Detection of acylated homoserine lactones in gram-negative proteolytic psychrotrophic bacteria isolated from cooled raw milk

Abstract

Through a mechanism called quorum sensing, bacteria are able to express specific genes in response to population density. Cell-to-cell communication in bacteria is mediated by signal molecules such as acylated homoserine lactones (AHLs). This work aimed to detect AHL production in Gram-negative psychrotrophic bacteria isolated from raw milk. A total of 84.9% of the bacteria were identified as AHL producers eliciting a diversity of responses in the AHL-monitor systems. These results demonstrate that AHL-production is common among psychrotrophic bacteria isolated from milk, and indicate that quorum sensing may play an important role in the spoilage of this product.

Full text

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Short communication
Detection of acylated homoserine lactones in gram-negative proteolytic
psychrotrophic bacteria isolated from cooled raw milk
Uelinton Manoel Pinto1, Eliseth de Souza Viana, Maurilio Lopes Martins,
Maria Cristina Dantas Vanetti*
Department of Microbiology, Federal University of Vic ¸osa, Vic ¸osa, MG, 36570-000, Brazil
Received 8 May 2006; received in revised form 6 September 2006; accepted 12 September 2006
Abstract
Through a mechanism called quorum sensing, bacteria are able to express specific genes in response to population density. Cell-to-cell
communication in bacteria is mediated by signal molecules such as acylated homoserine lactones (AHLs). This work aimed to detect
AHL production in Gram-negative psychrotrophic bacteria isolated from raw milk. A total of 84.9% of the bacteria were identified
as AHL producers eliciting a diversity of responses in the AHL-monitor systems. These results demonstrate that AHL-production is
common among psychrotrophic bacteria isolated from milk, and indicate that quorum sensing may play an important role in the spoilage
of this product.
? 2006 Elsevier Ltd. All rights reserved.
Keywords: Quorum sensing; Acylated homoserine lactones (AHL); Psychrotrophic bacteria; Raw milk
1. Introduction
Gram-negative proteolytic psychrotrophic bacteria are
the predominant microorganisms responsible for spoilage
of milk and milk products due to their ability to produce
thermostable proteases that hydrolyze casein and decrease
the yield and sensory quality of dairy products (Dogan &
Boor, 2003; Sørhaug & Stepaniak, 1997). Some bacteria
also secrete lecithinases and lipases that can play a signifi-
cant role in the spoilage of these products (Dogan & Boor,
2003). It is known that activity of hydrolytic enzymes is
detected at the end of logarithmic phase and at the begin-
ning of the stationary growth phase of these bacteria
(Matselis & Roussis, 1998; Rajmohan, Dodd, & Waites,
2002), conditions in which a high cell density is achieved.
Bacteria are able to regulate expression of phenotypic
characteristics as a function of cell density in a mechanism
termed quorum sensing (QS) (Fuqua, Winans, & Green-
berg, 1994). In Gram-negative bacteria, this regulation is
typically mediated by chemical signals such as N-acyl-L-
homoserine lactones (AHL) (Fuqua et al., 1994; White-
head, Barnard, Slater, Simpson, & Salmond, 2001). The
acyl side chain of different AHL can vary from 4 to 18
carbons in length, degree of substitution, and saturation
providing specificity to QS systems (Zhu, Chai, Zhong,
LI, & Winans, 2003). The key regulatory components of
these signaling systems are LuxI-type proteins which act
as AHL synthases, LuxR-type proteins which serve as
AHL receptors, and AHL-dependent transcription factors
(Fuqua & Greenberg, 2002). Examples of phenotypes reg-
ulated by AHLs include production of antibiotics, biofilm
development, competence for DNA uptake, cell differenti-
ation, bioluminescence, growth, pigment production, con-
jugal plasmid transfer, virulence gene expression, and
production of a range of degradative extracellular enzymes
(Smith, Fratamico, & Novak, 2004).
Several works have been done to elucidate the role of QS
in food spoilage. AHLs have been detected in some food
products such as cold-smoked salmon (Gram, Christensen,
0956-7135/$ - see front matter ? 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2006.09.005
*Corresponding author. Tel.: +55 31 38992954; fax: +55 3138992573.
E-mail address: mvanetti@ufv.br (M.C.D. Vanetti).
1Present address: 360 Wing Hall, Department of Microbiology, Cornell
University, Ithaca, NY 14853, USA.
www.elsevier.com/locate/foodcont
Food Control 18 (2007) 1322–1327

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Ravn, Molin, & Givskov, 1999), meat (Bruhn et al., 2004),
and bean sprouts (Rasch et al., 2005). In this last work, it
was shown that pectinase, protease, cellulase and sidero-
phore-mediated iron chelation were regulated by QS,
underlying the importance of this system in spoilage of
bean sprouts. Christensen et al. (2003) demonstrated that
several hydrolytic enzymes produced by Serratia protea-
maculans, a typical member of a food spoilage flora, are
regulated by QS. The involvement of QS in meat spoilage
process, and biofilm formation was suggested by Jay, Vilai,
and Hughes (2003). Furthermore, Liu and Griffths (2003)
pointed out that spoilage of milk by Pseudomonas fluores-
cens is correlated with its ability to produce AHLs and
the extracellular protease. However, currently, little is
known about signal molecule production, such as AHLs,
by bacteria isolated from food products.
The AHL detection is based on different bacterial bioas-
says. The use of reporter bacteria, in which the luxI homo-
logue gene responsible for AHL production had been
inactivated, has shown to be a valuable system for AHL
detection (Ravn, Christensen, Molin, & Givskov, 2001).
Then, the expression of a reporter gene is possible only in
the presence of exogenous AHLs. Plasmid reporter vectors
that respond to activation of LuxR homologues have also
been used in Escherichia coli strains (Winson et al., 1998).
Because of the specificity of each LuxR homologue, repor-
ter strains display specificity towards different AHL mole-
cules, allowing detection of a wide range of AHLs and
differentiation between AHL production patterns (Van
Houdt, Aerstsen, Jansen, Quintana, & Michiels, 2004).
For instance, LuxR of Vibrio fisheri, used in several repor-
ter plasmids such as pSB403, is activated by AHLs with
carbon chains of C6 or C8 with or without 3-oxo substitu-
tions (Winson et al., 1998). On the other hand, CviR of
Chromobacterium violaceum is sensitive to unsubstituted
chains varying in size from C4 to C8, and also is able to
detect long-chain AHLs by their ability to inhibit violacein
production if an activating AHL is incorporated to the
medium (McClean et al., 1997). The TraR of Agrobacte-
rium tumefaciens is sensitive to most 3-oxo AHLs (Shaw
et al., 1997).
In order to improve the understanding of the process of
cell-to-cell communication among bacteria found in milk,
this work aimed to evaluate the production of AHL in pro-
teolytic psychrotrophic bacteria isolated from cooled raw
milk.
2. Material and methods
2.1. Bacterial strains and culture conditions
Gram-negative psychrotrophic bacteria isolated from
cooled raw milk (Pinto, 2004) and strains from American
type culture collection – ATCC (Table 1) were investigated
for AHL-production. The bacteria were stored in brain
heart infusion (BHI) with the addition of glycerol to 20%
v/v and maintained at ?80 ?C. The strains were activated
in Luria Berthani broth (LB) and incubated at 25 ?C for
24 h with agitation (150 r.p.m.). Ultracompetent E. coli
DH5a was transformed with pSB403 (Winson et al.,
1998) using standard methods described by Sambrook,
Fritsch, and Maniats (1989). Chromobacterium violaceum
CV026 (McClean et al., 1997) and Agrobacterium tumefac-
iens a136 (Fuqua & Winans, 1996) were also used to detect
AHL-production. These monitor strains were grown in LB
supplemented with appropriate antibiotics (Ravn et al.,
2001), and incubated at 28 ?C during 24 h, with agitation
(150 r.p.m.), prior the assay. C. violaceum ATCC 6357
and P. aeruginosa 15442 were used as positive controls in
the experiments and the monitor strains as negative control
themselves.
2.2. Screening for AHL-production
The screening for AHL-production was performed
according to Ravn et al. (2001). The tested strains were
streaked in parallel to the monitor strains on LB agar
plates. E. coli (pSB403) is unable to produce luminescence
without exogenous AHL. The luminescence was observed
after approximately 3 min for adaptation of the eyes to a
darkened room, under weak red light, as a positive result
in response to AHL produced by the tested strain. C.
violaceum CV026 produces the pigment violacein only in
the presence of exogenous AHL. Thus, the pigment produc-
tion indicated a positive result. Testing for AHL production
against A. tumefaciens A136 assay, was done in a similar
assay supplementing the LB agar with 50 lg/ml 5-bromo-
4-chloro-3-indolyl-b-D-galactopyranoside
monitor strain has the lacZ gene fused to the promoter of
traI gene, which is regulated by autoinduction. The strain
produces a blue pigment in response to AHL when the med-
ium is supplemented with X-gal. All plates were incubated
for 24 h at 25 ?C, except the ones streaked with A. tumefac-
iens A136 that were incubated up to three days.
Long chains AHLs were detected observing the inhibi-
tion of the pigment production by the induced monitor
strain C. violaceum CV026. LB agar plates were supple-
mented with 75 nM of N-hexanoyl-L-homoserine lactone
(HHL) obtained commercially (Fluka, Switzerland). The
psychrotrophic isolates were streaked in the medium and
incubated for 24 h at 25 ?C. Then, the monitor strain was
streaked in parallel to the previous strains and the plates
were re-incubated at the same conditions. Experiments
were repeated at least twice.
(X-gal).This
2.3. Thin-layer chromatography (TLC)
Extracts for TLC were prepared from 100 ml cultures
after growth in LB medium for 20 h at 25 ?C with agitation
of 150 r.p.m. Bacteria were removed by centrifugation, the
supernatants were extracted twice with equal volumes of
ethyl acetate acidified with 0.5% of formic acid, and the
combined extracts were dried, filtered, and evaporated to
U.M. Pinto et al. / Food Control 18 (2007) 1322–1327
1323

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dryness. Cultures extract were dissolved in 400–600 ll of
HPLC-grade ethyl acetate.
Synthetic AHLs or extract samples dissolved in ethyl
acetate, in volumes of 10–20 ll were spotted onto c18
reversed-phase TLC plates (aluminum sheets 20 · 20 cm;
RP-18 F254 S, 1.05559. Merck 64271, Darmstadt, Ger-
many) and the chromatogram was developed using a sol-
vent system of methanol/water (60:40, v/v) as described
by Shaw et al. (1997). After development, the solvent
was evaporated, and the dried plates were overlaid with
a culture of monitor strain. A 30 ml overnight culture of
E coli (pSB403) or C. violaceum CV026 was used to inoc-
ulate 150 ml of LB medium, and the culture was spread
over the surface of the developed plates. After the agar
had solidified, the plates were incubated overnight at
30 ?C in a sterilized closed plastic container. AHLs were
visualized as bright spots in Eagle Eye II (Stratagene, La
Jolla,CA, USA)on plates
(pSB403), or purple spots for the ones with C. violaceum
CV026.
overlaid with
E. coli
3. Results and discussion
The psycrotrophic proteolytic strains elicited diverse
responses in the three AHL-monitor systems, which dem-
onstrate production of different AHL molecules. A total
of 45 (84.9%) of the psychrotrophic bacteria isolated from
milk were identified as AHL producers (Table 1). Because
of the specificity requirements of the R protein, the LuxR
homologues, most of the detection systems are limited in
the range of AHLs to which they respond (Shaw et al.,
1997). Therefore, it represents a limitation of the bioassay
since bacteria often produce more than one AHL molecule.
This limitation can be overcome by using multiple monitor
systems.
C. violaceum CV026 detected 40 (75.5%) of strains as
AHL producers (Table 1). Within the positive results, 11
(20.7%) produced short chain AHL as demonstrated by
the induction of the violacein production by the reporter
strain (Fig. 1A2), and 33 (62.3%) probably produced long
chain AHL (Fig. 1B1). The lack of the pigment in this
Table 1
Response of psychrotrophic proteolytic bacteria isolated from cooled raw milk using three-monitor systems: Chromobacterium violaceum CV 026,
Escherichia coli DH5a pSB403 and Agrobacterium tumefaciens A136
Species No. of evaluated strains No. responding in monitor system
CV 026pSB403A136
Induced Inhibited
Psychrotrophic proteolytic AHL producers
Acinetobacter lwoffi
Aeromonas hydrophila
Burkholderia pseudomallei
Cedecea lapagei
Chryseobacterium miningosepticum
Chryseomonas luteola
Enterobacter cloacae
Enterobacter gergoviae
Enterobacter sakasaki
Hafnia alvei
Klebsiella oxytoca
Klebsiella ozanae
Moraxella catharralis
Pantoea agllomerans
Pantoea spp
Providencia stuartii
Pseudomonas fluorescens
Pseudomonas putida
Pseudomonas stutzeri
Rhanella aquatilis
Serratia liquefaciens
Serratia odorifera
1
4
2
1
1
2
1
1
2
3
2
2
3
3
1
1
7
2
1
2
9
2
1
1
1
3
2
1
1
2
1
1
1
1
2
33
2
1
2
2
2
2
2
3
5
1
7
1
71
1
2
1
2
Total 5311 33159
ATCC strains
Aeromonas hydrophila ATCC 7966
Chromobacterium violaceum ATCC 6357
Enterobacter aerogenes ATCC 13048
Pseudomonas fluorescens ATCC 13525
Psedomonas aeruginosa ATCC 15442
Serratia liquefaciens ATCC 27592
1
1
1
1
1
1
11
1
1
1
1
1
1
11
Total71623
1324
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experiment is assumed to be due to specific interference
with the AHL-induced production of violacein (McClean
et al., 1997).
The proteolytic psychrotrophic bacteria isolated from
cooled raw milk that were positive in the induction assay
of C. violaceum CV026 may produce AHL compounds
with unsubstituted side chains from C4to C8in length,
since this monitor strain responds to these type of AHLs
(McClean et al., 1997). On the other hand, the strains,
which responded to the inhibition assay, probably produce
Fig. 1. Examples of agar plate assays for screening for AHL production in proteolytic psychrotrophic bacteria isolated from cooled raw milk. Monitor
strains were streaked parallel to the tested bacteria or to themselves as described in Section 2. The blue light seen on plate C2 refers to a positive
luminescence response. A1, B2, C1, and D1 refer to negative results. (A1) Chromobacterium violaceum CV026 parallel to itself; (A2) C. violaceum CV026
parallel to Pantoea agllomerans. B1 and B2 refer to C. violaceum CV026 inibition assay; (B1) C. violaceum CV026 parallel to Rhanella aquatilis as a positive
result for long chain AHL; (B2) C. violaceum CV026 parallel to Serratia odorifera as a negative result for long chain AHL. (C1) Escherichia coli (pSB403)
parallel to itself; (C2) E. coli (pSB403) parallel to Pantoea agllomerans. (D1) Agrobacterium tumefaciens A136 parallel to itself; (D2) A. tumefaciens A136
parallel to Pseudomonas fluorescens.
U.M. Pinto et al. / Food Control 18 (2007) 1322–1327
1325

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AHL with side chain from C10to C14in length (McClean
et al., 1997). Bacteria with positive response in E. coli
pSB403 (Fig. 1C2) may produce AHL with carbon side
chains of C6or C8with or without oxo-substitutions (Win-
son et al., 1998).
E. coli (pSB403) detected 15 (28.3%) and A. tumefaciens
A136 detected 9 (17.0%) psychrotrophic isolates as AHL
producers. Although Shaw et al. (1997) observed that an
A. tumefaciens monitor strain was able to detect AHL with
3-oxo, 3-hydroxy, and 3-unsubstituted side chain, the
majority of the isolates evaluated in this work did not
induce the A. tumefaciens A136 (Table 1), except the P.
fluorescens isolates which produced molecules that pre-
sented a weak response in the assay (Fig. 1D2). This result
indicates that the signal molecule(s) produced by P. fluores-
cens strains were in low concentration or they are different
from the cognate autoinducer that activates TraR protein
in A. tumefaciens. Once A. tumefaciens A136 responded
to substances present in culture media in some works per-
formed in our lab (unpublished data), we cannot discard a
false positive result. Holden et al. (1999) demonstrated that
other molecules besides AHLs could activate biosensors,
underlining the importance of chemical characterization
of the molecules identified in such bioassays.
The bacteria belonging to the Enterobacter genus,
including the ATCC 13048, showed a positive response
only in the inhibition assay using C. violaceum CV026.
The same result occurred with the majority of isolates
belonging to the species S. liquefaciens, P. fluorescens,
A. hydrophila, S. liquefaciens ATCC 27592, and P. fluores-
cens ATCC 13525 (Table 1) indicating that they may pro-
duce long side chain AHLs.
Hafnia alvei produced short chain AHLs once it induced
the reporter strains C. violaceum CV026 (induction assay),
and E. coli (pSB403). Bruhn et al. (2004) observed that
AHL-producing H. alvei may induce food quality-relevant
phenotypes in other bacterial species in the same environ-
ment. Therefore, H. alvei may influence spoilage of food
products in the presence of Enterobacteriacea.
To further confirm the agar assay results, TLC was per-
formed with AHL extracts from representative strains of
A. hydrophila, H. alvei, M. catharralis, Pantoea agllomer-
ans, and P. fluorescens, followed by revelation with
E. coli (pSB403) (Fig. 2) or C. violaceum CV026 (data
not shown). The TLC results indicate the presence of at
least one kind of AHL in the extracts of A. hydrophila,
H. alvei, M. catharralis, and P. agllomerans, confirming
the results of the agar assay.
Whan, Dunstall, and Rowe (2000) suggested the
involvement of QS in the growth of P. fluorescens, a known
spoilage organism in milk and milk products. In another
work, it was observed that N-benzoyloxycarbonyl-L-homo-
serine lactone and N-3-oxyhexanoyl-DL-homoserine lac-
tone significantly reduced the lag phase duration and
increased the exponential growth rate of three strains of
P. fluorescens (Dunstall, Rowe, Wisdom, & Kilpatrick,
2005). Christensen et al. (2003) demonstrated that AHLs
are involved in the production of spoilage characteristics
in milk samples inoculated with Serratia proteamaculans
B5a.
Our data demonstrated that AHL-production is com-
mon among proteolytic psychrotrophic bacteria isolated
from raw milk. Once these organisms were isolated from
a common source, the possibility of cross-communication
between them is relevant and raises the question of what
kind of phenotypes might be regulated when they are grow-
ing together, and also if these phenotypes have any relation
with food deterioration. The understanding of the role of
the QS mechanism in the regulation of spoilage phenotypes
in bacteria from food origin is relevant and may be used to
create new ways to preserve food products. The control of
cell-cell communicationusually
quenching has been studied and proved to be successful
in microorganisms isolated from non-food sources. In the
plant pathogen Erwinia carotovora, the attenuation of vir-
ulence was achieved by inactivating the signal molecule
with an enzyme called AiiA from Bacillus sp 240b1 (Dong,
Xu, Li, & Zhang, 2000). Some other works have shown
that halogenated furanones from alga Dalisea pulchra can
inhibit gene expression mediated by AHL by interfering
with the autoinducer binding to the LuxR homologue
(Manefield, Welch, Givskov, Salmond, & Kjelleberg,
2001; Rice, Givskov, Steingerg, & Kjelleberg, 1999). Ras-
mussen et al. (2005) screened several compounds for their
ability to inhibit quorum sensing and it was found that gar-
lic extract was one of the most effective being able to reduce
P. aeruginosa biofilm tolerance to tobramycin treatment as
well as virulence in a Caenorhabditis elegans pathogenesis
model. As far as this knowledge can be applied to improve
referredtoquorum
Fig. 2. A representative thin-layer chromatogram of the AHLs present in
cell free supernatants of proteolytic psychrotrophic bacteria isolated from
cooled raw milk. Samples were chromatographed on c18 reversed-phase
thin-layer plates, developed with methanol/water (60:40, vol/vol), and the
spots were visualized with the Escherichia coli (pSB403) reporter strain in
Eagle Eye II (Stratagene, La Jolla, CA, USA). Lane 1, extract from LB
medium as a negative control; lane 2, N-hexanoyl-DL-homoserine lactone
standard; lane 3–7, samples from culture extracts of the following: 3,
Aeromonas hydrophila; 4, Pseudomonas fluorescens; 5, Pantoea agllomer-
ans; 6, Moraxella catharralis; 7, Hafnia alvei.
1326
U.M. Pinto et al. / Food Control 18 (2007) 1322–1327

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food quality and safety more work needs to be done to
answer this question.
Acknowledgements
We thank Professor Paul Williams from Nottingham
University (UK) for the donation of plasmid pSB403 and
strain C. violaceum CV026, and Professor Stephen C. Win-
ans from Cornell University (USA) for the donation of
strain A. tumefaciens A136. Uelinton Manoel Pinto and
Maurilio Lopes Martins were supported by the Conselho
Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´gico
(CNPq) Brası ´lia, Brazil. Eliseth de Souza Viana was sup-
ported by the Coordenadoria de Pessoal de Nı ´vel Supe-
rior-Capes, Brazil.
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