ATCC 14579) of crude extracts. These
extracts were fractionated by silica column chromatography (15 g of
silica, 2 cm column diameter, particle size 0.035 to 0.070 mm), eluting
with hexane, ethyl acetate, methanol and mixtures of these solvents of
gradually increasing polarity. All fractions (10 mL) were monitored by
TLC. Additionally the fractions were dissolved in ethanol (2 mg/mL)
and monitored with A. tumefaciens NTL4(pZLR4) and C. violaceum
CV026 biosensors. These bioassays were performed by adding 20 μL
of a cell suspension of the biosensor to 20 μL of solution in test tubes
containing 2 mL of LB medium, which were incubated at 25 °C for
24 h. A control experiment for C. violaceum CV026 biosensor was
performed by adding 20 μL of cell suspension, grown for 24 h, to
20 μL of ethanolic solution of the synthetic (S)-N-heptanoyl-HSL
solution (2 mg/mL). The development of a violet color indicated the
presence of short chain acyl-HSL. The control experiment for A.
tumefaciens NTL4(pZLR4) biosensor was performed by adding 20 μL
of cell suspension grown for 24 h to 20 μL of synthetic (S)-N-
dodecanoyl-HSL solution (2 mg/mL). The development of a blue-
green color indicated the presence of acyl-HSL.
5
973 mass detector, equipped with a 30 m × 0.25 mm × 0.25 μm i.d.
DB5 fused silica capillary column. Mass spectra were recorded over the
4
flow rate of 1 mL/min. The injector temperature was kept at 250 °C.
The initial oven temperature was 100 °C, increasing at 10 °C/min to
0−600 amu range at 3.54 scans/s. Helium was the carrier gas at a
2
90 °C, then held for 10 min. Samples (1 mg/mL) of 1 μL were
injected using a 10:1 split ratio.
Chiral GC-FID Analyses. Chiral GC-FID analyses were carried
out with an Agilent 6890 chromatograph with a 25.0 m × 0.25 mm ×
0
.25 μm chiral capillary column (Chrompack CP Chirasil cyclodextrin
CB coating 7502). High purity hydrogen was the carrier gas at a flow
rate of 1 mL/min. Samples of 1 μL were injected, with a 1:100 split
ratio. For N-heptanoyl-HSL the temperature program was 50−180 °C
(
2 °C/min), held for 5 min at 180 °C, with the detector at 250 °C and
the injector at 220 °C. For N-dodecanoyl-HSL the isothermal
temperature was 180 °C for 150 min, the detector was at 240 °C
and the injector was at 280 °C. For N-tetradecanoyl-HSL the iso-
thermal temperature was 180 °C for 200 min, the detector was at
2
20 °C and the injector was at 240 °C.
ESI-MS Analyses. All experiments were performed using a LTQ-
Orbitrap hybrid mass spectrometer (Thermo Fischer Scientific,
Bremen, Germany). Nitrogen gas was used for nebulization, desolva-
tion and collision-induced dissociation (CID). The samples were
diluted in methanol, adding 0.1% of ammonium hydroxide for the
negative mode, and submitted to direct injection at atmospheric
pressure and at a flow rate of 10 μL/min. The positive ionization mode
was best to detect the (S)-N-dodecanoyl-HSL, sheat gas = 8, spray
voltage = 3.5 kV, capillary voltage = 43 V, capillary temperature =
2
75 °C. The negative ionization mode was more appropriate to
monitor the degradation of acyl-HSL, with sheat gas = 10, spray
voltage = 3.8 kV, capillary voltage at −38 V and capillary temperature
2
75 °C. Full scan experiments (range m/z 50−800) were performed in
both the linear trap as well as the Orbitrap. Masses were acquired as
profile data at a resolution of 30 000 at m/z 400. The automatic gain
control (AGC) ion population target in full scan MS was 30 000 for
LTQ-MS and 300 000 for Orbitrap-MS, and the ion population target
for MSn was set to 10 000 for LTQ-MS. Mass data were analyzed by
Xcalibur 2.0.5 software (Thermo Electron Corporation).
Bacteria Strains and Cultivation Media. E. sakazakii DUP
1
8623 was isolated from infant feeding bottles from a hospital nursery.
Coinjection of Synthetic Standards with Natural Products.
Structures of acyl-HSLs detected in the fractions of the E. sakazakii
crude extract by GC−MS were confirmed by coinjections with
synthetic standards previously synthesized and fully characterized in
B. cereus ATCC 14579 was obtained from the Laboratory of Hygiene
and Legislation of the State University of Campinas (UNICAMP).
Both strains were kept in Eppendorf tubes containing BHI (brain heart
infusion) and glycerol (80:20) at −80 °C. B. cereus CCT 4060 was
purchased from the Andre
Brazil, and was kept in Luria−Bertani medium (LB) adding 1%
2
8,29
our laboratory.
́
Tosello Culture Collection, Campinas,
Determination of the Absolute Configuration of Acyl-HSLs.
The absolute configuration of the natural acyl-HSLs was determined
5
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Journal of Agricultural and Food Chemistry
Article
by GC-FID equipped with a Chrompack Chirasil cyclodextrin column,
applying the chromatographical conditions previously described, to dis-
criminate both enantiomers of the racemic synthetic and (S) enantio-
mer standards: 66.54 min = (R)-N-heptanoyl-HSL, 66.75 min = (S)-N-
28
heptanoyl-HSL; 77.49 min = (R)-N-dodecanoyl-HSL, 78.75 min =
2
9
(
1
S)-N-dodecanoyl-HSL; and 169.81 min = (R)-N-tetradecanoyl-HSL,
29
72.60 min = (S)-N-tetradecanoyl-HSL. The natural product was
determined by injecting the fractions and standards using the same
conditions and by coinjecting with synthetic racemic product.
Figure 1. Bioassays with A. tumefaciens NTL4(pZLR4). (A) E. sakazakii
cell suspension. (B) B. cereus ATCC 14579 cell suspension. (C) E.
sakazakii and B. cereus ATCC 14579 mixed cell suspension. (D) Nega-
tive control. Assays in duplicate (two different batches). Blue-green
color reveals the expression of galactosidase due to the presence of
acyl-HSL. This enzyme hydrolyzes X-Gal added to the reaction
(
S)-N-Dodecanoyl-HSL Biotransformation by B. cereus ATCC
1
4579 and CCT 4060. B. cereus ATCC 14579 was inoculated into
test tubes containing 10 mL of LB liquid medium and kept in a BOD
at 30 °C for 24 h. After the incubation period, the test tubes were
centrifuged at 5000 rpm for 20 min to separate the cells. To 50 mL
Falcon tubes were added sterile phosphate buffer (5 mL, pH 7.0), wet
cells (100 mg) and (S)-N-dodecanoyl-HSL (1 mg dissolved in
ethanol). The bioreaction cell-free solution (filtrated) was monitored
for 8 h by ESI-MS−MS in the negative mode, but there was signal
suppression (sample D). Therefore, bioreactions using two different
strains of B. cereus (ATCC 14579 and CCT 4060) were monitored for
three hours and the resulting suspensions were stirred at 220 rpm and
3
0
medium producing a colored compound. Yellow is the color of the
natural medium.
no enzymes, yielding a blue-green color in the culture medium
after 24 h, confirming the presence of acyl-HSL, which is
consistent with the results employing cell suspensions (Figure 2a).
Negative control experiments with A. tumefaciens NTL4(pZLR4)
29 °C for 1 h and then extracted with ethyl acetate (3 × 5 mL). The
combined organic phases were dried over anhydrous magnesium
sulfate, filtered and the solvent was evaporated under reduced pressure
(
sample E). Control experiments were done with sterile phosphate
buffer (5 mL) and wet cells (100 mg) (control 1) and with sterile
phosphate buffer (5 mL) and substrate (1 mg dissolved in ethanol)
(
control 2), and were extracted with ethyl acetate as described above.
The sample E and control experiments were analyzed by electrospray
ionization mass spectrometry (ESI-MS) and also monitored by TLC.
The same procedure was repeated with B. cereus CCT 4060.
Samples for ESI-MS Analyses. Metanolic solutions A, B and C
containing homoserine lactone, (S)-N-dodecanoyl-homoserine lactone
and dodecanoic acid were prepared (Table 1) to optimize the ESI-MS
Figure 2. Bioassays with A. tumefaciens NTL4(pZLR4). (A) (S)-
dodecanoyl-HSL (positive control). (B) Negative control. (C) E. sakazakii
ethyl acetate extract. (D) E. sakazakii and B. cereus ATCC 14579 ethyl
acetate extract. Assays in duplicate (two different batches). Blue-green
color reveals the expression of galactosidase due to the presence of
acyl-HSL. This enzyme hydrolyzes X-Gal added to the reaction
2
Table 1. Samples Used in ESI-MS Analyses and MS
Optimization
a
b
c
d
sample
HSL
2
5
A
B
C
422.48
211.30
271.33
678.56
383.88
3
0
medium producing a colored compound. Yellow is the color of the
natural medium.
1026.65
192.00
a
b
c
Values in μmol/L. Homoserine lactone. (S)-N-Dodecanoyl-homo-
d
serine lactone. Dodecanoic acid.
were performed with cell suspensions in LB medium containing
X-gal, and the blue-green color was not observed.
2
analyses and MS (fragmentation pattern, daughter ions). Sample D
Notwithstanding this evidence there are limitations, and one
must be cautious in the interpretation of data based on acyl-
HSL biosensors. Therefore the isolation and identification of
the acyl-HSLs produced by E. sakazakii was undertaken.
Identification of E. sakazakii Acyl-HSLs. Fractions F50−
57, eluted with hexane/ethyl acetate (50%), and F58−64,
eluted with pure ethyl acetate, produced positive response with
A. tumefaciens NTL4(pZLR4). Additionally fractions F50 and
F56−62 responded to C. violaceum CV026, indicating the pre-
sence of short chain acyl-HSL.
was prepared by adding 500 μL of methanol to 500 μL of the
biotransformation reaction liquid medium (filtered, details in the
biotransformation reaction section). The resulting solution was split
into two samples of 500 μL for the negative and positive detection
modes. Sample E and the controls were prepared by adding 10 μL of a
stock solution (1 mg of ethyl acetate extract of the biotransformation
reaction, in 1 mL of methanol) to 100 μL of methanol (details in the
biotransformation reaction section).
RESULTS AND DISCUSSION
■
The bioactive fractions were analyzed by GC−MS, moni-
Detection of Acyl-HSL in E. sakazakii. The E. sakazakii
cell suspension furnished a blue-green solution in the presence
biosensor A. tumefaciens NTL4(pZLR4) and X-gal (Figure 1a).
This color suggested that A. tumefaciens NTL4(pZLR4)
detected E. sakazakii acyl-HSL by expressing a phenotypic ga-
lactosidase which hydrolyzed the 5-bromo-4-chloro-3-indolyl-β-
D-galactopyranoside (X-Gal) added to the reaction, yielding the
toring acyl-HSL characteristic fragments at m/z 185, 143, 128,
•
1
02, 101, 100 and 85. N-Heptanoyl-HSL (1) (Figure 3) (M+
m/z 213) was detected in fractions F56−F62 (Figure 1S of the
Supporting Information). An unsubstituted lateral chain was
suggested by the decreasing relative abundances of fragments
−
CH CH CH , at m/z 156 (13%), and −CH CH , at m/z 170
2
2
3
2
3
27,30
indigo blue derivative.
Since naturally occurring E. sakazakii
(4%). The base peak at m/z 143 was characteristic of the
acyl-HSL odd electron cation fragment formed during the
McLafferty rearrangement. N-dodecanoyl-HSL (2) (Figure 3)
β-galactosidase could hydrolyze X-gal and produce a false posi-
tive result, A. tumefaciens NTL4(pZLR4) biosensor was
employed with ethyl acetate extracts of E. sakazakii, carrying
+
•
(M m/z 283) was detected in fractions F56−F62 (Figure 2S
5
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Journal of Agricultural and Food Chemistry
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follow an N-acyl bond hydrolysis, thus demanding a better in-
vestigation of the acyl-HSL depletion mechanism.
Acyl-HSL Depletion Mechanism by B. cereus Enzymes. To
detect such enzymatic reactions we first selected and optimized
the ESI-MS−MS spectrometry methodology by using a mixture
of acyl-HSL, a fatty acid and homoserine in different con-
centrations (Table 1). The homoserine and acyl-HSL were best
detected in the positive mode at 422.48 and 271.33 μmol/L
levels. However, the fatty acid and, consequently, the opened
acyl-HSL (this standard was not available) were best detected
in the negative mode at 383.88 μmol/L. The bioreaction to
detect acyl-HSL lactonase and/or acyl-HSL acylase activities in
B. cereus to explain their degradation in mixed cultivation of
E. sakasaki and B. cereus was set with (S)-N-dodecanoyl-HSL
and B. cereus cells in buffer solution. The bioreaction cell-free
solution was monitored by ESI-MS−MS in the negative mode
for 8 h but (S)-N-dodecanoyl-HSL (2), N-dodecanoyl homo-
serine (4), dodecanoic acid (5) and homoserine (6) (Figure 4b)
were not detected due to signal suppression. Consequently
bioreactions using two different strains of B. cereus (ATCC
Figure 3. (S)-N-Acyl-homoserine lactones identified in E. sakazakii
(Cronobacter spp.) extracts: (S)-N-heptanoyl-HSL (1), (S)-N-dodec-
anoyl-HSL (2) and (S)-N-tetradecanoyl-HSL (3).
of the Supporting Information), and N-tetradecanoyl-HSL (3)
+
•
(
Figure 3) (M m/z 311) was present in F62−F63 (Figure 3S
of the Supporting Information), both displaying the acyl-HSL
27
characteristic fragmentation pattern at m/z 143. The detected
compounds were coinjected with synthetic standards, revealing
perfect chromatographic and mass fragmentation pattern
matchings (Figures 4S−6S of the Supporting Information).
Acyl-HSLs Absolute Configuration. The absolute config-
uration of the acyl-HSL produced by E. sakazakii (Cronobacter
spp.) was determined using a gas chromatography equipped
with a Chrompack chiral column (Chirasil cyclodextrin CB)
and a flame ionization detector. Racemic (±)-N-heptanoyl-HSL
standard was used to optimize the analytical conditions to have
a good enantiomeric resolution where (R) eluted in 66.54 min
and (S) eluted in 66.74 min. Synthetic (S)-N-heptanoyl-HSL
eluted in 66.78 min, and this was close to the elution time
of the N-heptanoyl-HSL in fraction F59 (66.73 min). A
coinjection of the natural product with the racemic synthetic
standard produced an enhancement of the relative abundance
of the (S) (66.73 min) peak in relation to the (R) (66.53 min)
enantiomer, confirming the presence of (S)-N-heptanoyl-HSL.
The (S) configuration of the N-dodecanoyl-HSL in F63 and
N-tetradecanoyl-HSL in F55 was also confirmed (Figure 7S of
the Supporting Information).
1
4579 and CCT 4060) were monitored for three hours, being
extracted with ethyl acetate prior to negative mode ESI-MS−MS
analyses.
The bioreaction with B. cereus CCT 4060 and (S)-N-
dodecanoyl-HSL (2) produced N-dodecanoyl homoserine (4),
suggested by the presence of the quasi-molecular ion (M − H)
at m/z 300.2183 (Figure 4b), indicating that 18 amu had been
added to the (S)-N-dodecanoyl-HSL by enzymatic action. This
is in agreement with N-dodecanoyl-homoserine (4), confirming
the presence of acyl-HSL lactonase (Figure 4c).
2
The MS fragmentation of the quasi-molecular ion m/z
3
00.2183 produced ions at m/z 282.2068, characteristic of loss
of a water molecule, at m/z 256.2275, assigned to the loss of
CO , at m/z 198.1857, assigned to N−C bond cleavage, and at
2
m/z 118.0504, corresponding to the loss of the homoserine
moiety (Figure 5a).
Detection of Acyl-HSL in Mixed Cultivation of
E. sakazakii and B. cereus ATCC 14579. A negative response
from A. tumefaciens NTL4(pZLR4) biosensor was obtained
with the B. cereus ATCC 14579 cell suspension (Figure 1b), a
Gram-positive bacterium employing cell-to-cell chemical
communication different from acyl-HSL. This biosensor had
an analogous negative response with the mixed cell suspension
and ethyl acetate extracts of both B. cereus and E. sakazakii
Additionally the quasi-molecular ions at m/z 199.1702 and
18.0507 revealed the formation of dodecanoic acid (5) and
homoserine (6), respectively, which are the expected products
of an acyl-homoserine acylase on N-dodecanoyl-homoserine
4) (Figure 4c). These compounds were not detected in the
ethyl acetate extracts of the control reactions with and without
1
(
B. cereus CCT 4060 cells in phosphate buffer (Figure 4a and
2
Figure 10S of the Supporting Information) The MS frag-
(
Figures 1c and 2d). These results were interpreted as deple-
mentation of the ion at m/z 118.0507 produced the ion at m/z
00.0401 by loss of a water molecule (Figure 5c). These results
are also in agreement with the presence of an acyl-homoserine
acylase (Figure 4c).
tion of the E. sakazaki acyl-HSLs by B. cereus enzymes. To
confirm this hypothesis we have searched for acyl-HSL in the
ethyl acetate extract of the E. sakazakii and B. cereus ATCC
1
14579 mixed culture and investigated hydrolase activity in
Furthermore the genes coding for acyl-HSL inactivating
18
different strains of B. cereus.
enzymes were detected and studied, showing that they are
present in various microorganisms; moreover, mining
sequenced genome databases revealed organisms possessing
conserved domains for acyl-HSL lactonases and acylases
(Deinococcus, Streptomyces, Hyphomonas, Photorhabdus and
Ralstonia), however, Bacillus showed domain only for acyl-
A dereplication strategy was applied to the chromatographic
fractions with A. tumefaciens NTL4(pZLR4) and C. violaceum
CV026 revealing that fraction M42 had a positive response with
A. tumefaciens NTL4(pZLR4) (Figure 8S of the Supporting
Information). The GC−MS analysis in the SIM mode (single
ion monitoring, m/z 143) revealed the presence of trace amounts
of N-dodecanoyl-HSL (2) (Figure 9S of the Supporting
Information), confirming that most acyl-HSL were depleted by
B. cereus ATCC 14579. This degradation phenomenon of Gram-
negative bacterium quorum signal molecules, acyl-HSLs, by
B. cereus strains of (Gram-positive) producing acyl-HSL lactonases
was already reported in the literature for Erwinia carotovora and
31
HSL lactonase. The acylase activity has never been reported
32
in Bacillus before, but, based on enzymatic promiscuity, that
refers to one enzyme accepting more than one substrate, and
16
on the fact that peptidases were detected in B. cereus genome,
our results are novel but not unexpected.
Finally, in B. cereus ATCC 14579 acyl-HSL lactonase was
detected, however, no acyl-homoserine acylase activity was
observed.
19
Yersinia enterocolitica. However acyl-HSL degradation could
5
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Figure 4. Bioreaction monitoring with ESI-MS. (a) Spectrum of control experiment (ethyl acetate extract of sterile phosphate buffer and wet cells).
b) Spectrum and compounds 2, 4, 5 and 6 indicated with arrows. (c) Schematic origin of 4, 5 and 6.
(
In conclusion, this work revealed that the E. sakazakii strain
however, further research will be necessary to identify the
produces homoserine lactones (S)-N-heptanoyl-HSL, (S)-N-
dodecanoyl-HSL and (S)-N-tetradecanoyl-HSL, results not
before reported in the literature. The identification and char-
acterization of these structures was the first important step,
functions regulated by these molecules in E. sakazakii.
The present work also provides evidence of acyl-HSL-degrading
capacities during the cocultivation of a Gram-negative bac-
terium (E. sakazakii) with a Gram-positive bacterium
5
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Journal of Agricultural and Food Chemistry
Article
2
2
2
Figure 5. (a) MS of fragment at m/z 300.2183; (b) MS of fragment at m/z 199.1702 and (c) MS of fragment at m/z 118.0507.
(
B. cereus). This phenomenon was further investigated
synchronized operation are responsible for degradation by
B. cereus, leading to a disruption of the acyl-HSL communication
system.
revealing that a cascade of enzymatic reactions with an acyl-
HSL lactonase and acyl-homoserine acylase (or peptidase) in
5
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Article
(
11) Dong, Y. H.; Zhang, L. H. Quorum sensing and quorum
ASSOCIATED CONTENT
Supporting Information
Additional chromatograms and mass spectra, and bioassay with
A. tumefaciens NTL4(pZLR4) for E. sakazakii and B. cereus
ATCC 14579 mixed culture extract fractions (Figures 1S−9S).
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
quenching enzymes. J. Microbiol. 2005, 43, 101−109.
(
lactone-dependent quorum sensing by enzymatic disruption of signal
molecules. Acta Biochim. Pol. 2009, 56, 1−16.
(
dialogues: from natural autoinducers to non-natural analogues that
modulate quorum sensing in Gram-negative bacteria. Chem. Soc. Rev.
*
S
12) Czajkowski1, R.; Jafra, S. Quenching of acyl-homoserine
13) Geske, G. D.; O’Neill, J. C.; Blackwell, H. E. Expanding
2
008, 37, 1432−1447.
AUTHOR INFORMATION
Corresponding Author
Tel: +55 19 35213067. Fax: +55 19 35213023. E-mail: anita@
iqm.unicamp.br.
(14) Dong, Y. H.; Wang, L. H.; Xu, J. L.; Zhang, H. B.; Zhang, X. F.;
Zhang, L. H. Quenching quorum-sensing-dependent bacterial
infection by an N-acyl homoserine lactonase. Nature 2001, 411, 813−
■
*
8
(
17.
15) Leadbetter, J. R.; Greenberg, E. P. Metabolism of acyl-
Present Address
Department of Bromatology, Faculty of Pharmacy, Fluminense
Federal University, Niteroi, RJ, Brazil.
homoserine lactone quorum-sensing signals by Variovorax paradoxus.
J. Bacteriol. 2000, 182, 6921−6926.
(16) Tran, S. L.; Guillemet, E.; Gohar, M.; Lereclus, D.; Ramarao, N.
CwpFM (EntFM) is a Bacillus cereus potential cell wall peptidase
implicated in adhesion, biofilm formation, and virulence. J. Bacteriol.
§
Funding
The authors acknowledge CNPq and FAPESP for financial
support.
2
(
010, 192, 2638−2642.
17) Medina-Martínez, M. S.; Uyttendaele, M.; Rajkovic, A.; Nadal,
P.; Debevere, J. Degradation of N-acyl-L-homoserine lactones by
Bacillus cereus in culture media and pork extract. Appl. Environ.
Microbiol. 2007, 73, 2329−2332.
ACKNOWLEDGMENTS
The authors acknowledge Carol H. Collins (IQ/UNICAMP)
for revising the manuscript.
■
(18) Dong, Y.; Gusti, A. R.; Zhang, Q.; Xu, J.; Zhang, L. Iden-
tification of quorum-quenching N-acyl homoserine lactonases from
Bacillus species. Appl. Environ. Microbiol. 2002, 68, 1754−1759.
ABBREVIATIONS USED
HSL, homoserine lactone; X-gal, 5-bromine-4-chloro-3-indolyl-
β-D-galactopyranoside; BOD, biochemical oxygen demand
■
(19) Zhao, C.; Zeng, H.; Yu, Z.; Sun, M. N-Acyl homoserine
lactonase promotes prevention of Erwinia virulence with zwittermicin
a-producing strain Bacillus cereus. Biotechnol. Bioeng. 2008, 100, 599−
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